|
1
|
Capdevila DA, Rondón JJ, Edmonds KA, Rocchio JS, Dujovne MV, Giedroc DP. Bacterial Metallostasis: Metal Sensing, Metalloproteome Remodeling, and Metal Trafficking. Chem Rev 2024; 124:13574-13659. [PMID: 39658019 DOI: 10.1021/acs.chemrev.4c00264] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2024]
Abstract
Transition metals function as structural and catalytic cofactors for a large diversity of proteins and enzymes that collectively comprise the metalloproteome. Metallostasis considers all cellular processes, notably metal sensing, metalloproteome remodeling, and trafficking (or allocation) of metals that collectively ensure the functional integrity and adaptability of the metalloproteome. Bacteria employ both protein and RNA-based mechanisms that sense intracellular transition metal bioavailability and orchestrate systems-level outputs that maintain metallostasis. In this review, we contextualize metallostasis by briefly discussing the metalloproteome and specialized roles that metals play in biology. We then offer a comprehensive perspective on the diversity of metalloregulatory proteins and metal-sensing riboswitches, defining general principles within each sensor superfamily that capture how specificity is encoded in the sequence, and how selectivity can be leveraged in downstream synthetic biology and biotechnology applications. This is followed by a discussion of recent work that highlights selected metalloregulatory outputs, including metalloproteome remodeling and metal allocation by metallochaperones to both client proteins and compartments. We close by briefly discussing places where more work is needed to fill in gaps in our understanding of metallostasis.
Collapse
Affiliation(s)
- Daiana A Capdevila
- Fundación Instituto Leloir, Instituto de Investigaciones Bioquímicas de Buenos Aires (IIBBA-CONICET), C1405 BWE Buenos Aires, Argentina
| | - Johnma J Rondón
- Fundación Instituto Leloir, Instituto de Investigaciones Bioquímicas de Buenos Aires (IIBBA-CONICET), C1405 BWE Buenos Aires, Argentina
| | - Katherine A Edmonds
- Department of Chemistry, Indiana University, Bloomington, Indiana 47405-7102, United States
| | - Joseph S Rocchio
- Department of Chemistry, Indiana University, Bloomington, Indiana 47405-7102, United States
| | - Matias Villarruel Dujovne
- Fundación Instituto Leloir, Instituto de Investigaciones Bioquímicas de Buenos Aires (IIBBA-CONICET), C1405 BWE Buenos Aires, Argentina
| | - David P Giedroc
- Department of Chemistry, Indiana University, Bloomington, Indiana 47405-7102, United States
| |
Collapse
|
|
2
|
Trost CN, Yang J, Garcia B, Hidalgo-Reyes Y, Fung BCM, Wang J, Lu WT, Maxwell KL, Wang Y, Davidson AR. An anti-CRISPR that pulls apart a CRISPR-Cas complex. Nature 2024; 632:375-382. [PMID: 38961300 DOI: 10.1038/s41586-024-07642-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2022] [Accepted: 05/31/2024] [Indexed: 07/05/2024]
Abstract
In biological systems, the activities of macromolecular complexes must sometimes be turned off. Thus, a wide variety of protein inhibitors has evolved for this purpose. These inhibitors function through diverse mechanisms, including steric blocking of crucial interactions, enzymatic modification of key residues or substrates, and perturbation of post-translational modifications1. Anti-CRISPRs-proteins that block the activity of CRISPR-Cas systems-are one of the largest groups of inhibitors described, with more than 90 families that function through diverse mechanisms2-4. Here, we characterize the anti-CRISPR AcrIF25, and we show that it inhibits the type I-F CRISPR-Cas system by pulling apart the fully assembled effector complex. AcrIF25 binds to the predominant CRISPR RNA-binding components of this complex, comprising six Cas7 subunits, and strips them from the RNA. Structural and biochemical studies indicate that AcrIF25 removes one Cas7 subunit at a time, starting at one end of the complex. Notably, this feat is achieved with no apparent enzymatic activity. To our knowledge, AcrIF25 is the first example of a protein that disassembles a large and stable macromolecular complex in the absence of an external energy source. As such, AcrIF25 establishes a paradigm for macromolecular complex inhibitors that may be used for biotechnological applications.
Collapse
Affiliation(s)
- Chantel N Trost
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
- Genetics and Genome Biology Program, The Hospital for Sick Children, Toronto, Ontario, Canada
| | - Jing Yang
- Key Laboratory of RNA Science and Engineering, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
- Institute of Life Sciences, Jiangsu University, Zhenjiang, China
| | - Bianca Garcia
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
| | - Yurima Hidalgo-Reyes
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
| | - Beatrice C M Fung
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
| | - Jiuyu Wang
- Key Laboratory of RNA Science and Engineering, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
| | - Wang-Ting Lu
- Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada
| | - Karen L Maxwell
- Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada
| | - Yanli Wang
- Key Laboratory of RNA Science and Engineering, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
- University of the Chinese Academy of Sciences, Beijing, China.
| | - Alan R Davidson
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
- Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.
| |
Collapse
|
|
3
|
Rakesh S, Aravind L, Krishnan A. Reappraisal of the DNA phosphorothioate modification machinery: uncovering neglected functional modalities and identification of new counter-invader defense systems. Nucleic Acids Res 2024; 52:1005-1026. [PMID: 38163645 PMCID: PMC10853773 DOI: 10.1093/nar/gkad1213] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2023] [Revised: 12/03/2023] [Accepted: 12/10/2023] [Indexed: 01/03/2024] Open
Abstract
The DndABCDE systems catalysing the unusual phosphorothioate (PT) DNA backbone modification, and the DndFGH systems, which restrict invasive DNA, have enigmatic and paradoxical features. Using comparative genomics and sequence-structure analyses, we show that the DndABCDE module is commonly functionally decoupled from the DndFGH module. However, the modification gene-neighborhoods encode other nucleases, potentially acting as the actual restriction components or suicide effectors limiting propagation of the selfish elements. The modification module's core consists of a coevolving gene-pair encoding the DNA-scanning apparatus - a DndD/CxC-clade ABC ATPase and DndE with two ribbon-helix-helix (MetJ/Arc) DNA-binding domains. Diversification of DndE's DNA-binding interface suggests a multiplicity of target specificities. Additionally, many systems feature DNA cytosine methylase genes instead of PT modification, indicating the DndDE core can recruit other nucleobase modifications. We show that DndFGH is a distinct counter-invader system with several previously uncharacterized domains, including a nucleotide kinase. These likely trigger its restriction endonuclease domain in response to multiple stimuli, like nucleotides, while blocking protective modifications by invader methylases. Remarkably, different DndH variants contain a HerA/FtsK ATPase domain acquired from multiple sources, including cellular genome-segregation systems and mobile elements. Thus, we uncovered novel HerA/FtsK-dependent defense systems that might intercept invasive DNA during replication, conjugation, or packaging.
Collapse
Affiliation(s)
- Siuli Rakesh
- Department of Biological Sciences, Indian Institute of Science Education and Research Berhampur (IISER Berhampur), Berhampur 760010, India
| | - L Aravind
- National Center for Biotechnology Information (NCBI), National Library of Medicine (NLM), National Institutes of Health (NIH), Bethesda, MD 20894, USA
| | - Arunkumar Krishnan
- Department of Biological Sciences, Indian Institute of Science Education and Research Berhampur (IISER Berhampur), Berhampur 760010, India
| |
Collapse
|
|
4
|
Wang ZQ, Yang Y, Zhang JY, Zeng X, Zhang CC. Global translational control by the transcriptional repressor TrcR in the filamentous cyanobacterium Anabaena sp. PCC 7120. Commun Biol 2023; 6:643. [PMID: 37322092 PMCID: PMC10272220 DOI: 10.1038/s42003-023-05012-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2023] [Accepted: 06/02/2023] [Indexed: 06/17/2023] Open
Abstract
Transcriptional and translational regulations are important mechanisms for cell adaptation to environmental conditions. In addition to house-keeping tRNAs, the genome of the filamentous cyanobacterium Anabaena sp. strain PCC 7120 (Anabaena) has a long tRNA operon (trn operon) consisting of 26 genes present on a megaplasmid. The trn operon is repressed under standard culture conditions, but is activated under translational stress in the presence of antibiotics targeting translation. Using the toxic amino acid analog β-N-methylamino-L-alanine (BMAA) as a tool, we isolated and characterized several BMAA-resistance mutants from Anabaena, and identified one gene of unknown function, all0854, named as trcR, encoding a transcription factor belonging to the ribbon-helix-helix (RHH) family. We provide evidence that TrcR represses the expression of the trn operon and is thus the missing link between the trn operon and translational stress response. TrcR represses the expression of several other genes involved in translational control, and is required for maintaining translational fidelity. TrcR, as well as its binding sites, are highly conserved in cyanobacteria, and its functions represent an important mechanism for the coupling of the transcriptional and translational regulations in cyanobacteria.
Collapse
Affiliation(s)
- Zi-Qian Wang
- State Key Laboratory of Freshwater Ecology and Biotechnology and Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, 430072, People's Republic of China.
- University of Chinese Academy of Sciences, Beijing, 100049, People's Republic of China.
- State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
| | - Yiling Yang
- State Key Laboratory of Freshwater Ecology and Biotechnology and Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, 430072, People's Republic of China
| | - Ju-Yuan Zhang
- State Key Laboratory of Freshwater Ecology and Biotechnology and Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, 430072, People's Republic of China
| | - Xiaoli Zeng
- State Key Laboratory of Freshwater Ecology and Biotechnology and Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, 430072, People's Republic of China
| | - Cheng-Cai Zhang
- State Key Laboratory of Freshwater Ecology and Biotechnology and Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, 430072, People's Republic of China.
- Institute AMU-WUT, Aix-Marseille Université and Wuhan University of Technology, Wuhan, Hubei, People's Republic of China.
| |
Collapse
|
|
5
|
Pothulapadu CAS, Jayaraj A, N S, Priyanka RN, Sivaraman G. Novel Benzothiazole-Based Highly Selective Ratiometric Fluorescent Turn-On Sensors for Zn 2+ and Colorimetric Chemosensors for Zn 2+, Cu 2+, and Ni 2+ Ions. ACS OMEGA 2021; 6:24473-24483. [PMID: 34604629 PMCID: PMC8482408 DOI: 10.1021/acsomega.1c02855] [Citation(s) in RCA: 31] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/01/2021] [Indexed: 05/17/2023]
Abstract
Metal ions play a very important role in environmental as well as biological fields. The detection of specific metal ions at a minute level caught much attention, and hence, several probes are available in the literature. Even though benzothiazole-based molecules have a special place in the medicinal field, only very few chemosensors are reported based on this moiety. The current work describes the design and synthesis of the benzothiazole-based chemosensor for a highly selective and sensitive detection of biologically important metal ions such as Zn2+, Cu2+, and Ni2+. The sensing studies of compound-1 showed a ratiometric as well as colorimetric response toward Zn2+, Cu2+, and Ni2+ ions and color changes from colorless to yellow and is found to be insensitive toward various metal ions (Cd2+, Cr3+, Mn2+, Pb2+, Ba2+, Al3+, Ca2+, Fe2+, Fe3+, Mg2+, K+, and Na+). Further, compound-1 exhibited ratiometric as well as turn-on-enhanced fluorescence response toward Zn2+ ions and turn off response for Cu2+ and Ni2+ ions. The Job plots revealed that the binding stoichiometry of compound-1 and metal ions is 2:1. The detection limits were found to be 0.25 ppm for Zn2+, while it was 0.30 ppm and 0.34 ppm for Ni2+ and Cu2+, respectively. In addition, density functional theory results strongly support the colorimetric response of metals, and the reversibility studies suggested that compound-1 can be used as a powerful chemosensor for the detection of Zn2+, Cu2+, and Ni2+ ions. The bioimaging data illustrated that compound-1 is a very effective ratiometric sensor for Zn2+ ions in live cells.
