Structural and functional insights into corrinoid iron-sulfur protein from human pathogen Clostridium difficile

Journal of Inorganic Biochemistry 170 (2017) 26–33

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Structural and functional insights into corrinoid iron-sulfur protein from human pathogen Clostridium difficile Yaozhu Wei, Xiaofei Zhu, Sixue Zhang, Xiangshi Tan ⁎ Department of Chemistry, Institutes of Biomedical Sciences & Shanghai Key Laboratory of Chemical Biology for Protein Research, Fudan University, Shanghai 200433, China

a r t i c l e

i n f o

Article history: Received 31 August 2016 Received in revised form 21 January 2017 Accepted 9 February 2017 Available online 13 February 2017 Keywords: Corrinoid iron-sulfur protein CoFeSPcd Clostridium difficile Metalloprotein Structure and function

a b s t r a c t The human pathogen Clostridium difficile infection (CDI) is one of the most important healthcare-associated infections. The Wood-Ljungdahl pathway, which is responsible for Acetyl-CoA biosynthesis, is essential for the survival of the pathogen and is absent in humans. The key proteins and enzymes involved in the pathway are attractive targets for the treatment of CDI. Corrinoid iron-sulfur protein (CoFeSP) is a key protein and acts as a methyl transformer in the Wood-Ljungdahl pathway. In this study, CoFeSP from Clostridium difficile (CoFeSPCd) was cloned, expressed in E. coli and characterized for the first time. The structure and function of CoFeSPCd were investigated using homology structure modeling, spectroscopy, electrochemistry, steady state/pre-steady state kinetics and molecular docking. The two metal centers of CoFeSPCd, corrinoid cofactor and [4Fe-4S] cluster, were characterized using metal analysis, structural modeling, UV–Vis, EPR and direct electrochemistry. The methyl transfer activity between CH3-H4folate (CH3-THF) and CoFeSPCd catalyzed by methyl transferase (MeTrCd) was determined by kinetic studies. These results provide a molecular basis for innovative drug design and development to treat human CDI. © 2017 Published by Elsevier Inc.

1. Introduction Clostridium difficile is a spore-forming anaerobic pathogen, which can produce toxins A and B and then result in acute illnesses called C. difficile infection (CDI) in humans, ranging from severe diarrhea, antibiotic-associated colitis, pseudomembranous colitis, toxic megacolon, intestinal perforations, and even death [1]. CDI with high morbidity and mortality broke out in North American and European at the earliest time. And with the appearance of a high virulent strain (027/NAP1/BI), the incidence and severity of CDI is increasing around the world [2,3]. Due to the emergence of new drug resistant strains, the morbidity, recurrence rate, severity and mortality resulting from CDI have increased significantly. At present, there are three main treatments for CDI, including antibiotics [4], immune regulation [5] and probiotics [6]. The current antibiotic treatments for CDI are several broad-spectrum antibiotics such as vancomycin and metronidazole, which can easily lead to antibiotics resistance and disease recurrence [4,7,8,9]. Therefore, new drugs or new targets for the treatment of CDI are desperately needed [10]. The genome of Clostridium difficile was sequenced in 2006, and all the genes involved in Wood-Ljungdahl pathway, which is responsible for acetyl-CoA biosynthesis, were identified [11]. The pathway for catabolism and anabolism of the pathogen involves a series of critical

⁎ Corresponding author. E-mail address: [email protected] (X. Tan).

http://dx.doi.org/10.1016/j.jinorgbio.2017.02.005 0162-0134/© 2017 Published by Elsevier Inc.

proteins and enzymes, such as formate dehydrogenase [12], corrinoiddependent methyl transferase (MeTr) [13], corrinoid iron-sulfur protein (CoFeSP) [14,15], Acetyl-CoA synthase (ACS) and carbon monoxide dehydrogenase (CODH) [16]. As Acetyl-CoA is essential for survival of the pathogen and the Wood-Ljungdahl pathway is absent in humans, the proteins and enzymes involved in this pathway are attractive targets for the treatment of CDI. In previous research, the structures and functions of MeTr, CoFeSP and ACS from bacteria (but not from Clostridium difficile) had been well studied [17,18]. CoFeSP, which acts as a methyl transformer in the Wood-Ljungdahl pathway, can accept methyl group from CH3-H4folate catalyzed by MeTr (Reaction 1), and then transfer the methyl group to ACS (Reaction 2). CH3 −H4 folate þ Co1þ FeSP⇌H4 folate þ CH3 −Co3þ FeSP

ð1Þ

CH3 −Co3þ FeSP þ ACS⇌Co1þ FeSP þ CH3 −ACS

ð2Þ

Up to now, CoFeSP proteins from three kinds of bacteria, including the acetogenic bacterium Moorella thermoacetica (CoFeSPMt), the methanogenic archaeon Methanosarcina thermophila and the hydrogenogenic bacterium Carboxydothermus hydrogenoformans (CoFeSP Ch ), have been isolated. These heteromeric CoFeSPs share sequence identities of 35%–53%. And there is about 38% identity between CoFeSPCd and that from other bacteria.

