Subscriber access provided by University of Otago Library
Article
The Mechanism of Selective Nickel Transfer From HypB to HypA, E. coli [NiFe]-Hydrogenase Accessory Proteins Michael J. Lacasse, Colin D. Douglas, and Deborah Beth Zamble Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00706 • Publication Date (Web): 09 Nov 2016 Downloaded from http://pubs.acs.org on November 12, 2016
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 46
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biochemistry
The Mechanism of Selective Nickel Transfer From HypB to HypA, E. coli [NiFe]-Hydrogenase Accessory Proteins B. Funding Source Statement This work was supported in part by funding from the Natural Science and Engineering Research Council (Canada), including an NSERC Postgraduate Scholarship (MJL), and the Canadian Institutes of Health Research. B. Byline
Michael J. Lacasse †1, Colin D. Douglas †1, Deborah B. Zamble*1,2 1
Department of Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 3H6 and
2
Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8 *Correspondence to Deborah Zamble:
[email protected], 416-978-3568
† These authors contributed equally to this work.
Page 1 of 42
ACS Paragon Plus Environment
Biochemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Abbreviations and Textual Footnotes GDP: guanosine 5′-diphosphate, GppCp: βγ-methyleneguanosine 5′-triphosphate, GFC: gel filtration chromatography, DTT: dithiothreitol, EDTA: ethylenediaminetetraacetic acid, ESI-MS: electrosprayionization mass spectrometry, HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, IPTG: isopropyl β-D-1-thiogalactopyranoside, MF2: Mag-Fura-2, PAR: 4-(2-pyridylazo)resorcinol, PMB: phydroxymercuribenzoate, PMSF: phenylmethanesulfonyl fluoride, TCEP: tris (2-carboxyethyl) phosphine, and TRIS: tris(hydroxymethyl)aminomethane.
Page 2 of 42
ACS Paragon Plus Environment
Page 2 of 46
Page 3 of 46
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biochemistry
Abstract [NiFe]-hydrogenase enzymes catalyze the reversible reduction of protons to molecular hydrogen and serve as a vital component of the anaerobic metabolism of many pathogens. The synthesis of the bimetallic catalytic center requires a suite of accessory proteins and the penultimate step, nickel insertion, is facilitated by the metallochaperones HypA and HypB. In Escherichia coli, nickel moves from a site in the GTPase domain of HypB to HypA in a process accelerated by GDP. To determine how the transfer of nickel is controlled, the impact of HypA and nucleotides on the properties of HypB were examined. Integral to this work was His2Gln HypA, a mutant with attenuated nickel affinity that does not support hydrogenase production in E. coli. This mutation inhibits nickel translocation from HypB. H2Q-HypA does not modulate the apparent metal affinity of HypB, but the stoichiometry and stability of the HypB-nickel complex are modulated by nucleotide. Furthermore, the HypA-HypB interaction was detected by gel filtration chromatography if HypB was loaded with GDP, but not the GTP analog, and the protein complex dissociated upon nickel binding to His2 of HypA. In contrast, nucleotide does not modulate zinc binding to HypB, and loading zinc into the GTPase domain of HypB inhibits complex formation with HypA. These results demonstrate that GTP hydrolysis controls both metal binding and protein-protein interactions, conferring selective and directional nickel transfer during [NiFe]hydrogenase biosynthesis.