Collapse
Affiliation(s)
- Chinna Ayya Swamy Pothulapadu
- Main
Group Organometallics Materials, Supramolecular Chemistry and Catalysis
Lab, Department of Chemistry, National Institute
of Technology, Calicut 673601, India
| | - Anjitha Jayaraj
- Main
Group Organometallics Materials, Supramolecular Chemistry and Catalysis
Lab, Department of Chemistry, National Institute
of Technology, Calicut 673601, India
| | - Swathi N
- Maharani
Lakshmi Ammanni College for Women (Autonomous), Bangalore 560012, India
| | - Ragam N. Priyanka
- School
of Chemical Sciences, Mahatma Gandhi University, Kottayam 686560, India
| | - Gandhi Sivaraman
- Department
of Chemistry, Gandhigram Rural Institute
(Deemed to be University), Gandhigram 624302, India
| |
Collapse
|
|
6
|
Baksh KA, Zamble DB. Allosteric control of metal-responsive transcriptional regulators in bacteria. J Biol Chem 2020; 295:1673-1684. [PMID: 31857375 PMCID: PMC7008368 DOI: 10.1074/jbc.rev119.011444] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Many transition metals are essential trace nutrients for living organisms, but they are also cytotoxic in high concentrations. Bacteria maintain the delicate balance between metal starvation and toxicity through a complex network of metal homeostasis pathways. These systems are coordinated by the activities of metal-responsive transcription factors-also known as metal-sensor proteins or metalloregulators-that are tuned to sense the bioavailability of specific metals in the cell in order to regulate the expression of genes encoding proteins that contribute to metal homeostasis. Metal binding to a metalloregulator allosterically influences its ability to bind specific DNA sequences through a variety of intricate mechanisms that lie on a continuum between large conformational changes and subtle changes in internal dynamics. This review summarizes recent advances in our understanding of how metal sensor proteins respond to intracellular metal concentrations. In particular, we highlight the allosteric mechanisms used for metal-responsive regulation of several prokaryotic single-component metalloregulators, and we briefly discuss current open questions of how metalloregulators function in bacterial cells. Understanding the regulation and function of metal-responsive transcription factors is a fundamental aspect of metallobiochemistry and is important for gaining insights into bacterial growth and virulence.
Collapse
Affiliation(s)
- Karina A Baksh
- Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada
| | - Deborah B Zamble
- Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada; Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada.
| |
Collapse
|
|
7
|
Abstract
Nickel is essential for the survival of many pathogenic bacteria. E. coli and H. pylori require nickel for [NiFe]-hydrogenases. H. pylori also requires nickel for urease. At high concentrations nickel can be toxic to the cell, therefore, nickel concentrations are tightly regulated. Metalloregulators help to maintain nickel concentration in the cell by regulating the expression of the genes associated with nickel import and export. Nickel import into the cell, delivery of nickel to target proteins, and export of nickel from the cell is a very intricate and well-choreographed process. The delivery of nickel to [NiFe]-hydrogenase and urease is complex and involves several chaperones and accessory proteins. A combination of biochemical, crystallographic, and spectroscopic techniques has been utilized to study the structures of these proteins, as well as protein-protein interactions resulting in an expansion of our knowledge regarding how these proteins sense and bind nickel. In this review, recent advances in the field will be discussed, focusing on the metal site structures of nickel bound to metalloregulators and chaperones.
Collapse
|
|
8
|
Sala D, Musiani F, Rosato A. Application of Molecular Dynamics to the Investigation of Metalloproteins Involved in Metal Homeostasis. Eur J Inorg Chem 2018. [DOI: 10.1002/ejic.201800602] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Affiliation(s)
- Davide Sala
- Magnetic Resonance Center (CERM); University of Florence; Via Luigi Sacconi 6 50019 Sesto Fiorentino Italy
| | - Francesco Musiani
- Laboratory of Bioinorganic Chemistry; Department of Pharmacy and Biotechnology; University of Bologna; Viale Giuseppe Fanin 40, I 40127 Bologna Italy
| | - Antonio Rosato
- Magnetic Resonance Center (CERM); University of Florence; Via Luigi Sacconi 6 50019 Sesto Fiorentino Italy
- Consorzio Interuniversitario di Risonanze Magnetiche di Metallo Proteine; Via Luigi Sacconi 6 50019 Sesto Fiorentino Italy
- Department of Chemistry; University of Florence; Via della Lastruccia 3 50019 Sesto Fiorentino Italy
| |
Collapse
|
|
9
|
Carr CE, Foster AW, Maroney MJ. An XAS investigation of the nickel site structure in the transcriptional regulator InrS. J Inorg Biochem 2017; 177:352-358. [PMID: 28844329 PMCID: PMC5741488 DOI: 10.1016/j.jinorgbio.2017.08.003] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2017] [Revised: 07/10/2017] [Accepted: 08/05/2017] [Indexed: 12/29/2022]
Abstract
InrS (Internal nickel-responsive Sensor) is a transcriptional repressor of the nickel exporter NrsD and de-represses expression of the exporter upon binding Ni(II) ions. Although a crystal structure of apo-InrS has been reported, no structure of the protein with metal ions bound is available. Herein we report the results of metal site structural investigations of Ni(II) and Cu(II) complexes of InrS using X-ray absorption spectroscopy (XAS) that are complementary to data available from the apo-InrS crystal structure, and are consistent with a planar four-coordinate [Ni(His)2(Cys)2] structure, where the ligands are derived from the side chains of His21, Cys53, His78, and Cys82. Coordination of Cu(II) to InrS forms a nearly identical planar four-coordinate complex that is consistent with a simple replacement of the Ni(II) center by Cu(II).
Collapse
Affiliation(s)
- Carolyn E Carr
- Chemistry Department, University of Massachusetts Amherst, MA 01003, USA
| | - Andrew W Foster
- Department of Biosciences, Durham University, Durham, UK; Department of Chemistry, Durham University, Durham, UK
| | - Michael J Maroney
- Chemistry Department, University of Massachusetts Amherst, MA 01003, USA; Program in Molecular and Cellular Biology, University of Massachusetts Amherst, MA 01003, USA.
| |
Collapse
|
|
10
|
Abstract
BACKGROUND Helicobacter pylori is well adapted to colonize the epithelial surface of the human gastric mucosa and can cause persistent infections. In order to infect the gastric mucosa, it has to survive in the gastric acidic pH. This organism has well developed mechanisms to neutralize the effects of acidic pH. OBJECTIVE This review article was designed to summarize the various functional and molecular aspects by which the bacterium can combat and survive the gastric acidic pH in order to establish the persistent infections. METHODS We used the keywords (acid acclimation, gastric acidic environment, H. pylori and survival) in combination or alone for pubmed search of recent scientific literatures. One hundred and forty one papers published between 1989 and 2016 were sorted out. The articles published with only abstracts, other than in English language, case reports and reviews were excluded. RESULTS Many literatures describing the role of several factors in acid survival were found. Recently, the role of several other factors has been claimed to participate in acid survival. CONCLUSION In conclusion, this organism has well characterized mechanisms for acid survival.
Collapse
Affiliation(s)
- Shamshul Ansari
- Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, Yufu, Japan
| | - Yoshio Yamaoka
- Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, Yufu, Japan,Department of Medicine-Gastroenterology, Baylor College of Medicine, Houston, Texas, USA,Corresponding author: Yoshio Yamaoka, MD, PhD, Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, 1-1 Idaigaoka, Hasama-machi, Yufu-City, Oita 879-5593, Japan, Tel: +81-97-586-5740; Fax: +81-97-586-5749,
| |
Collapse
|
|
11
|
Carr CE, Musiani F, Huang HT, Chivers PT, Ciurli S, Maroney MJ. Glutamate Ligation in the Ni(II)- and Co(II)-Responsive Escherichia coli Transcriptional Regulator, RcnR. Inorg Chem 2017; 56:6459-6476. [PMID: 28517938 DOI: 10.1021/acs.inorgchem.7b00527] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Escherichia coli RcnR (resistance to cobalt and nickel regulator, EcRcnR) is a metal-responsive repressor of the genes encoding the Ni(II) and Co(II) exporter proteins RcnAB by binding to PRcnAB. The DNA binding affinity is weakened when the cognate ions Ni(II) and Co(II) bind to EcRcnR in a six-coordinate site that features a (N/O)5S ligand donor-atom set in distinct sites: while both metal ions are bound by the N terminus, Cys35, and His64, Co(II) is additionally bound by His3. On the other hand, the noncognate Zn(II) and Cu(I) ions feature a lower coordination number, have a solvent-accessible binding site, and coordinate protein ligands that do not include the N-terminal amine. A molecular model of apo-EcRcnR suggested potential roles for Glu34 and Glu63 in binding Ni(II) and Co(II) to EcRcnR. The roles of Glu34 and Glu63 in metal binding, metal selectivity, and function were therefore investigated using a structure/function approach. X-ray absorption spectroscopy was used to assess the structural changes in the Ni(II), Co(II), and Zn(II) binding sites of Glu → Ala and Glu → Cys variants at both positions. The effect of these structural alterations on the regulation of PrcnA by EcRcnR in response to metal binding was explored using LacZ reporter assays. These combined studies indicate that while Glu63 is a ligand for both metal ions, Glu34 is a ligand for Co(II) but possibly not for Ni(II). The Glu34 variants affect the structure of the cognate metal sites, but they have no effect on the transcriptional response. In contrast, the Glu63 variants affect both the structure and transcriptional response, although they do not completely abolish the function of EcRcnR. The structure of the Zn(II) site is not significantly perturbed by any of the glutamic acid variations. The spectroscopic and functional data obtained on the mutants were used to calculate models of the metal-site structures of EcRcnR bound to Ni(II), Co(II), and Zn(II). The results are interpreted in terms of a switch mechanism, in which a subset of the metal-binding ligands is responsible for the allosteric response required for DNA release.