Y. Wei et al. / Journal of Inorganic Biochemistry 170 (2017) 26–33

CoFeSP contains two metal centers, a cobalt-containing corrinoid cofactor and a single [4Fe-4S] cluster [19,20,21]. The corrinoid cofactor is the active center of CoFeSP, and cycles between the Co1+ and the methyl-Co3+ states during catalysis. And the [4Fe-4S] cluster was proved to be necessary to facilitate the reactivation of oxidatively inactivated Co2+ FeSP to the active Co1+ FeSP [22,23]. In this study, the CoFeSPCd from Clostridium difficile 630 in the acetylcoenzyme A synthesis pathway was cloned, expressed, and purified. Its structure and function were investigated using spectroscopy, electrochemistry, enzyme kinetics, homology structure modeling and molecular docking. 2. Materials and methods 2.1. Materials The genomic DNA of Clostridium difficile 630 was purchased from American Type Culture Collection (ATCC) (#9689D-5), USA. MBPHTmCherry2 vector was a kind gift from Prof. Guan Zhu (Texas A&M University). The 6 × His-TEV protease expression vector pRK1043, fusion protein vector pETDuet-1 were obtained from Novagen. The E. coli strains Trans 10 and BL21 (DE3) were obtained from TransGen. Pfu DNA polymerase, T4 DNA ligase, dNTP and restriction enzymes were purchased from New England Biolabs. Oligonucleotide PCR primer pairs were synthesized and DNA sequencing reactions were performed by Shanghai Sangon Biotech Co. Ltd. The plasmid purification kit, gel extraction kit and nickel nitrilotriacetic acid (Ni-NTA) resin were purchased from Qiagen (Chatsworth, CA). Superdex™ 200 HiLoadTM 10/ 300 gel filtration column was from Pharmacia. All proteins were purified anaerobically in a glove box (Braun, MB100, O2 concentration was b1 ppm). Unless otherwise indicated, biochemical agents used in the following procedures were brought from Sigma. Oligonucleotide PCR primer pairs, labeled as P1, P2, P3 and P4, were synthesized by Shanghai Sangon Biotech Co. Ltd. The primer pairs were as follows: P1: [5′-CGCGGATCCGGCGGCGGCAGCGGTATGGCATTAAAAGCTTTAG3′5′-CCGCTCGAGTTATTAGTTTGTTTGGCATG-3′] P2: [5′-CGGGATCCATGGCATTTAAAATGTCTACTC-3′5′CCCAAGCTTTTATTAAGCTAATGCGTTAACCAATT-3′] P3: [5′-CGCGGATCCGATGGCATTAAAAGCTTTAGATATAT-3′5′GAACTGCAGTTAGAAAGTTTGGCATGCTTTTTCTTGG-3′] P4: [5′-GGAATTCCATATGATGGCATTTAAAATGTCTACTC-3′5′CCTCTCGAGTTAAGCTAATGCGTTAACC-3′] 2.2. Sequence and structure analysis The gene sequences of CoFeSPs (CoFeSPCd, CoFeSPCh and CoFeSPMt) were retrieved from NCBI GenBank after BLAST search using CoFeSPCd protein sequences as query. Sequence alignments were performed automatically with ClustalW [24]. The 3D structures of CoFeSPCd was predicted based on the 3D structure of CoFeSPCh (PDB: 2YCL) in the Protein Data Bank using the amino acids homology modeling (Swiss-Model) on http://www.swissmodel.expasy.org/. The 3D ribbon figures were prepared using PyMOL software. 2.3. Gene cloning Two schemes are designed to obtain CoFeSPCd protein. One way is to express and purify the subunit gamma (γ subunit) and subunit delta (δ subunit) respectively, and then the recombinant γ subunit and δ subunit are mixed to obtain a holo-CoFeSPCd. The other way is to construct a co-expression system. Firstly, the gene which encodes γ subunit of CoFeSPCd was amplified from the genomic DNA of clostridium difficile 630 using primer pair P1, and a linker consisted of five amino acid residues (GGGSG) was introduced. The resulting PCR (polymerase chain reaction) products were