Page 3 of 42
ACS Paragon Plus Environment
Biochemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Introduction Metalloenzymes are vital for the survival of living organisms, but the transition metals that serve as necessary catalytic cofactors can be toxic if unregulated.1-3 To control the distribution and availability of these crucial nutrients, organisms deploy networks of transporters, storage proteins, regulators, and metallochaperones.1, 4-6 These systems keep metals appropriately compartmentalized, minimizing the amounts of "free" metal and ensuring correct metallation of metalloenzymes in the face of the relative thermodynamic affinities dictated by the Irving-Williams series.7 One strategy to achieve these goals is directed and selective metal transfer between the metallochaperones that funnel the cognate metal to the metalloenzyme active sites; however, the mechanisms behind these processes are not well defined. Hydrogenases are metalloenzymes that catalyze the reversible interconversion between molecular hydrogen and protons and electrons, and they make crucial contributions to the metabolism of many microorganisms.8, 9 Canonically, hydrogenases are divided into three categories based on the metals at the active site: [FeFe], [Fe], and [NiFe].9 The biosynthesis and function of [NiFe]-hydrogenases are of interest due to the drive to understand fundamental metal metabolism, the obligatory role of these systems in infections by bacteria such as Escherichia coli and Helicobacter pylori, as well as potential applications to the hydrogen economy.8, 10-14 Maturation of the E. coli [NiFe]-hydrogenase isoenzymes involves at least seven proteins, most of which are encoded on the hyp operon.12, 15-17 Two of these maturation proteins, HypA (or the homologous HybF) and HypB, are metallochaperones required for nickel delivery to the hydrogenase precursor protein.12, 15 Escherichia coli HypB is a 31 kDa metal-binding protein with GTPase activity.18, 19 Although it has been clearly demonstrated that GTP hydrolysis by HypB is required for nickel delivery to the hydrogenase accessory protein,20 the biological purpose of this activity is not known. Escherichia coli HypB has two distinct metal-binding sites. The N-terminus contains the “high-affinity site”, a seven-
Page 4 of 42
ACS Paragon Plus Environment
Page 4 of 46
Page 5 of 46
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biochemistry
residue sequence that binds nickel with sub-picomolar affinity.19, 21, 22 Mutation of metal-binding residues at the high-affinity site of E. coli HypB impairs its hydrogenase maturation function,23 but this site is only partially conserved amongst HypB orthologs.16 A second metal-binding site, also required for hydrogenase maturation in E. coli,23 is located in the C-terminal GTPase domain (G-domain) of HypB and is conserved across virtually all bacterial species containing this gene.19, 24-27 Nickel binds to this site with mid-micromolar affinity while zinc binds an order of magnitude tighter.19 Metal binding to the G-domain of HypB is linked to the GTPase activity because hydrolysis of GTP in E. coli HypB is significantly reduced when nickel or zinc is bound in the G-domain.25, 27, 28 This relationship may be bidirectional, as biochemical analysis of H. pylori HypB demonstrated that the nucleotide-loaded state of the protein impacts the metal-binding site.25, 26 HypA also has two metal-binding domains: a zinc-binding domain and a nickel-binding domain. The zinc-binding domain is comprised of a structural tetrathiolate site and is thought to mediate proteinprotein interactions.29-32 Studies of HypA from H. pylori, where HypA and HypB also moonlight in the production of the nickel enzyme urease,33 suggested that this site can act as a pH sensor and direct nickel to the appropriate metalloenzyme based on the acidity of the cytosol.34, 35 The other domain binds nickel, with mid-nanomolar affinity measured for E. coli HypA,36 and includes residues from the Nterminus.29, 30, 32, 37 Biochemical, NMR, and X-ray absorption spectroscopy data indicate that the highly conserved His-2 and Glu-3 residues in conjunction with backbone nitrogens are likely coordinating the nickel, but the complete site is currently undefined.29, 32, 34, 37 Purified E. coli HypA and HypB proteins form a complex in vitro,30 an interaction that was also observed by using pull-down experiments from crude E. coli cell lysates.38 HypA and HypB proteins were detected in a complex with HycE, the large subunit of hydrogenase 3, but a hypA knockout prevented HypB from being pulled down with HycE.38 Taken together, the properties of HypA are consistent with the hypothesis that HypA/HybF (the latter functionally replacing HypA for maturation of hydrogenases 1
Page 5 of 42
ACS Paragon Plus Environment
Biochemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
and 239) act as ‘adaptors’ that dock the nickel delivery complex onto the target hydrogenase precursor protein during metallocenter assembly.38 As predicted by the thermodynamics of nickel binding to the separate proteins, nickel was observed to move from the G-domain site of HypB to HypA within minutes when the two proteins were mixed together.36 When the same experiment was performed in the presence of GDP, the transfer of nickel to HypA was accelerated by several orders of magnitude, but the transfer of nickel from HypB to small molecule chelators was slower.36 Furthermore, mutations in HypB that disrupted the interaction with HypA also retarded nickel transfer in vitro and hydrogenase production in vivo.36, 40 These observations support the model in which the HypB-HypA complex is a key component of the nickel delivery stage of hydrogenase maturation, with nickel passing from one protein to the other. However, the specific molecular mechanisms by which this transfer is controlled were not known. In this study, we examined how nucleotide loading and complex formation with HypA impact the metal-binding properties of the G-domain site of E. coli HypB, allowing us to probe the changes in HypB during the various steps of the GTPase cycle. We discovered that GTP and GDP affect nickel binding to HypB in a manner consistent with the protein first acting as a nickel acceptor and then as a nickel source. Furthermore, only GDP promotes complex formation with HypA, and the protein complex dissociates once HypA is loaded with nickel. In contrast, zinc appears to inhibit the protein-protein interaction. These experiments demonstrate directional and specific nickel translocation and shed light on how this process is controlled by GTP hydrolysis during the maturation of [NiFe]-hydrogenase in E. coli.