Collapse
Affiliation(s)
- Carolyn E Carr
- Department of Chemistry, University of Massachusetts , Amherst, Massachusetts 01003, United States
| | - Francesco Musiani
- Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology, University of Bologna , Bologna 40126, Italy
| | - Hsin-Ting Huang
- Department of Chemistry, University of Massachusetts , Amherst, Massachusetts 01003, United States
| | - Peter T Chivers
- Departments of Biosciences and Chemistry, Durham University , Durham DH1 3LE, United Kingdom
| | - Stefano Ciurli
- Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology, University of Bologna , Bologna 40126, Italy
| | - Michael J Maroney
- Department of Chemistry, University of Massachusetts , Amherst, Massachusetts 01003, United States
| |
Collapse
|
|
12
|
Maillard AP, Künnemann S, Große C, Volbeda A, Schleuder G, Petit-Härtlein I, de Rosny E, Nies DH, Covès J. Response of CnrX from Cupriavidus metallidurans CH34 to nickel binding. Metallomics 2016; 7:622-31. [PMID: 25628016 DOI: 10.1039/c4mt00293h] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
Resistance to high concentration of nickel ions is mediated in Cupriavidus metallidurans by the CnrCBA transenvelope efflux complex. Expression of the cnrCBA genes is regulated by the transmembrane signal transduction complex CnrYXH. Together, the metal sensor CnrX and the transmembrane antisigma factor CnrY control the availability of the extracytoplasmic function sigma factor CnrH. Release of CnrH from sequestration by CnrY at the cytoplasmic side of the membrane depends essentially on the binding of the agonist metal ion Ni(ii) to the periplasmic metal sensor domain of CnrX. CnrH availability leads to transcription initiation at the promoters cnrYp and cnrCp and to the expression of the genes in the cnrYXHCBA nickel resistance determinant. The first steps of signal propagation by CnrX rely on subtle metal-dependent allosteric modifications. To study the nickel-mediated triggering process by CnrX, we have altered selected residues, F66, M123, and Y135, and explored the physiological consequences of these changes with respect to metal resistance, expression of a cnrCBA-lacZ reporter fusion and protein production. M123C- and Y135F-CnrXs have been further characterized in vitro by metal affinity measurements and crystallographic structure analysis. Atomic-resolution structures of metal-bound M123C- and Y135F-CnrXs showed that Ni(ii) binds two of the three canonical conformations identified and that Ni(ii) sensing likely proceeds by conformation selection.
Collapse
Affiliation(s)
- Antoine P Maillard
- Institut de Biologie Structurale, UMR 5075 CNRS-CEA-Université Grenoble-Alpes, 71, Avenue des Martyrs, 38044 Grenoble Cedex 9, France
| | | | | | | | | | | | | | | | | |
Collapse
|
|
13
|
Abstract
In Escherichia coli, hydrogen metabolism plays a prominent role in anaerobic physiology. The genome contains the capability to produce and assemble up to four [NiFe]-hydrogenases, each of which are known, or predicted, to contribute to different aspects of cellular metabolism. In recent years, there have been major advances in the understanding of the structure, function, and roles of the E. coli [NiFe]-hydrogenases. The membrane-bound, periplasmically oriented, respiratory Hyd-1 isoenzyme has become one of the most important paradigm systems for understanding an important class of oxygen-tolerant enzymes, as well as providing key information on the mechanism of hydrogen activation per se. The membrane-bound, periplasmically oriented, Hyd-2 isoenzyme has emerged as an unusual, bidirectional redox valve able to link hydrogen oxidation to quinone reduction during anaerobic respiration, or to allow disposal of excess reducing equivalents as hydrogen gas. The membrane-bound, cytoplasmically oriented, Hyd-3 isoenzyme is part of the formate hydrogenlyase complex, which acts to detoxify excess formic acid under anaerobic fermentative conditions and is geared towards hydrogen production under those conditions. Sequence identity between some Hyd-3 subunits and those of the respiratory NADH dehydrogenases has led to hypotheses that the activity of this isoenzyme may be tightly coupled to the formation of transmembrane ion gradients. Finally, the E. coli genome encodes a homologue of Hyd-3, termed Hyd-4, however strong evidence for a physiological role for E. coli Hyd-4 remains elusive. In this review, the versatile hydrogen metabolism of E. coli will be discussed and the roles and potential applications of the spectrum of different types of [NiFe]-hydrogenases available will be explored.
Collapse
|
|
14
|
Abstract
This chapter focuses on transition metals. All transition metal cations are toxic-those that are essential for Escherichia coli and belong to the first transition period of the periodic system of the element and also the "toxic-only" metals with higher atomic numbers. Common themes are visible in the metabolism of these ions. First, there is transport. High-rate but low-affinity uptake systems provide a variety of cations and anions to the cells. Control of the respective systems seems to be mainly through regulation of transport activity (flux control), with control of gene expression playing only a minor role. If these systems do not provide sufficient amounts of a needed ion to the cell, genes for ATP-hydrolyzing high-affinity but low-rate uptake systems are induced, e.g., ABC transport systems or P-type ATPases. On the other hand, if the amount of an ion is in surplus, genes for efflux systems are induced. By combining different kinds of uptake and efflux systems with regulation at the levels of gene expression and transport activity, the concentration of a single ion in the cytoplasm and the composition of the cellular ion "bouquet" can be rapidly adjusted and carefully controlled. The toxicity threshold of an ion is defined by its ability to produce radicals (copper, iron, chromate), to bind to sulfide and thiol groups (copper, zinc, all cations of the second and third transition period), or to interfere with the metabolism of other ions. Iron poses an exceptional metabolic problem due its metabolic importance and the low solubility of Fe(III) compounds, combined with the ability to cause dangerous Fenton reactions. This dilemma for the cells led to the evolution of sophisticated multi-channel iron uptake and storage pathways to prevent the occurrence of unbound iron in the cytoplasm. Toxic metals like Cd2+ bind to thiols and sulfide, preventing assembly of iron complexes and releasing the metal from iron-sulfur clusters. In the unique case of mercury, the cation can be reduced to the volatile metallic form. Interference of nickel and cobalt with iron is prevented by the low abundance of these metals in the cytoplasm and their sequestration by metal chaperones, in the case of nickel, or by B12 and its derivatives, in the case of cobalt. The most dangerous metal, copper, catalyzes Fenton-like reactions, binds to thiol groups, and interferes with iron metabolism. E. coli solves this problem probably by preventing copper uptake, combined with rapid efflux if the metal happens to enter the cytoplasm.
Collapse
|
|
15
|
Musiani F, Ciurli S. Evolution of Macromolecular Docking Techniques: The Case Study of Nickel and Iron Metabolism in Pathogenic Bacteria. Molecules 2015; 20:14265-92. [PMID: 26251891 PMCID: PMC6332059 DOI: 10.3390/molecules200814265] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2015] [Revised: 07/23/2015] [Accepted: 07/28/2015] [Indexed: 11/24/2022] Open
Abstract
The interaction between macromolecules is a fundamental aspect of most biological processes. The computational techniques used to study protein-protein and protein-nucleic acid interactions have evolved in the last few years because of the development of new algorithms that allow the a priori incorporation, in the docking process, of experimentally derived information, together with the possibility of accounting for the flexibility of the interacting molecules. Here we review the results and the evolution of the techniques used to study the interaction between metallo-proteins and DNA operators, all involved in the nickel and iron metabolism of pathogenic bacteria, focusing in particular on Helicobacter pylori (Hp). In the first part of the article we discuss the methods used to calculate the structure of complexes of proteins involved in the activation of the nickel-dependent enzyme urease. In the second part of the article, we concentrate on two applications of protein-DNA docking conducted on the transcription factors HpFur (ferric uptake regulator) and HpNikR (nickel regulator). In both cases we discuss the technical expedients used to take into account the conformational variability of the multi-domain proteins involved in the calculations.
Collapse
Affiliation(s)
- Francesco Musiani
- Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology, University of Bologna, Viale G. Fanin 40, Bologna I-40127, Italy.
| | - Stefano Ciurli
- Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology, University of Bologna, Viale G. Fanin 40, Bologna I-40127, Italy.
| |
Collapse
|
|
16
|
Mazzei L, Dobrovolska O, Musiani F, Zambelli B, Ciurli S. On the interaction of Helicobacter pylori NikR, a Ni(II)-responsive transcription factor, with the urease operator: in solution and in silico studies. J Biol Inorg Chem 2015. [PMID: 26204982 DOI: 10.1007/s00775-015-1284-0] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Helicobacter pylori (Hp) is a carcinogen that relies on Ni(II) to survive in the extreme pH conditions of the human guts. The regulation of genes coding for Ni(II) enzymes and proteins is effected by the nickel-responsive transcription factor NikR, composed of a DNA-binding domain (DBD) and a metal-binding domain (MBD). The scope of this study is to obtain the molecular details of the HpNikR interaction with the urease operator OP ureA , in solution. The size of the full-length protein prevents the characterization of the HpNikR-OP ureA interaction using NMR. We thus investigated the two separate domains of HpNikR. The conservation of their oligomeric state was established by multiple-angle light scattering. Isothermal calorimetric titrations indicated that the thermodynamics of Ni(II) binding to the isolated MBD is independent of the presence of the adjacent DBDs. The NMR spectra of the isolated DBD support considerable conservation of its structural properties. The spectral perturbations induced on the DBD by OP ureA provided information useful to calculate a structural model of the HpNikR-OP ureA complex using a docking computational protocol. The NMR assignment of the residues involved in the protein-DNA interaction represents a starting point for the development of drugs potentially able to eradicate H. pylori infections. All evidences so far collected, in this and previous studies, consistently indicate that binding of Ni(II) to the MBD increases the HpNikR-DNA affinity by modulating the dynamic, and not the structural, properties of the protein, suggesting that the formation of a stable complex relies upon an induced fit mechanism.