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purified, digested with BamH I and Xho I, ligated to the expression vector MBPHT-mCherry2, and then transformed into the E. coli Trans10 strain in order to attain enough copies of the recombinant plasmids. A similar strategy was used to construct the CoFeSPCd subunit delta (δ subunit). Primer pair P2, which introduce BamH I and Xho I respectively, were used to amplify the target gene from the genomic DNA. The resulting PCR products were purified, digested with BamH I and Xho I, ligated to the expression vector pETDuet-1, and then transformed into the E. coli Trans10 strain. The resulting two fusion proteins are 6 × His tag-Maltose binding protein (MBP) tag-linker-γ (named CoFeSP-γ) [25] and 6 × His tag-δ (named CoFeSP-δ), respectively. To construct the co-expression system, pETDuet-1 was chosen. The gene that encodes the gamma subunit of CoFeSPCd was amplified using primer pair P3. The resulting PCR products were purified and digested with BamH I and Pst I, ligated to the multiple cloning sites-1 (MCS1) of pETDuet-1. A similar strategy was used to construct the delta subunit of CoFeSPCd. Primer pair P4 were used to amplify the target gene from the genomic DNA. The resulting PCR products were purified and digested with Nde I and Xho I, ligated to the MCS2 of pETDuet-1, and then transformed into the E. coli Trans10 strain in order to attain enough copies of the recombinant plasmid. The resulting fusion protein is 6 × His tag-CoFeSPCd (named co-expressed CoFeSPCd). All the cloned regions were sequenced (Sangon Biotech, Shanghai Co. Ltd.) to verify the fidelity of the PCR reactions. 2.4. Expression and purification CoFeSP-γ was expressed in E. coli BL21 (DE3) with the bacteriophage T7 RNA polymerase/promoter system. Freshly transformed cells were pooled, inoculated into 50 ml of LB plus ampicillin medium, and incubated for 3 h at 37 °C and 270 rpm. This culture was used to inoculate an 8-l fermentor maintained at 37 °C. Cells were grown anaerobically in 2YT (Tryptone 16 g/L, 10 g/L yeast extraction, 5 g/L NaCl) plus ampicillin medium containing a phosphate buffer system (16 mM KH2PO4, 73 mM K2HPO4, pH = 7.6). Once cells reached an A600 of about 0.6, T7 RNA polymerase dependent transcription was induced with 0.1 mM isopropyl-g-D-thiogalactopyranoside (IPTG). The culture was then cooled to 25 °C, added with 5 g/L glucose, and incubated overnight. The culture was centrifuged at 5000 × g for 30 min at 4 °C. The cell pellets were frozen by liquid nitrogen and stored at −80 °C. All remaining steps in the purification were performed at 21 °C in a glove box mentioned above. The Tris-HCl buffer used in the following purification contains 50 mM Tris-HCl with a pH of 7.6 and 5 mM β-mercaptoethanol. The cell pellets were thawed in 100 mL Tris-HCl buffer containing 1 mM henylmethyl sulfonylfluoride, 25 U Deoxyribonuclease I (Bio Basic Inc.), 40 U Lysozyme (Bio Basic Inc.), stirred for 20 min, and disrupted by ultrasonication. The lysate was purified by nickel-nitrilotriacetic acid (Ni–NTA) column. The 6 × His tag-MBP tag of the purified fusion protein was cleaved off by 6 × His tag-Tobacco Etch Virus (TEV) protease. The protein was reloaded to the Ni–NTA column to remove the tag, protease and none cleaved proteins. The target protein was further purified by Superdex™ G-75 (GE). The lysate that containing CoFeSP-δ was obtained in a similar way to CoFeSP-γ. The lysate was centrifuged at 10,000 × g for 30 min at 4 °C. The pellet, containing inclusion bodies, was washed twice in an equal volume of Tris-HCl buffer. Protein aggregates were solubilized in 30 mL Tris buffer containing 6 M urea, and then centrifuged at 10,000 × g for 20 min at 4 °C to remove insoluble components. The denatured protein was purified by Ni–NTA column and renatured by diluting 20-fold in Tris-HCl buffer containing 50 mM NaCl. The remaining urea was removed by dialysis. The target protein was further purified by Superdex™ 200 (GE). Co-expressed CoFeSPCd were expressed in E. coli strain BL21(DE3). Freshly transformed cells were pooled, inoculated into 50 ml of LB plus ampicillin medium, and incubated for 3 h at 37 °C and 270 rpm. This culture was used to inoculate a 8-l fermentor maintained at 37 °C.