Page 6 of 42
ACS Paragon Plus Environment
Page 6 of 46
Page 7 of 46
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biochemistry
Materials and Methods PAR (4-(2-pyridylazo)-resorcinol), PMB (para-mercury-benzenesulfonic acid), DTNB (5,5′-dithiobis(2nitrobenzoic acid), GppCp (βγ-methyleneguanosine 5′-triphosphate), benzyl viologen, sodium formate, arabinose, and GDP (Guanosine diphosphate) were purchased from Sigma-Aldrich. Metal salts (NiSO4, ZnSO4, and MgSO4) were trace metal grade and were purchased from Sigma-Aldrich. MF2 (Mag-Fura-2) was purchased from Invitrogen. All other chemicals were obtained from BioShop, Canada as either biology grade or certified ACS reagents, unless otherwise described. Nitrogen gas and hydrogen gas mix were supplied by Praxair. Solutions for metal assays were prepared with Milli-Q water and treated with Chelex-100 (Bio-Rad) to minimize trace metal contamination. Electronic absorption spectra were recorded on an Agilent 8453 spectrophotometer at room temperature. Cloning, Expression and Purification The H2Q mutation was introduced to both strA-pET24b and strA-pBAD18-kan plasmids38 in one step by using Quikchange mutagenesis to create H2QstrA-pET24b and H2QstrA-pBAD18-kan respectively. Forward (5'-GGAGATATACATATGCAGGAAATAACCCTCTGCCAACGGGCACTGG-3’) and reverse (5'CCAGTGCCCGTTGGCAGAGGGTTATTTCCTGCATATGTATATCTCC-3’) primers were used to create the mutation in pET24b. Forward (5'-GGAGGAATTCACCATGCAGGAAATAACCCTCTGCC-3’) and reverse (5'GGCAGAGGGTTATTTCCTGCATGGTGAATTCCTCC-3’) primers were used to create the mutation in pBAD18-kan. Primers were purchased from Integrated DNA Technologies. All mutations were verified by sequencing (ACGT, Toronto). HypAStr and H2Q-HypAStr were expressed in BL21(DE3) E. coli and separated from cell lysates as previously described,36 using Strep-Tactin Sepharose affinity purification (IBA Life Sciences, Goettingen, Germany). After purification through Strep Tactin Sepharose resin, HypAStr and H2Q-HypAStr were dialyzed into 20 mM Tris, pH 7.5, 1 mM TCEP and either stored at -80°C if
Page 7 of 42
ACS Paragon Plus Environment
Biochemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
the stoichiometric zinc was measured (PAR assay) or further purified through a HiTrap Q HP column (GE Healthcare) to remove apo-protein. SDS-PAGE with Coomassie blue stain was used to screen the chromatography fractions. The molecular weights of purified proteins were verified by ESI-MS, performed by AIMS Laboratory (University of Toronto, Toronto). HypAStr and H2Q-HypAStr were purified with > 90% zinc as verified by non-denaturing ESI-MS (described below) and PAR assays.36 Wild type (WT) E. coli HypB and mutant versions were prepared as previously described.19, 36 WT-HypB was purified with the N-terminal metal-biding site occupied with nickel. To prepare mTM-HypB, a combination of monomeric HypB (L242A, L246A) and triple mutant HypB (C2A, C5A, C7A), the plasmid, mTmhypB-pET24b was constructed from a previously prepared pET24b-HypB C2A, C5A, C7A plasmid.19 The L242A and L246A mutations were introduced in one step by using QuikChange mutagenesis. The forward (5'-GCTCAACAAAGTTGACGCGTTGCCGTATGCCAACTTTGACG-3’) and reverse (5'CGTCAAAGTTGGCATACGGCAACGCGTCAACTTTGTTGAGC-3’) primers were used. All mutations were verified by sequencing (ACGT, Toronto). Non-Denaturing Electrospray Mass Spectrometry Analysis HypAStr and H2Q-HypAStr protein samples were buffer exchanged into 10 mM ammonium acetate, pH=7.5, using Amicon Ultra 3 kDa molecular weight cut-off centrifugal filters (Millipore) under an anaerobic atmosphere (95% N2, 5% H2), and then diluted to 5 µM in the same buffer. For nickel binding analysis, the protein was briefly (