Collapse
Affiliation(s)
- Luca Mazzei
- Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology, University of Bologna, Bologna, 40127, Italy
| | | | | | | | | |
Collapse
|
|
17
|
Carpenter BM, West AL, Gancz H, Servetas SL, Pich OQ, Gilbreath JJ, Hallinger DR, Forsyth MH, Merrell DS, Michel SLJ. Crosstalk between the HpArsRS two-component system and HpNikR is necessary for maximal activation of urease transcription. Front Microbiol 2015; 6:558. [PMID: 26124751 PMCID: PMC4464171 DOI: 10.3389/fmicb.2015.00558] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2015] [Accepted: 05/20/2015] [Indexed: 12/14/2022] Open
Abstract
Helicobacter pylori NikR (HpNikR) is a nickel dependent transcription factor that directly regulates a number of genes in this important gastric pathogen. One key gene that is regulated by HpNikR is ureA, which encodes for the urease enzyme. In vitro DNA binding studies of HpNikR with the ureA promoter (PureA) previously identified a recognition site that is required for high affinity protein/DNA binding. As a means to determine the in vivo significance of this recognition site and to identify the key DNA sequence determinants required for ureA transcription, herein, we have translated these in vitro results to analysis directly within H. pylori. Using a series of GFP reporter constructs in which the PureA DNA target was altered, in combination with mutant H. pylori strains deficient in key regulatory proteins, we confirmed the importance of the previously identified HpNikR recognition sequence for HpNikR-dependent ureA transcription. Moreover, we identified a second factor, the HpArsRS two-component system that was required for maximum transcription of ureA. While HpArsRS is known to regulate ureA in response to acid shock, it was previously thought to function independently of HpNikR and to have no role at neutral pH. However, our qPCR analysis of ureA expression in wildtype, ΔnikR and ΔarsS single mutants as well as a ΔarsS/nikR double mutant strain background showed reduced basal level expression of ureA when arsS was absent. Additionally, we determined that both HpNikR and HpArsRS were necessary for maximal expression of ureA under nickel, low pH and combined nickel and low pH stresses. In vitro studies of HpArsR-P with the PureA DNA target using florescence anisotropy confirmed a direct protein/DNA binding interaction. Together, these data support a model in which HpArsRS and HpNikR cooperatively interact to regulate ureA transcription under various environmental conditions. This is the first time that direct “cross-talk” between HpArsRS and HpNikR at neutral pH has been demonstrated.
Collapse
Affiliation(s)
- Beth M Carpenter
- Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences Bethesda, MD, USA
| | - Abby L West
- Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland Baltimore, Maryland, USA
| | - Hanan Gancz
- Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences Bethesda, MD, USA
| | - Stephanie L Servetas
- Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences Bethesda, MD, USA
| | - Oscar Q Pich
- Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences Bethesda, MD, USA
| | - Jeremy J Gilbreath
- Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences Bethesda, MD, USA
| | - Daniel R Hallinger
- Department of Biology, The College of William and Mary Williamsburg, VA, USA
| | - Mark H Forsyth
- Department of Biology, The College of William and Mary Williamsburg, VA, USA
| | - D Scott Merrell
- Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences Bethesda, MD, USA
| | - Sarah L J Michel
- Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland Baltimore, Maryland, USA
| |
Collapse
|
|
18
|
Musiani F, Zambelli B, Bazzani M, Mazzei L, Ciurli S. Nickel-responsive transcriptional regulators. Metallomics 2015; 7:1305-18. [DOI: 10.1039/c5mt00072f] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The structural features, metal coordination modes and metal binding thermodynamics of known Ni(ii)-dependent transcriptional regulators are highlighted and discussed.
Collapse
Affiliation(s)
- Francesco Musiani
- Laboratory of Bioinorganic Chemistry
- Department of Pharmacy and Biotechnology
- University of Bologna
- 40127 Bologna, Italy
| | - Barbara Zambelli
- Laboratory of Bioinorganic Chemistry
- Department of Pharmacy and Biotechnology
- University of Bologna
- 40127 Bologna, Italy
| | - Micaela Bazzani
- Laboratory of Bioinorganic Chemistry
- Department of Pharmacy and Biotechnology
- University of Bologna
- 40127 Bologna, Italy
| | - Luca Mazzei
- Laboratory of Bioinorganic Chemistry
- Department of Pharmacy and Biotechnology
- University of Bologna
- 40127 Bologna, Italy
| | - Stefano Ciurli
- Laboratory of Bioinorganic Chemistry
- Department of Pharmacy and Biotechnology
- University of Bologna
- 40127 Bologna, Italy
| |
Collapse
|
|
19
|
Functional domain analysis of the cell division inhibitor EzrA. PLoS One 2014; 9:e102616. [PMID: 25068683 PMCID: PMC4113482 DOI: 10.1371/journal.pone.0102616] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2012] [Accepted: 06/21/2014] [Indexed: 11/18/2022] Open
Abstract
The precise spatial and temporal control of bacterial cell division is achieved through the balanced actions of factors that inhibit assembly of the tubulin-like protein FtsZ at aberrant subcellular locations or promote its assembly at the future sites of division. In Bacillus subtilis, the membrane anchored cell division protein EzrA, interacts directly with FtsZ to prevent aberrant FtsZ assembly at cell poles and contributes to the inherently dynamic nature of the cytokinetic ring. Recent work suggests EzrA also serves as a scaffolding protein to coordinate lateral growth with cell wall biosynthesis through interactions with a host of proteins, a finding consistent with EzrA's four extensive coiled-coil domains. In a previous study we identified a conserved patch of residues near EzrA's C-terminus (the QNR motif) that are critical for maintenance of a dynamic cytokinetic ring, but dispensable for EzrA-mediated inhibition of FtsZ assembly at cell poles. In an extension of this work, here we report that EzrA's two C-terminal coiled-coils function in concert with the QNR motif to mediate interactions with FtsZ and maintain the dynamic nature of the cytokinetic ring. In contrast, EzrA's two N-terminal coiled-coils are dispensable for interaction between EzrA and FtsZ in vitro and in vivo, but required for EzrA mediated inhibition of FtsZ assembly at cell poles. Finally, chimeric analysis indicates that EzrA's transmembrane anchor plays a generic role: concentrating EzrA at the plasma membrane where presumably it can most effectively modulate FtsZ assembly.
Collapse
|
|
20
|
Chivers PT. Cobalt and Nickel. BINDING, TRANSPORT AND STORAGE OF METAL IONS IN BIOLOGICAL CELLS 2014. [DOI: 10.1039/9781849739979-00381] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
Cobalt and nickel play key roles in biological systems as cofactors in a small number of important enzymes. The majority of these are found in microbes. Evidence for direct roles for Ni(II) and Co(II) enzymes in higher organisms is limited, with the exception of the well-known requirement for the cobalt-containing vitamin B12 cofactor and the Ni-dependent urease in plants. Nonetheless, nickel in particular plays a key role in human health because of its essential role in microbes that inhabit various growth niches within the body. These roles can be beneficial, as can be seen with the anaerobic production and consumption of H2 in the digestive tract by bacteria and archaea that results in increased yields of short-chain fatty acids. In other cases, nickel has an established role in the establishment of pathogenic infection (Helicobacter pylori urease and colonization of the stomach). The synthesis of Co- and Ni-containing enzymes requires metal import from the extracellular milieu followed by the targeting of these metals to the appropriate protein and enzymes involved in metallocluster or cofactor biosynthesis. These metals are toxic in excess so their levels must be regulated carefully. This complex pathway of metalloenzyme synthesis and intracellular homeostasis requires proteins that can specifically recognize these metals in a hierarchical manner. This chapter focuses on quantitative and structural details of the cobalt and nickel binding sites in transport, trafficking and regulatory proteins involved in cobalt and nickel metabolism in microbes.
Collapse
Affiliation(s)
- Peter T. Chivers
- Department of Chemistry, School of Biological and Biomedical Sciences, and Biophysical Sciences Institute, Durham University Durham UK
| |
Collapse
|
|
21
|
Yamamoto K. The hierarchic network of metal-response transcription factors in Escherichia coli. Biosci Biotechnol Biochem 2014; 78:737-47. [PMID: 25035972 DOI: 10.1080/09168451.2014.915731] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
Enterobacteria such as Escherichia coli are able to survive under various environments within host animals by changes of the expression pattern of its genome. The selective expression of genes in its genome takes place by controlling the promoter recognition properties of RNA polymerase by protein-protein interplays with transcription factors. In this review, I describe the regulatory network formed by the metal-sensing transcription factors in E. coli. Comprehensive analyses identify the set of regulation targets for a total of 13 metal-response transcription factors, indicating that nine species of transcription factors are local regulators while four species of transcription factors are global regulators. The signal transduction pathways for these metal-response regulons show not only the complex cross-talks but also the hierarchic multi-regulatory network. This regulatory network seems to play a role for E. coli survival to colonize in a large intestine within host animals.
Collapse
Affiliation(s)
- Kaneyoshi Yamamoto
- a Department of Frontier Bioscience and Micro-Nano Technology Research Center , Hosei University , Koganei, Tokyo , Japan
| |
Collapse
|
|
22
|
Ge RG, Wang DX, Hao MC, Sun XS. Nickel trafficking system responsible for urease maturation in Helicobacter pylori. World J Gastroenterol 2013; 19:8211-8218. [PMID: 24363511 PMCID: PMC3857443 DOI: 10.3748/wjg.v19.i45.8211] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/06/2013] [Revised: 10/17/2013] [Accepted: 11/03/2013] [Indexed: 02/06/2023] Open
Abstract
Helicobacter pylori (H. pylori) is a common human pathogen responsible for various gastric diseases. This bacterium relies on the production of urease and hydrogenase to inhabit the acidic environment of the stomach. Nickel is an essential cofactor for urease and hydrogenase. H. pylori has to uptake sufficient nickel ions for the maturation of urease, and on the other way, to prevent the toxic effects of excessive nickel ions. Therefore, H. pylori has to strike a delicate balance between the import of nickel ions, its efficient intracellular storage, and delivery to nickel-dependent metalloenzymes when required. The assembly and maturation of the urease enzyme is a complex and timely ordered process, requiring various regulatory, uptake, chaperone and accessory proteins. In this review, we focus on several nickel trafficking proteins involved in urease maturation: NikR, NixA, HypAB, UreEFGH, HspA, Hpn and Hpnl. The work will deepen our understanding of how this pathogenic bacterium adapts to severe habitant environments in the host.
Collapse
|
|
23
|
|
|
24
|
Higgins KA, Hu HQ, Chivers PT, Maroney MJ. Effects of select histidine to cysteine mutations on transcriptional regulation by Escherichia coli RcnR. Biochemistry 2012; 52:84-97. [PMID: 23215580 DOI: 10.1021/bi300886q] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The RcnR metalloregulator represses the transcription of the Co(II) and Ni(II) exporter, RcnAB. Previous studies have shown that Co(II) and Ni(II) bind to RcnR in six-coordinate sites, resulting in derepression. Here, the roles of His60, His64, and His67 in specific metal recognition are examined. His60 and His64 correspond to ligands that are important for Cu(I) binding in the homologous Cu(I)-responsive metalloregulator, CsoR. These residues are known to be functionally important in RcnR transcriptional regulation. X-ray absorption spectroscopy (XAS) was used to examine the structure of bound cognate and noncognate metal ions, and lacZ reporter assays were used to assess the transcription of rcnA in response to metal binding in the three His → Cys mutations, H60C, H64C, and H67C. These studies confirm that both Ni(II) and Co(II) use His64 as a ligand. H64C-RcnR is also the only known mutant that retains a Co(II) response while eliminating the response to Ni(II) binding. XAS data indicate that His60 and His67 are potential Co(II) ligands. The effects of the mutations of His60, His64, and His67 on the structures of the noncognate metal ions [Zn(II) and Cu(I)] reveal that these residues have distinctive roles in binding noncognate metals. None of the His → Cys mutants in RcnR confer any response to Cu(I) binding, including H64C-RcnR, where the ligands involved in Cu(I) binding in CsoR are present. These data indicate that while the secondary, tertiary, and quaternary structures of CsoR and RcnR are quite similar, small changes in primary sequence reveal that the specific mechanisms involved in metal recognition are quite different.