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Cells were grown anaerobically in 2YT (Tryptone 16 g/L, 10 g/L yeast extraction, 5 g/L NaCl) plus ampicillin medium. When OD600 had reached 0.6, the expression was induced with 0.1 mM IPTG, and 0.3 mM FeSO4 and Na2S were added. The culture was further cultivated overnight at 25 °C. All the purification steps were performed in a glove box mentioned above. Cells were re-suspended in buffer A (50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 10 mM β-mercaptoethanol) and disrupted by sonication. After centrifugation for 30 min at 12,000 rpm at 4 °C, the supernatant was loaded onto a Ni-NTA agarose column equilibrated with buffer A. The column was washed with 200 mL buffer A containing 10 mM imidazole, and brown CoFeSPCd was eluted with buffer A containing 100 mM imidazole. Then fractions were diluted with buffer B (50 mM Tris-HCl, pH 8.0, 10 mM β-mercaptoethanol) and loaded onto DEAE-Sepharose anionexchange column. Fractions were eluted by applying a linear gradient of buffer B containing 0–0.6 M NaCl. After concentration, CoFeSPCd was subjected to a Superdex™ 200 prep-grade gel-filtration column equilibrated with buffer C (20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10 mM β-mercaptoethanol). All the purified proteins were found to be at least 90% pure based on the denaturing polyacrylamide gels stained with Coomassie Brilliant Blue (Figs. S3, S4, S5). 2.5. Reconstitution of CoFeSPCd As the as-isolated CoFeSP-γ and CoFeSP-δ lacked metal centers, reconstitution of the holo protein of CoFeSP-γ and CoFeSP-δ with metal centers was performed. To 100 μM as-isolated CoFeSP-δ, 10 equivalent hydroxocobalamin was added. The mixture was stirred for 1 h on ice. The unbound cobalamin cofactor was separated from protein by a Sephadex G-25 column equilibrated with Tris-HCl buffer. Protein-containing fractions were concentrated to 1 mL by ultrafiltration using a Centricon YM-30 (Amicon-Millipore). Samples were protected from light during all these manipulations. To 100 μM as-isolated CoFeSP-γ, 10 equivalent Fe(NH4)2(SO4)2 was added firstly, stirred for 30 min on ice. Then, 10-fold of Na2S was added and stirred for 1 h on ice. The protein-containing fractions were purified and concentrated to 1 mL in a similar way to CoFeSP-δ. Subsequently, the two reconstituted subunits were mixed together and stirred for 1 h in a dark condition, and the obtained protein was named as overexpressed CoFeSPCd. The as-isolated co-expressed CoFeSPCd contained a [4Fe-4S] cluster but lacked the corrinoid cofactor. For reconstitution, 10-fold of hydroxocobalamin was added, and the mixture was stirred for 1 h on ice. The unbound cofactor was separated from the protein by a Sephadex G-25 column equilibrated with buffer C. Samples were protected from light during all the manipulations. All the reconstitution steps were performed in a glove box mentioned above. 2.6. Metal analysis Protein samples for metal analysis were digested with metal free nitric acid and heated overnight at 65 °C. The denatured protein samples were diluted with metal free water to analyze on a Pekin-Elmer Optima 3000 DV inductively coupled plasma optical emission spectrometer (ICP-OES). All the data were gained from an average of three independent protein preparations. Buffer was also determined as control. 2.7. UV–Visible spectroscopy UV–Vis spectra were recorded on a HP 8453 UV/Vis spectrophotometer using a quartz cuvette with 1 cm path length at 25 °C. All samples in buffer C were transferred into a cuvette closed with a rubber serum stopper in a glove box and transferred outside the glove box for experiments. As-isolated CoFeSPCd was used to measure the [4Fe-4S]-only spectrum. The reduction spectra of the [4Fe–4S] cluster were obtained by adding various concentrations of Na2S2O4 using a syringe to keep