Collapse
Affiliation(s)
- Khadine A Higgins
- Department of Chemistry, University of Massachusetts, Amherst, MA 01003, USA
| | | | | | | |
Collapse
|
|
25
|
Benoit SL, Seshadri S, Lamichhane-Khadka R, Maier RJ. Helicobacter hepaticus NikR controls urease and hydrogenase activities via the NikABDE and HH0418 putative nickel import proteins. MICROBIOLOGY-SGM 2012; 159:136-146. [PMID: 23139401 DOI: 10.1099/mic.0.062976-0] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Helicobacter hepaticus open reading frame HH0352 was identified as a nickel-responsive regulator NikR. The gene was disrupted by insertion of an erythromycin resistance cassette. The H. hepaticus nikR mutant had five- to sixfold higher urease activity and at least twofold greater hydrogenase activity than the wild-type strain. However, the urease apo-protein levels were similar in both the wild-type and the mutant, suggesting the increase in urease activity in the mutant was due to enhanced Ni-maturation of the urease. Compared with the wild-type strain, the nikR strain had increased cytoplasmic nickel levels. Transcription of nikABDE (putative inner membrane Ni transport system) and hh0418 (putative outer membrane Ni transporter) was nickel- and NikR-repressed. Electrophoretic mobility shift assays (EMSAs) revealed that purified HhNikR could bind to the nikABDE promoter (P(nikA)), but not to the urease or the hydrogenase promoter; NikR-P(nikA) binding was enhanced in the presence of nickel. Also, qRT-PCR and EMSAs indicated that neither nikR nor the exbB-exbD-tonB were under the control of the NikR regulator, in contrast with their Helicobacter pylori homologues. Taken together, our results suggest that HhNikR modulates urease and hydrogenase activities by repressing the nickel transport/nickel internalization systems in H. hepaticus, without direct regulation of the Ni-enzyme genes (the latter is the case for H. pylori). Finally, the nikR strain had a two- to threefold lower growth yield than the parent, suggesting that the regulatory protein might play additional roles in the mouse liver pathogen.
Collapse
Affiliation(s)
| | | | | | - Robert J Maier
- Department of Microbiology, University of Georgia, Athens, GA, USA
| |
Collapse
|
|
26
|
Higgins KA, Carr CE, Maroney MJ. Specific metal recognition in nickel trafficking. Biochemistry 2012; 51:7816-32. [PMID: 22970729 DOI: 10.1021/bi300981m] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Nickel is an essential metal for a number of bacterial species that have developed systems for acquiring, delivering, and incorporating the metal into target enzymes and controlling the levels of nickel in cells to prevent toxic effects. As with other transition metals, these trafficking systems must be able to distinguish between the desired metal and other transition metal ions with similar physical and chemical properties. Because there are few enzymes (targets) that require nickel for activity (e.g., Escherichia coli transports nickel for hydrogenases made under anaerobic conditions, and Helicobacter pylori requires nickel for hydrogenase and urease that are essential for acid viability), the "traffic pattern" for nickel is relatively simple, and nickel trafficking therefore presents an opportunity to examine a system for the mechanisms that are used to distinguish nickel from other metals. In this review, we describe the details known for examples of uptake permeases, metallochaperones and proteins involved in metallocenter assembly, and nickel metalloregulators. We also illustrate a variety of mechanisms, including molecular recognition in the case of NikA protein and examples of allosteric regulation for HypA, NikR, and RcnR, employed to generate specific biological responses to nickel ions.
Collapse
Affiliation(s)
- Khadine A Higgins
- Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA
| | | | | |
Collapse
|
|
27
|
Krecisz S, Jones MD, Zamble DB. Nonspecific interactions between Escherichia coli NikR and DNA are critical for nickel-activated DNA binding. Biochemistry 2012; 51:7873-9. [PMID: 22971172 DOI: 10.1021/bi300510z] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
The Escherichia coli transcription factor NikR is responsible for nickel-mediated repression of the operon encoding the Nik uptake transporter. The crystal structure of Ni(II)-NikR bound to the nik operator sequence revealed that residues in the loop preceding helix α3 in the metal-binding domain, which becomes structurally ordered upon stoichiometric nickel binding, interact with the DNA backbone. Here, we show that mutating both of these residues that make the nonspecific contacts, K64 and R65, abolishes DNA binding in vitro and nickel-responsive transcriptional repression of the nik promoter in vivo. In contrast, mutation of Q118, which forms a bridge between R65 and a potassium site, does not impact the activities of NikR. These data support the model that the nonspecific interactions between the metal-binding domain of the protein and the DNA phosphodiester backbone are critical for the Ni(II)-responsive activity of E. coli NikR.
Collapse
Affiliation(s)
- Sandra Krecisz
- Department of Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 3H6
| | | | | |
Collapse
|
|
28
|
Evans SE, Michel SLJ. Dissecting the role of DNA sequence in Helicobacter pylori NikR/DNA recognition. Dalton Trans 2012; 41:7946-51. [PMID: 22549756 DOI: 10.1039/c2dt30504f] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
HpNikR is a prokaryotic nickel binding transcription factor found in Helicobacter pylori, where it functions as a regulator of multiple genes including those involved in nickel ion homeostasis and acid adaptation. The target operator sequences of the genes that are regulated by HpNikR do not have symmetric recognition sites, and the mechanism by which HpNikR distinguishes between the genes it regulates is not well understood. HpNikR utilizes a two-tiered mode of DNA binding in which some target sequences are bound with high affinity (K(d) of nM) and others with low affinity (K(d) of μM). An alignment of the high affinity and low affinity binder sequences identified a consensus binding sequence. The consensus sequence was conserved to a greater degree for the high affinity binder sequences compared to the low affinity binder sequences. The exact bases within the consensus sequence that are crucial for a high affinity binding interaction have not been identified. Here we sought to identify key residues from the consensus sequence that are crucial for a tight binding interaction using a competitive fluorescence anisotropy assay. Systematic mutations were made to a weak binder operator sequence, P(nikR), so that it more closely resembled the consensus sequence and the effect of these mutations on protein-DNA binding was measured. Similarly, mutations that disrupted the consensus sequence were made to a tight binder operator sequence, P(ureA), and their effects on protein-DNA binding were measured. Taken together, these studies implicate thymine 10, located on the 3' end of the palindrome, as crucial for tight binding.
Collapse
Affiliation(s)
- Sarah E Evans
- Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201, USA
| | | |
Collapse
|
|
29
|
Higgins KA, Chivers PT, Maroney MJ. Role of the N-terminus in determining metal-specific responses in the E. coli Ni- and Co-responsive metalloregulator, RcnR. J Am Chem Soc 2012; 134:7081-93. [PMID: 22471551 PMCID: PMC3375346 DOI: 10.1021/ja300834b] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/28/2022]
Abstract
RcnR (resistance to cobalt and nickel regulator) is a 40-kDa homotetrameric protein and metalloregulator that controls the transcription of the Co(II) and Ni(II) exporter, RcnAB, by binding to DNA as an apoprotein and releasing DNA in response to specifically binding Co(II) and Ni(II) ions. Using X-ray absorption spectroscopy (XAS) to examine the structure of metals bound and lacZ reporter assays of the transcription of RcnA in response to metal binding, in WT and mutant proteins, the roles of coordination number, ligand selection, and residues in the N-terminus of the protein were examined as determinants in metal ion recognition. The studies show that the cognate metal ions, Co(II) and Ni(II), which bind in (N/O)(5)S six-coordinate sites, are distinguished from non-cognate metal ions (Cu(I) and Zn(II)), which bind only three protein ligands and one anion from the buffer, by coordination number and ligand selection. Using mutations of residues near the N-terminus, the N-terminal amine is shown to be a ligand of the cognate metal ions that is missing in the complexes with non-cognate metal ions. The side chain of His3 is also shown to play an important role in distinguishing metal ions. The imidazole group is shown to be a ligand in the Co(II) RcnR complex, but not in the Zn(II) complex. Further, His3 does not appear to bind to Ni(II), providing a structural basis for the differential regulation of RcnAB by the two cognate ions. The Zn(II) complexes change coordination number in response to the residue in position three. In H3C-RcnR, the Zn(II) complex is five-coordinate, and in H3E-RcnR the Zn(II) ion is bound to six protein ligands. The metric parameters of this unusual Zn(II) structure resemble those of the WT-Ni(II) complex, and the mutant protein is able to regulate expression of RcnAB in response to binding the non-cognate ion. The results are discussed within a protein allosteric model for gene regulation by metalloregulators.
Collapse
Affiliation(s)
- Khadine A. Higgins
- Department of Chemistry , University of Massachusetts, Amherst, Massachusetts 01003
| | - Peter T. Chivers
- Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis MO 63110
| | - Michael J. Maroney
- Department of Chemistry , University of Massachusetts, Amherst, Massachusetts 01003
| |
Collapse
|
|
30
|
Witkowska D, Rowinska-Zyrek M, Valensin G, Kozlowski H. Specific poly-histidyl and poly-cysteil protein sites involved in Ni2+ homeostasis in Helicobacter pylori. Impact of Bi3+ ions on Ni2+ binding to proteins. Structural and thermodynamic aspects. Coord Chem Rev 2012. [DOI: 10.1016/j.ccr.2011.06.014] [Citation(s) in RCA: 44] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
|
|
31
|
Abstract
The dramatic changes in the environmental conditions that organisms encountered during evolution and adaptation to life in specific niches, have influenced intracellular and extracellular metal ion contents and, as a consequence, the cellular ability to sense and utilize different metal ions. This metal-driven differentiation is reflected in the specific panels of metal-responsive transcriptional regulators found in different organisms, which finely tune the intracellular metal ion content and all metal-dependent processes. In order to understand the processes underlying this complex metal homeostasis network, the study of the molecular processes that determine the protein-metal ion recognition, as well as how this event is transduced into a transcriptional output, is necessary. This chapter describes how metal ion binding to specific proteins influences protein interaction with DNA and how this event can influence the fate of genetic expression, leading to specific transcriptional outputs. The features of representative metal-responsive transcriptional regulators, as well as the molecular basis of metal-protein and protein-DNA interactions, are discussed on the basis of the structural information available. An overview of the recent advances in the understanding of how these proteins choose specific metal ions among the intracellular metal ion pool, as well as how they allosterically respond to their effector binding, is given.