the anaerobic environment of the closed cuvette. The re-oxidized spectrum was obtained from a re-oxidized CoFeSPCd, which was obtained by adding K3[Fe(CN)6] to the reduced CoFeSPCd in the glove box. To avoid the interference of K3[Fe(CN)6] to the spectrum, the excess oxidant was separated by a Sephadex G-25 column and the concentration of the protein was adjusted to be consistent with the reduced protein. Reconstituted CoFeSPCd was used to measure the spectrum of Co2 + state, and the spectrum of Co1 + state was generated by adding Na2S2O4 to Co2+-CoFeSPCd. Then the Co1+-CoFeSPCd was methylated by CH3-H4folate as a methyl donor in the reaction catalyzed by MeTrCd, and the spectrum of CH3-Co3+-CoFeSPCd was detected. All the samples used to determine the corrinoid center were prepared in the glove box and protected from light until determination. 2.8. Electron paramagnetic resonance (EPR) spectroscopy Low-temperature EPR spectra were recorded with a Bruker EMX Xband spectrometer equipped with an Oxford-910 cryostat and ITC-503 temperature controller (Oxford Instruments Ltd.). All data were analyzed with the Bruker WinEPR software. All the samples were transferred into quartz EPR tubes in an anaerobic glove box. Then, the samples were transferred outside the glove box, and frozen in liquid N2 immediately. The reduced protein was achieved by anaerobic addition of excessive Na2S2O4 to the protein in the glove box. EPR simulation was performed with the EasySpin software [32], using a spin Hamiltonian taking into account the electron Zeeman (g-value) and hyperfine (Avalue) interactions, as implemented in the software. Spin concentrations were measured by comparing the double integrals (using supplied Bruker software) of the spectra with those of a 1 mM copper perchlorate standard. All the samples were protected from light until the determination. 2.9. Electrochemistry The electrochemical measurements were carried out using CHI 660A electrochemical workstation (Chenhua, Shanghai, China). Differential pulse voltammetry (DPV) was performed at 25 °C under nitrogen-saturated atmosphere. A standard three electrode cell was utilized with a saturated calomel reference electrode (SCE), a Pt wire auxiliary electrode, and a protein/MPA-modified Au disk working electrode (0.2 cm2). MPA (3-mercaptopropionic acid) modified Au electrodes were prepared using a previously reported method [26]. The freshly prepared Au electrodes were soaked in a 20 mM MPA solution for 1 h. The electrodes were then rinsed with ethanol to remove the nonchemisorbed MPA prior to use in electrochemical experiments. CoFeSPCd/MPA-modified Au electrodes were prepared by soaking the MPA-modified Au electrodes in 0.1 mM CoFeSPCd for 6 h. The as-prepared electrodes were then rinsed with water and stored at 4 °C when not in use. The supporting electrolyte solution was 0.1 M phosphate buffer solution (PBS) pH 7.0. DPV scans were obtained at step potential and modulation amplitude of 4 mV (modulation time 0.02 s, interval time 0.2 s). All potentials were cited versus the normal hydrogen electrode (NHE) using a correction of +242 mV (according to the SCE electrode manufacturer) for the potential of the reference electrode. 2.10. Enzyme activity assay Stopped-flow experiments were performed at 25 °C using a SF-61 DX2 Double-Mixing Stopped-Flow instrument (Hi-Tech Limited, UK) installed in a glove box (Braun, MB100 with an O2 analyzer to make sure that the O2 concentration was b1 ppm). The methyl group transfer reactions between CH3-THF and Co1+ FeSPCd catalyzed by MeTrCd were monitored at 390 nm and 450 nm under anaerobic conditions. Ti3+-citrate solutions were prepared as described before and standardized by titration against K3[Fe(CN)6] [27]. The reaction mixture solution containing 60 μM CH3-THF, 1 μM MeTrCd and 1 mM Ti3+-citrate in 0.1 M