Collapse
|
|
32
|
Abstract
Nickel has long been known to be an important human toxicant, including having the ability to form carcinomas, but until recently nickel was believed to be an issue only to microorganisms living in nickel-rich serpentine soils or areas contaminated by industrial pollution. This assumption was overturned by the discovery of a nickel defense system (RcnR/RcnA) found in microorganisms that live in a wide range of environmental niches, suggesting that nickel homeostasis is a general biological concern. To date, the mechanisms of nickel toxicity in microorganisms and higher eukaryotes are poorly understood. In this review, we summarize nickel homeostasis processes used by microorganisms and highlight in vivo and in vitro effects of exposure to elevated concentrations of nickel. On the basis of this evidence we propose four mechanisms of nickel toxicity: (1) nickel replaces the essential metal of metalloproteins, (2) nickel binds to catalytic residues of non-metalloenzymes; (3) nickel binds outside the catalytic site of an enzyme to inhibit allosterically and (4) nickel indirectly causes oxidative stress.
Collapse
Affiliation(s)
- Lee Macomber
- Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824-4320, USA
| | - Robert P. Hausinger
- Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824-4320, USA
- Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319, USA
| |
Collapse
|
|
33
|
Most mutant OccR proteins that are defective in positive control hold operator DNA in a locked high-angle bend. J Bacteriol 2011; 193:5442-9. [PMID: 21804007 DOI: 10.1128/jb.05352-11] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023] Open
Abstract
OccR is a LysR-type transcriptional regulator of Agrobacterium tumefaciens that positively regulates the octopine catabolism operon of the Ti plasmid. Positive control of the occ genes occurs in response to octopine, a nutrient released from crown gall tumors. OccR also functions as an autorepressor in the presence or absence of octopine. OccR binds to a site between occQ and occR in the presence or absence of octopine, although octopine triggers a conformational change that shortens the DNA footprint and relaxes a DNA bend. In order to determine the roles of this conformational change in transcriptional activation, we isolated 11 OccR mutants that were defective in activation of the occQ promoter but were still capable of autorepression. The mutations in these mutants spanned most of the length of the protein. Two additional positive-control mutants were isolated using site-directed mutagenesis. Twelve mutant proteins displayed a high-angle DNA bend in the presence or absence of octopine. One mutant, the L26A mutant, showed ligand-responsive DNA binding similar to that of wild-type OccR and therefore must be impaired in a subsequent step in activation.
Collapse
|
|
34
|
Muller C, Bahlawane C, Aubert S, Delay CM, Schauer K, Michaud-Soret I, De Reuse H. Hierarchical regulation of the NikR-mediated nickel response in Helicobacter pylori. Nucleic Acids Res 2011; 39:7564-75. [PMID: 21666253 PMCID: PMC3177205 DOI: 10.1093/nar/gkr460] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
Nickel is an essential metal for Helicobacter pylori, as it is the co-factor of two enzymes crucial for colonization, urease and hydrogenase. Nickel is taken up by specific transporters and its intracellular homeostasis depends on nickel-binding proteins to avoid toxicity. Nickel trafficking is controlled by the Ni(II)-dependent transcriptional regulator NikR. In contrast to other NikR proteins, NikR from H. pylori is a pleiotropic regulator that depending on the target gene acts as an activator or a repressor. We systematically quantified the in vivo Ni2+-NikR response of 11 direct NikR targets that encode functions related to nickel metabolism, four activated and seven repressed genes. Among these, four targets were characterized for the first time (hpn, hpn-like, hydA and hspA) and NikR binding to their promoter regions was demonstrated by electrophoretic mobility shift assays. We found that NikR-dependent repression was generally set up at higher nickel concentrations than activation. Kinetics of the regulation revealed a gradual and temporal NikR-mediated response to nickel where activation of nickel-protection mechanisms takes place before repression of nickel uptake. Our in vivo study demonstrates, for the first time, a chronological hierarchy in the NikR-dependent transcriptional response to nickel that is coherent with the control of nickel homeostasis in H. pylori.
Collapse
Affiliation(s)
- Cécile Muller
- Département de Microbiologie, Institut Pasteur, Unité Pathogenèse de Helicobacter, Paris Cedex 15, France
| | | | | | | | | | | | | |
Collapse
|
|
35
|
Benanti EL, Chivers PT. Helicobacter pylori NikR protein exhibits distinct conformations when bound to different promoters. J Biol Chem 2011; 286:15728-37. [PMID: 21393642 DOI: 10.1074/jbc.m110.196055] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
Helicobacter pylori NikR (HpNikR) is a ribbon-helix-helix (RHH) DNA-binding protein that binds to several different promoter regions. The binding site sequences are not absolutely conserved. The ability of HpNikR to discriminate specific DNA sites resides partly in its nine-amino acid N-terminal arm. Previously, indirect evidence indicated that the arm exists in different conformations when HpNikR is bound to the nixA and ureA promoters. Here, we directly examined HpNikR conformation when it was bound to nixA and ureA DNA fragments by tethering (S)-1{[bis(carboxymethyl)amino]methyl}-2-{4-[(2-bromoacetyl)amino]phenylethyl}(carboxymethyl)amino]acetic acid, iron(III) to different positions in the N-terminal arm and RHH DNA binding domain. Different cleavage patterns at each promoter directly demonstrated that both the RHH domain and the arm adopt different conformations on the nixA and ureA promoters. Additionally, the two RHH domain dimers of the HpNikR tetramer are in distinct conformations at ureA. Site-directed mutagenesis identified an interchain salt bridge (Lys(48)-Glu(47')) in the RHH domain remote from the DNA binding interface that is required for high affinity binding to ureA but not nixA. Finally, DNA affinity measurements of wild-type HpNikR and a salt bridge mutant (K48A) to hybrid nixA-ureA promoters demonstrated that inverted repeat half-sites, spacers, and flanking DNA are all required for sequence-specific DNA binding by HpNikR. Notably, the spacer region made the largest contribution to DNA affinity. HpNikR exhibits a substantially expanded regulon compared with other NikR proteins. The results presented here provide a molecular basis for understanding regulatory network expansion by NikR as well as other prokaryotic regulatory proteins.
Collapse
Affiliation(s)
- Erin L Benanti
- Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA
| | | |
Collapse
|
|
36
|
Segura-Cabrera A, Guo X, Rojo-Domínguez A, Rodríguez-Pérez MA. Integrative computational protocol for the discovery of inhibitors of the Helicobacter pylori nickel response regulator (NikR). J Mol Model 2011; 17:3075-84. [PMID: 21360181 DOI: 10.1007/s00894-011-0962-2] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2010] [Accepted: 01/05/2011] [Indexed: 12/21/2022]
Abstract
In order to identify novel inhibitors of the Helicobacter pylori nickel response regulator (HpNikR) an integrative protocol was performed for half a million compounds retrieved from the ZINC database. We firstly implement a structure-based virtual screening to build a library of potential inhibitors against the HpNikR using a docking analysis (AutoDock Vina). The library was then used to perform a hierarchical clustering of docking poses, based on protein-contact footprints calculation from the multiple conformations given by the AutoDock Vina software, and the drug-protein interaction analyses to identify and remove potential promiscuous compounds likely interacting with human proteins, hence causing drug side effects. 250 drug-like compounds were finally proposed as non-promicuous potential inhibitors for HpNikR. These compounds target the DNA-binding sites of HpNikR so that HpNikR-compound binding could be able to mimic key interactions in the DNA-protein recognition process. HpNikR inhibitors with promising potential against H. pylori could also act against other human bacterial pathogens due to the conservation of targeting motif of NikR involved in DNA-protein interaction.
Collapse
Affiliation(s)
- Aldo Segura-Cabrera
- Laboratorio de Bioinformática, Centro de Biotecnología Genómica, Instituto Politécnico Nacional, Boulevard del Maestro esquina Elías Piña, Colonia Narciso Mendoza, 88710 Ciudad Reynosa, Tamaulipas, Mexico.
| | | | | | | |
Collapse
|
|
37
|
West AL, St John F, Lopes PEM, MacKerell AD, Pozharski E, Michel SLJ. Holo-Ni(II)HpNikR is an asymmetric tetramer containing two different nickel-binding sites. J Am Chem Soc 2011; 132:14447-56. [PMID: 20863122 DOI: 10.1021/ja104118r] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The metalloregulatory protein NikR from Helicobacter pylori (HpNikR) is a master regulator of gene expression which both activates and represses specific genes in response to nickel availability. Here, we report the first crystal structure (at 2.37 Å resolution) of Ni(II)HpNikR prepared directly from the holo protein. The protein contains four nickel ions located in two distinct coordination environments. Two nickel ions are bound to sites in a four-coordinate square-planar geometry as predicted on the basis of the structures of NikR from Escherichia coli and Pyrococcus horikoshii . The remaining two nickel ions are bound to sites with unexpected 5- or 6-coordination geometries which were previously thought to be involved in nickel incorporation into the protein. The nickel with 5-/6-coordination geometry utilizes three histidines from two separate monomeric HpNikR units along with two or three water molecules as ligands. The spatial location of the nickel in the 5-/6-coordinate site is within approximately 5 Å of the expected site if a 4-coordinate square-planar geometry occurred. Two of the histidines that participate as ligands in the 5-/6-coordinate site would also participate as ligands if the 4-coordinate site was occupied, making it impossible for both sites to be occupied simultaneously. DFT calculations show that the 5-/6-coordinate geometries are energetically favorable when the local protein environment is included in the calculations. The presence of two distinct coordination environments in HpNikR is suggested to be related to the specificity and binding affinity of this transcription factor for DNA.
Collapse
Affiliation(s)
- Abby L West
- Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland 21201-1180, USA
| | | | | | | | | | | |
Collapse
|
|
38
|
In vivo recognition of the fecA3 target promoter by Helicobacter pylori NikR. J Bacteriol 2011; 193:1131-41. [PMID: 21216998 DOI: 10.1128/jb.01153-10] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
In Helicobacter pylori, the transcriptional regulator HpNikR represses transcription of the fecA3 gene by binding to two adjacent operators spanning a region of almost 80 nucleotides along the fecA3 promoter in a nickel-dependent manner. By employing hydroxyl radical footprinting, we mapped the protected nucleotides within each operator. Three short sequences rich in A and T nucleotides were identified within each operator, comprising just 24 bases for both operators, with 4 or 5 protected bases interspaced by 4 to 7 free nucleotides, with no center of symmetry. Base substitutions at any site strongly reduced the affinity of HpNikR for the operators and also affected the stability of the DNA-protein complex, when the promoter-regulator interaction was analyzed in vitro. The effect of these substitutions was remarkably different when transcription of the mutant promoters was analyzed in vivo. Base changes introduced at the farthest subsites impaired the HpNikR-dependent repression, with the mutations closer to +1 completely abolishing the repression, the more distal one still allowing almost 50% of transcription, and the mutations in the middle being ineffective. The data presented here show that HpNikR may first select its targets by identifying sequences within the previously defined consensus and subsequently establish base-specific contacts to firmly bind DNA. In particular, HpNikR seems to interact in an asymmetric mode with the fecA3 target to repress its transcription.