Y. Wei et al. / Journal of Inorganic Biochemistry 170 (2017) 26–33

MES pH 5.1, containing 0.1 M NaCl, was rapidly mixed with 10 μM Co1+ FeSP in 0.1 MES pH 5.1, containing 0.1 M NaCl, which was pre-reduced with 1 mM Ti3+-citrate. To determine steady-state kinetics, the concentration of CH3-THF was fixed at 60 μM and CoFeSPCd concentration was varied. The traces monitored at 390 nm and 450 nm, respectively, were fit to single-exponential decay equations. The data were then fit to the Michaelis-Menten equation, yielding values of kcat for MeTrCd and Km for CoFeSPCd. 2.11. Molecular docking Z-DOCK program [28] implanted into the Accelrys Discovery studio 2.5 was used to fit CoFeSPCd onto MeTrCd. As the 3D structures of CoFeSPCd and MeTrCd are not yet known, they were predicted using homology modeling (Swiss-Model). Before docking, the structures of the generated 3D structures were further set for validation run using Ramachandran Plot and profile-3D server from Accelrys Discovery studio. Then Z-DOCK performs a systematic search of a uniform sample of docked protein poses and uses an internal scoring algorithm to predict optimal interactions. 54,000 docked poses of CoFeSPCd-MeTrCd were initially produced, which were further filtered and re-ranked to obtain top 200 poses based on ZRank score using electrostatic and desolvation energy and non-deterministic FFT optimization. These 200 poses were then visually scrutinized and judged by Z-Score. The most reasonable poses as the model for the CoFeSPCd-MeTrCd complexes were finally chosen. 3. Results and discussion 3.1. Gene sequence and structure analysis The gene sequence alignment analysis of CoFeSPCd, CoFeSPMt and CoFeSPCh indicated that CoFeSPCd shared significant sequence homology (about 38% identities) with CoFeSPMt and CoFeSPCh (Figs. S1 and S2), and the residues at the active metal centers binding sites were highly conserved (Table S1) [19,29]. The homology modeling structure of CoFeSPCd (Fig. 1), created based on the 3D structure of CoFeSPCh (PDB: 2YCL), consists of two subunits. The small subunit (δ subunit) contains a single domain, which folds into a TIM barrel. The large subunit (γ subunit) contains three domains joined by two linkers. The N-terminal [4Fe-4S] activation domain (residues 1–55) contains the binding motif for the [4Fe-4S] cluster (motif Cys17X2Cys20X4Cys25X16Cys42). The middle TIM barrel domain (residues 63–315) is the dominating structural element of γ subunit and is

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connected by a proline-rich linker (named M-C linker) to the C-terminal B12-binding domain (residues 330–455). The corrinoid cofactor is sandwiched between δ subunit and the C-terminal domain of γ subunit. Five conserved residues (Gly373, Ser375 and Thr378 come from γ subunit, Tyr219 and Glu249 come from δ subunit) are found to interact with the corrin macrocycle by hydrogen bond. The overall structure of CoFeSPCd is very similar to CoFeSPCh, and both the [4Fe-4S] clusters and the corrinoid cofactor are conserved, which allows us to predict that the properties and functions of CoFeSPCd are similar to those of CoFeSPCh. 3.2. Protein preparation At first, the γ subunit and δ subunit of CoFeSPCd were expressed and purified respectively. Then, reconstitution was performed by adding hydroxocobalamin to δ subunit and Fe(NH4)2(SO4)2 and Na2S to γ subunit. The mixture of two reconstituted subunits yielded an overexpressed CoFeSPCd, which contained ~4 (3.5 ± 0.1) Fe atoms per monomer molecule but lacked the corrinoid cofactor. Therefore, a coexpression method of CoFeSPCd was applied. The genes encoding two subunits of CoFeSPCd were cloned into a co-expression vector and the co-expressed protein was overexpressed in E. coli. A three-step purification system was established to produce a His-tag fusion co-expressed CoFeSPCd. The as-isolated protein was eluted as a single peak from gelfiltration column, which corresponded in size to the heterodimeric CoFeSPCd complex (Fig. S5). The complex was 95% pure and the equal molar ratio of the two subunits was identified according to the densitometric analysis of SDS-PAGE gels (Fig. S5). Metal analysis of the as-isolated protein indicated that the co-expressed CoFeSPCd contained ~ 4 (3.5 ± 0.1) Fe atoms per monomer molecule but lacked the corrinoid cofactor, which was consistent with the previous report of heterologously produced CoFeSPCh from Carboxydothermus hydrogenoformans [19]. Then, reconstitution was performed by adding hydroxocobalamin without any unfolding procedure, and ~ 1 (1.1 ± 0.1) Co atoms per monomer molecule was yielded, which indicated the formation of corrinoid cofactor. So, the following experiments were performed using the co-expressed CoFeSPCd, which was just named as CoFeSPCd in the following work. 3.3. UV–Vis spectroscopy The electronic absorption spectra of CoFeSPCd in different forms were presented in Fig. 2. As expected, as-isolated recombinant CoFeSPCd exhibited a broad absorption at around 420 nm, which is a characteristic

Fig. 1. Ribbon cartoon diagram of the CoFeSPCd protein structure using homology modeling (made with Pymol software). The δ subunit is shown in yellow. The three domains of the γ subunit are shown in blue (N-terminal domain), green (middle domain), and red (C-terminal domain). Two linker regions are denoted as the N-M linker (pink) and the M-C linker (cyan). The [4Fe-4S] cluster is shown as a ball-and-stick model. The carbon atoms of the corrinoid cofactor are shown in cyan.

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Y. Wei et al. / Journal of Inorganic Biochemistry 170 (2017) 26–33

Fig. 2. UV–Vis spectra of as-isolated CoFeSPCd (dash dot), Co2+ FeSPCd (solid), Co+ FeSPCd (dash) and CH3-Co3+ FeSPCd (dot).

feature of [4Fe-4S]2+ cluster containing protein and confirms the existence of the [4Fe-4S]2+ cubane in CoFeSPCd [19], while no contribution of cobalamin was observed. With the addition of dithionite, absorption of the broad feature at around 420 nm rapidly declined. And with the oxidation of the reduced as-isolated CoFeSPCd by K3[Fe(CN)6], the absorption around 420 nm recovered again (Fig. S6). These results indicated that the [4Fe-4S] cluster was redox-active in the absence of corrinoid cofactor [19]. After hydroxocobalamin reconstitution, absorption bands at 308 nm, 420 nm and 470 nm appeared in the spectra, among which the band at around 470 nm is characteristic of corrinoids in the bassoff Co2+ form and is also observed in CoFeSPCh and CoFeSPMt.[19,29] Reportedly, reduction of CoFeSPCd is required prior to the methylation reaction [30]. The hydroxocobalamin reconstituted Co2+ FeSPCd was reduced by dithionite at pH 7.4, and the corresponding spectrum showed the distinct absorption peak characteristic of the Co+ FeSPCd at 390 nm, which is also closely related to the spectra reports for Co+ FeSPCh and Co+ FeSPMt.[19,29] Co+ FeSPCd was methylated by CH3-THF as the methyl donor in a reaction catalyzed by MeTrCd. The 390 nm absorption declined upon methylation, while the absorption at 450 nm increased, indicating the generation of CH3-Co3+ FeSPCd. These results suggest that CoFeSPCd contains a [4Fe-4S] cluster and a base-off cob(II)-amide, which are similar to CoFeSPCh and CoFeSPMt.

3.4. EPR spectroscopy The EPR spectra of CoFeSPCd at 70 K was nearly identical to those of CoFeSPCh and CoFeSPMt [20,22]. Spectral simulation indicated a S = 1/2 system with g∥ = 1.998 and g⊥ = 2.228, which was typical of low spin “base-off” cob(ΙΙ)alamin (Fig. 3A). The g∥ resonance was split into eight lines by hyperfine interaction between the unpaired electron and the cobalt nucleus. In corrinoid proteins that have imidazole or benzimidazole base base-on, each of the eight lines was split into three components by super hyperfine interactions with nitrogen nucleus of the base. CoFeSPCd exhibit a large cobalt hyperfine splitting pattern (A∥,Co = 147 G) and lack the super hyperfine interactions, which indicated that the Co2+ center was in “base-off” structure. Thus, the coordination environment of the corrinoid cofactor in CoFeSPCd appeared to be identical to those of CoFeSPCh and CoFeSPMt [20,22,31]. Double integration of the Co2 + spectrum yielded 0.9 spins/mol of CoFeSPCd. When CoFeSPCd was reduced by dithionite, an EPR signal arising from iron-sulfur cluster was obtained at 4 K (Fig. 3B). The g values at 2.05, 1.94 and 1.85 resemble the reported [4Fe-4S]1+ cluster signals in other CoFeSPs [20,31]. The EPR spectrum of the reduced CoFeSPCd appears to consist of several species, which might be caused by some heterogeneity in the cluster environment or some damage of the iron-sulfur cluster in preparation. This phenomenon features characteristic of other CoFeSPs

Fig. 3. EPR spectra of CoFeSPCd. A, 20 mg/ml CoFeSPCd in 20 mM Tris-HCl buffer, pH 7.6, temperature, 70 K. The red dashed line was obtained by simulation; B, reduced CoFeSPCd in 20 mM Tris-HCl buffer, temperature, 4 K. (For interpretation of the references to color in this table, the reader is referred to the web version of this article.)