Collapse
|
|
39
|
Lovley DR, Ueki T, Zhang T, Malvankar NS, Shrestha PM, Flanagan KA, Aklujkar M, Butler JE, Giloteaux L, Rotaru AE, Holmes DE, Franks AE, Orellana R, Risso C, Nevin KP. Geobacter: the microbe electric's physiology, ecology, and practical applications. Adv Microb Physiol 2011; 59:1-100. [PMID: 22114840 DOI: 10.1016/b978-0-12-387661-4.00004-5] [Citation(s) in RCA: 412] [Impact Index Per Article: 29.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Geobacter species specialize in making electrical contacts with extracellular electron acceptors and other organisms. This permits Geobacter species to fill important niches in a diversity of anaerobic environments. Geobacter species appear to be the primary agents for coupling the oxidation of organic compounds to the reduction of insoluble Fe(III) and Mn(IV) oxides in many soils and sediments, a process of global biogeochemical significance. Some Geobacter species can anaerobically oxidize aromatic hydrocarbons and play an important role in aromatic hydrocarbon removal from contaminated aquifers. The ability of Geobacter species to reductively precipitate uranium and related contaminants has led to the development of bioremediation strategies for contaminated environments. Geobacter species produce higher current densities than any other known organism in microbial fuel cells and are common colonizers of electrodes harvesting electricity from organic wastes and aquatic sediments. Direct interspecies electron exchange between Geobacter species and syntrophic partners appears to be an important process in anaerobic wastewater digesters. Functional and comparative genomic studies have begun to reveal important aspects of Geobacter physiology and regulation, but much remains unexplored. Quantifying key gene transcripts and proteins of subsurface Geobacter communities has proven to be a powerful approach to diagnose the in situ physiological status of Geobacter species during groundwater bioremediation. The growth and activity of Geobacter species in the subsurface and their biogeochemical impact under different environmental conditions can be predicted with a systems biology approach in which genome-scale metabolic models are coupled with appropriate physical/chemical models. The proficiency of Geobacter species in transferring electrons to insoluble minerals, electrodes, and possibly other microorganisms can be attributed to their unique "microbial nanowires," pili that conduct electrons along their length with metallic-like conductivity. Surprisingly, the abundant c-type cytochromes of Geobacter species do not contribute to this long-range electron transport, but cytochromes are important for making the terminal electrical connections with Fe(III) oxides and electrodes and also function as capacitors, storing charge to permit continued respiration when extracellular electron acceptors are temporarily unavailable. The high conductivity of Geobacter pili and biofilms and the ability of biofilms to function as supercapacitors are novel properties that might contribute to the field of bioelectronics. The study of Geobacter species has revealed a remarkable number of microbial physiological properties that had not previously been described in any microorganism. Further investigation of these environmentally relevant and physiologically unique organisms is warranted.
Collapse
Affiliation(s)
- Derek R Lovley
- Department of Microbiology and Environmental Biotechnology Center, University of Massachusetts, Amherst, Massachusetts, USA
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
40
|
Phillips CM, Stultz CM, Drennan CL. Searching for the Nik operon: how a ligand-responsive transcription factor hunts for its DNA binding site. Biochemistry 2010; 49:7757-63. [PMID: 20712334 PMCID: PMC2934762 DOI: 10.1021/bi100947k] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
![]()
Transcription factors regulate a wide variety of genes in the cell and play a crucial role in maintaining cellular homeostasis. A major unresolved issue is how transcription factors find their specific DNA binding sequence in the vast expanse of the cell and how they do so at rates that appear faster than the diffusion limit. Here, we relate an atomic-detail model that has been developed to describe the transcription factor NikR’s mechanism of DNA binding to the broader theories of how transcription factors find their binding sites on DNA. NikR is the nickel regulatory transcription factor for many bacteria, and NikR from Escherichia coli is one of the best studied ligand-mediated transcription factors. For the E. coli NikR protein, there is a wide variety of structural, biochemical, and computational studies that provide significant insight into the NikR−DNA binding mechanism. We find that the two models, the atomic-level model for E. coli NikR and the cellular model for transcription factors in general, are in agreement, and the details laid out by the NikR system may lend additional credence to the current models for transcription factors searching for DNA.
Collapse
Affiliation(s)
- Christine M Phillips
- Department of Chemistry, Massachusetts Institute of Technology,Cambridge, Massachusetts 02139, USA
| | | | | |
Collapse
|
|
41
|
Phillips CM, Schreiter ER, Stultz CM, Drennan CL. Structural basis of low-affinity nickel binding to the nickel-responsive transcription factor NikR from Escherichia coli. Biochemistry 2010; 49:7830-8. [PMID: 20704276 PMCID: PMC2934763 DOI: 10.1021/bi100923j] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
![]()
Escherichia coli NikR regulates cellular nickel uptake by binding to the nik operon in the presence of nickel and blocking transcription of genes encoding the nickel uptake transporter. NikR has two binding affinities for the nik operon: a nanomolar dissociation constant with stoichiometric nickel and a picomolar dissociation constant with excess nickel [Bloom, S. L., and Zamble, D. B. (2004) Biochemistry 43, 10029−10038; Chivers, P. T., and Sauer, R. T. (2002) Chem. Biol. 9, 1141−1148]. While it is known that the stoichiometric nickel ions bind at the NikR tetrameric interface [Schreiter, E. R., et al. (2003) Nat. Struct. Biol. 10, 794−799; Schreiter, E. R., et al. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 13676−13681], the binding sites for excess nickel ions have not been fully described. Here we have determined the crystal structure of NikR in the presence of excess nickel to 2.6 Å resolution and have obtained nickel anomalous data (1.4845 Å) in the presence of excess nickel for both NikR alone and NikR cocrystallized with a 30-nucleotide piece of double-stranded DNA containing the nik operon. These anomalous data show that excess nickel ions do not bind to a single location on NikR but instead reveal a total of 22 possible low-affinity nickel sites on the NikR tetramer. These sites, for which there are six different types, are all on the surface of NikR, and most are found in both the NikR alone and NikR−DNA structures. Using a combination of crystallographic data and molecular dynamics simulations, the nickel sites can be described as preferring octahedral geometry, utilizing one to three protein ligands (typically histidine) and at least two water molecules.
Collapse
Affiliation(s)
- Christine M Phillips
- Department of Chemistry, Massachusetts Institute of Technology,Cambridge, Massachusetts 02139, USA
| | | | | | | |
Collapse
|
|
42
|
Musiani F, Bertoša B, Magistrato A, Zambelli B, Turano P, Losasso V, Micheletti C, Ciurli S, Carloni P. Computational Study of the DNA-Binding Protein Helicobacter pylori NikR: The Role of Ni2+ 2 Francesco Musiani and Branimir Bertoša contributed equally to the simulations presented here. J Chem Theory Comput 2010; 6:3503-15. [DOI: 10.1021/ct900635z] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Affiliation(s)
- Francesco Musiani
- Laboratory of Bioinorganic Chemistry, University of Bologna, Viale G. Fanin 40, 40127 Bologna, Italy, International School for Advanced Studies (SISSA) and CNR-IOM-DEMOCRITOS National Simulation Center, via Bonomea 265, 34136 Trieste, Italy, Ruder Bošković Institute, Bijeniěka 54, 10000 Zagreb, Croatia, German Research School for Simulation Science, FZ-Jülichand RWTH, Wilhelm-Johnen-Strasse, 52428 Jülich, Germany, Center for Magnetic Resonance (CERM), University of Florence, Via Luigi Sacconi 6, 50019
| | - Branimir Bertoša
- Laboratory of Bioinorganic Chemistry, University of Bologna, Viale G. Fanin 40, 40127 Bologna, Italy, International School for Advanced Studies (SISSA) and CNR-IOM-DEMOCRITOS National Simulation Center, via Bonomea 265, 34136 Trieste, Italy, Ruder Bošković Institute, Bijeniěka 54, 10000 Zagreb, Croatia, German Research School for Simulation Science, FZ-Jülichand RWTH, Wilhelm-Johnen-Strasse, 52428 Jülich, Germany, Center for Magnetic Resonance (CERM), University of Florence, Via Luigi Sacconi 6, 50019
| | - Alessandra Magistrato
- Laboratory of Bioinorganic Chemistry, University of Bologna, Viale G. Fanin 40, 40127 Bologna, Italy, International School for Advanced Studies (SISSA) and CNR-IOM-DEMOCRITOS National Simulation Center, via Bonomea 265, 34136 Trieste, Italy, Ruder Bošković Institute, Bijeniěka 54, 10000 Zagreb, Croatia, German Research School for Simulation Science, FZ-Jülichand RWTH, Wilhelm-Johnen-Strasse, 52428 Jülich, Germany, Center for Magnetic Resonance (CERM), University of Florence, Via Luigi Sacconi 6, 50019
| | - Barbara Zambelli
- Laboratory of Bioinorganic Chemistry, University of Bologna, Viale G. Fanin 40, 40127 Bologna, Italy, International School for Advanced Studies (SISSA) and CNR-IOM-DEMOCRITOS National Simulation Center, via Bonomea 265, 34136 Trieste, Italy, Ruder Bošković Institute, Bijeniěka 54, 10000 Zagreb, Croatia, German Research School for Simulation Science, FZ-Jülichand RWTH, Wilhelm-Johnen-Strasse, 52428 Jülich, Germany, Center for Magnetic Resonance (CERM), University of Florence, Via Luigi Sacconi 6, 50019
| | - Paola Turano
- Laboratory of Bioinorganic Chemistry, University of Bologna, Viale G. Fanin 40, 40127 Bologna, Italy, International School for Advanced Studies (SISSA) and CNR-IOM-DEMOCRITOS National Simulation Center, via Bonomea 265, 34136 Trieste, Italy, Ruder Bošković Institute, Bijeniěka 54, 10000 Zagreb, Croatia, German Research School for Simulation Science, FZ-Jülichand RWTH, Wilhelm-Johnen-Strasse, 52428 Jülich, Germany, Center for Magnetic Resonance (CERM), University of Florence, Via Luigi Sacconi 6, 50019
| | - Valeria Losasso
- Laboratory of Bioinorganic Chemistry, University of Bologna, Viale G. Fanin 40, 40127 Bologna, Italy, International School for Advanced Studies (SISSA) and CNR-IOM-DEMOCRITOS National Simulation Center, via Bonomea 265, 34136 Trieste, Italy, Ruder Bošković Institute, Bijeniěka 54, 10000 Zagreb, Croatia, German Research School for Simulation Science, FZ-Jülichand RWTH, Wilhelm-Johnen-Strasse, 52428 Jülich, Germany, Center for Magnetic Resonance (CERM), University of Florence, Via Luigi Sacconi 6, 50019
| | - Cristian Micheletti
- Laboratory of Bioinorganic Chemistry, University of Bologna, Viale G. Fanin 40, 40127 Bologna, Italy, International School for Advanced Studies (SISSA) and CNR-IOM-DEMOCRITOS National Simulation Center, via Bonomea 265, 34136 Trieste, Italy, Ruder Bošković Institute, Bijeniěka 54, 10000 Zagreb, Croatia, German Research School for Simulation Science, FZ-Jülichand RWTH, Wilhelm-Johnen-Strasse, 52428 Jülich, Germany, Center for Magnetic Resonance (CERM), University of Florence, Via Luigi Sacconi 6, 50019
| | - Stefano Ciurli
- Laboratory of Bioinorganic Chemistry, University of Bologna, Viale G. Fanin 40, 40127 Bologna, Italy, International School for Advanced Studies (SISSA) and CNR-IOM-DEMOCRITOS National Simulation Center, via Bonomea 265, 34136 Trieste, Italy, Ruder Bošković Institute, Bijeniěka 54, 10000 Zagreb, Croatia, German Research School for Simulation Science, FZ-Jülichand RWTH, Wilhelm-Johnen-Strasse, 52428 Jülich, Germany, Center for Magnetic Resonance (CERM), University of Florence, Via Luigi Sacconi 6, 50019
| | - Paolo Carloni
- Laboratory of Bioinorganic Chemistry, University of Bologna, Viale G. Fanin 40, 40127 Bologna, Italy, International School for Advanced Studies (SISSA) and CNR-IOM-DEMOCRITOS National Simulation Center, via Bonomea 265, 34136 Trieste, Italy, Ruder Bošković Institute, Bijeniěka 54, 10000 Zagreb, Croatia, German Research School for Simulation Science, FZ-Jülichand RWTH, Wilhelm-Johnen-Strasse, 52428 Jülich, Germany, Center for Magnetic Resonance (CERM), University of Florence, Via Luigi Sacconi 6, 50019
| |
Collapse
|
|
43
|
Wang SC, Li Y, Ho M, Bernal ME, Sydor AM, Kagzi WR, Zamble DB. The response of Escherichia coli NikR to nickel: a second nickel-binding site. Biochemistry 2010; 49:6635-45. [PMID: 20583753 DOI: 10.1021/bi100685k] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
The Escherichia coli transcription factor NikR mediates two levels of regulatory control of Ni(II) uptake in response to changes in the levels of available nickel. Despite the evidence that metal binding to two distinct sites on NikR, referred to as the high- and low-affinity Ni(II) sites, is required for Ni(II)-selective DNA binding by the protein, the role of the latter set of Ni(II) ions in the activation of NikR remains controversial, and the position of the putative low-affinity Ni(II)-binding site(s) on NikR has not been determined. In this study we confirm that NikR has a high-affinity Ni(II)-binding site that is maintained upon DNA binding. The ligands of the low-affinity Ni(II)-binding site were examined by using selective chemical modification and mass spectrometry performed in the presence of excess Ni(II) and DNA. We localized this Ni(II) site to a region at the interface between the metal- and DNA-binding domains and identified His48 and His110 as residues that participate in the low-affinity Ni(II)-binding response. Mutation of His48 and His110 to asparagines reduces significantly both NikR's tendency to precipitate in the presence of excess Ni(II) and the affinity of the DNA-bound complex in the presence of excess Ni(II). A complete scheme involving all of the metal-binding sites that contribute to the regulatory function of E. coli NikR in nickel homeostasis is described.
Collapse
Affiliation(s)
- Sheila C Wang
- Department of Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 3H6
| | | | | | | | | | | | | |
Collapse
|
|
44
|
Geobacter uraniireducens NikR displays a DNA binding mode distinct from other members of the NikR family. J Bacteriol 2010; 192:4327-36. [PMID: 20581212 DOI: 10.1128/jb.00152-10] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
NikR is a nickel-responsive ribbon-helix-helix transcription factor present in many bacteria and archaea. The DNA binding properties of Escherichia coli and Helicobacter pylori NikR (factors EcNikR and HpNikR, respectively) have revealed variable features of DNA recognition. EcNikR represses a single operon by binding to a perfect inverted repeat sequence, whereas HpNikR binds to promoters from multiple genes that contain poorly conserved inverted repeats. These differences are due in large part to variations in the amino acid sequences of the DNA-contacting beta-sheets, as well as residues preceding the beta-sheets of these two proteins. We present here evidence of another variation in DNA recognition by the NikR protein from Geobacter uraniireducens (GuNikR). GuNikR has an Arg-Gly-Ser beta-sheet that binds specifically to an inverted repeat sequence distinct from those recognized by Ec- or HpNikR. The N-terminal residues that precede the GuNikR beta-sheet residues are required for high-affinity DNA binding. Mutation of individual arm residues dramatically reduced the affinity of GuNikR for specific DNA. Interestingly, GuNikR tetramers are capable of binding cooperatively to the promoter regions of two different genes, nik(MN)1 and nik(MN)2. Cooperativity was not observed for the closely related G. bemidjiensis NikR, which recognizes the same operator sequence. The cooperative mode of DNA binding displayed by GuNikR could affect the sensitivity of transporter gene expression to changes in intracellular nickel levels.
Collapse
|
|
45
|
Dodani SC, He Q, Chang CJ. A turn-on fluorescent sensor for detecting nickel in living cells. J Am Chem Soc 2010; 131:18020-1. [PMID: 19950946 DOI: 10.1021/ja906500m] [Citation(s) in RCA: 182] [Impact Index Per Article: 12.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
We present the synthesis and properties of Nickelsensor-1 (NS1), a new water-soluble, turn-on fluorescent sensor that is capable of selectively responding to Ni(2+) in aqueous solution and in living cells. NS1 combines a BODIPY chromophore and a mixed N/O/S receptor to provide good selectivity for Ni(2+) over a range of biologically abundant metal ions in aqueous solution. In addition to these characteristics, confocal microscopy experiments further show that NS1 can be delivered into living cells and report changes in intracellular Ni(2+) levels in a respiratory cell model.
Collapse
Affiliation(s)
- Sheel C Dodani
- Department of Chemistry and the Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA
| | | | | |
Collapse
|
|
46
|
Danielli A, Scarlato V. Regulatory circuits in Helicobacter pylori : network motifs and regulators involved in metal-dependent responses. FEMS Microbiol Rev 2010; 34:738-52. [PMID: 20579104 DOI: 10.1111/j.1574-6976.2010.00233.x] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
The ability of Helicobacter pylori, one of the most successful human bacterial pathogens, to colonize the acidic gastric niche persistently, depends on the proper homeostasis of intracellular metal ions, needed as cofactors of essential metallo-proteins involved in acid acclimation, respiration and detoxification. This fundamental task is controlled at the transcriptional level mainly by the regulators Fur and NikR, involved in iron homeostasis and nickel response, respectively. Herein, we review the molecular mechanisms that underlie the activity of these key pleiotropic regulators. In addition, we will focus on their involvement in the transcriptional regulatory network of the bacterium, pinpointing a surprising complexity of network motifs that interconnects them and their gene targets. These motifs appear to confer versatile dynamics of metal-dependent responses by extensive horizontal connections between the regulators and feedback control of metal-cofactor availability.
Collapse
|
|
47
|
A new member of the ribbon-helix-helix transcription factor superfamily from the plant pathogen Xanthomonas axonopodis pv. citri. J Struct Biol 2010; 170:21-31. [DOI: 10.1016/j.jsb.2009.12.022] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2009] [Revised: 11/11/2009] [Accepted: 12/22/2009] [Indexed: 11/19/2022]
|
|
48
|
Iwig JS, Chivers PT. Coordinating intracellular nickel-metal-site structure-function relationships and the NikR and RcnR repressors. Nat Prod Rep 2010; 27:658-67. [PMID: 20442957 DOI: 10.1039/b906683g] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Metalloregulator function requires both sensitivity and selectivity to ensure metal-specific activity without interfering with intracellular metal trafficking pathways. Here, we examine the role of metal coordination geometry in the function of NikR and RcnR, two widely conserved nickel-responsive regulators that are both present in E. coli. The available data suggest an emerging trend in which coordination number is linked to metal-binding affinity, and thus regulatory function. The differences in coordination geometry also suggest that the kinetic mechanisms of metal-association and dissociation will contribute to metalloregulator function. We also discuss ways in which the ligand binding properties of metalloregulators may be tuned to alter the regulatory response.
Collapse
Affiliation(s)
- Jeffrey S Iwig
- Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, USA
| | | |
Collapse
|
|
49
|
Sindhikara DJ, Roitberg AE, Merz KM. Apo and nickel-bound forms of the Pyrococcus horikoshii species of the metalloregulatory protein: NikR characterized by molecular dynamics simulations. Biochemistry 2010; 48:12024-33. [PMID: 19891498 DOI: 10.1021/bi9013352] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
NikR is a homotetrameric nickel regulatory protein whose binding to free Ni(2+) increases its binding affinity for a gene that codes for a nickel transporter protein. It is comprised of a tetrameric nickel-binding domain, flanked by two dimeric DNA-binding domains. Though X-ray crystallography data for various species (Escherichia coli, Heliobacter pylori, and Pyrococcus horikoshii) of NikR reveal large conformational differences between nickel-bound, DNA-bound, and unbound forms, transitions between them have never been observed. We have run all-atom molecular dynamics simulations of three forms of the Pyrococcus horikoshii species of NikR including two apo-forms and one nickel-bound form. Though all 552 residues of this species occur naturally, quantum-mechanics-based force-field parametrization was required to accurately represent the four nickel-centers in the nickel-bound form. Global conformational analysis of the three 100-ns-long simulations indicates slow conformational kinetics and independent DNA binding domain motion. Correlation and flexibility analysis revealed regions of high structural and dynamical importance. A striking relationship was observed between regions with high levels of structural importance and regions with known biological importance. Mutation of key regions of P. horikoshii and analogous regions in both E. coli and H. pylori are suggested that might inhibit DNA-binding activity while not affecting nickel-binding.
Collapse
Affiliation(s)
- Daniel J Sindhikara
- University of Florida, Department of Chemistry Quantum Theory Project, 2328 New Physics Building, P.O. Box 118435, Gainesville, Florida 32611-8435, USA
| | | | | |
Collapse
|
|
50
|
Ma Z, Jacobsen FE, Giedroc DP. Coordination chemistry of bacterial metal transport and sensing. Chem Rev 2009; 109:4644-81. [PMID: 19788177 PMCID: PMC2783614 DOI: 10.1021/cr900077w] [Citation(s) in RCA: 452] [Impact Index Per Article: 28.3] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Affiliation(s)
- Zhen Ma
- Department of Chemistry, Indiana University, Bloomington, IN 47401-7005 USA
- Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128 USA
| | - Faith E. Jacobsen
- Department of Chemistry, Indiana University, Bloomington, IN 47401-7005 USA
| | - David P. Giedroc
- Department of Chemistry, Indiana University, Bloomington, IN 47401-7005 USA
| |
Collapse
|