as well [20,31,33]. The results above proved that CoFeSPCd contains a base-off cob(II)amide and a [4Fe-4S] cluster, similar to other CoFeSPs. 3.5. Direct electrochemistry measurements Direct electrochemistry of the [4Fe-4S] cluster and corrinoid cofactor of CoFeSPCd were investigated in this study. As the CoFeSPCd molecule is very large and the corrinoid cofactor is buried deeply in the protein chains, no direct electrochemistry result about CoFeSP has been reported. To promote direct electron transfer at electrode surface, gold electrodes modified with self-assembled monolayer of MPA were used as working electrodes [26]. DPV was chosen to measure the redox potentials of the [4Fe-4S] cluster and corrinoid cofactor. Compared with cyclic voltammetry, which is more frequently used, this technique allowed us to increase the sensitivity of measurements by minimizing the effect of charging current by the application of a pulse. According to the previous EPR titration results [20], the redox potential values of the [4Fe-4S] cluster and the corrinoid cofactor in CoFeSP are very close. To avoid the overlap of the signals, DPV measurements of the two metal centers were performed respectively. Firstly, the as-isolated CoFeSPCd, which only contained the [4Fe-4S] cluster, was loaded on gold electrode. DPV result showed a wave at − 439 mV (vs NHE) for [4Fe-4S]2 +/1 + (Fig. 4A). Then, the destroyed as-isolated CoFeSPCd, which was exposed to aerobic environment for 2 h to damage the [4Fe-4S] cluster, was measured. The DPV only produced a wave at ~ 0 mV (vs NHE), which come from the oxidized [3Fe-4S] cluster (Fig. S7) [22]. Lastly, CoFeSPCd , which contains [4Fe-4S] cluster and corrinoid cofactor, was also exposed to aerobic environment for 2 h, and the DPV measurement produced two waves at 0 mV (vs NHE) for oxidized [3Fe-4S] cluster and − 423 mV (vs NHE) for Co2+/1+ (Fig. 4B). DPV results showed that the redox potential value of [4Fe-4S] cluster in CoFeSPCd was ~20 mV more negative than corrinoid cofactor. The disparity between two redox potential data was consistent with other CoFeSPs (Table 1). Mutational studies indicated that the [4Fe-4S] cluster in CoFeSPs could facilitate one electron reductive activation of

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Fig. 5. Stopped-flow reaction between CH3-THF (60 μM) and CoFeSPCd (10 μM) catalyzed by MeTrCd (1 μM) at 25 °C in 0.1 M MES at pH 5.1. Rate constants were measured at 390 nm and 450 nm.

Fig. 4. Differential pulse voltammetry at MPA-modified Au electrode in 100 mM PBS (pH 7.0) containing: A, as-isolated CoFeSPCd; B, reconstituted CoFeSPCd that was exposed to aerobic environment for 2 h.

oxidatively inactivated Cob(II)amide to the active Co(I) state [22,23]. Thus, the [4Fe-4S] cluster in CoFeSPCd might play the same role in methyl transfer reactions. Besides, the redox potential values of CoFeSPCd observed in this study were 60–80 mV more positive than previously reported CoFeSPs (Table 1). Considering that the redox potential values were determined in solutions with different pH values, DPV was reexamined. The redox potentials of CoFeSPCd were found to be dependent on pH value of the solution (Fig. S8). It decreased linearly with increasing pH, with a slope of ~− 60 mV/pH from pH 5.2 to pH 7.0, indicating a one electron transfer process. Therefore, when the solution pH is 7.8, the calculated redox potentials of [4Fe-4S]2+/1+ and Co2+/1+ are 487 mV vs NHE and 471 mV vs NHE. These values were almost same with that of CoFeSPMt within the error range.

decay of Co+ FeSPCd, while 450 nm was also monitored for the rise of CH3-Co3 + FeSPCd. The curves were fit to a single-exponential decay equation (Fig. 5). Clearly, CoFeSPCd had the ability to accept methyl group from CH3-THF catalyzed by MeTrCd. Then, a series of single-turn over stopped-flow experiments at different CoFeSPCd concentrations were performed for the steady-state kinetics study. The methyl transfer traces at 390 nm were fitted with a single-exponential decay equation to obtain the rate constant at each substrate concentration. These data, averaged from five independent experiments, were fit to the Michaelis-Menten equation (Fig. 6), yielding values of kcat = 43.2 s− 1, Km = 7.9 μM and kcat/Km = 5.47 μM−1 s−1 for the methyl transfer reaction between CH3-THF and CoFeSPCd catalyzed by MeTrCd (Table 1). In previous studies, the kinetic parameters of the methyl group transfer reactions between CH3-THF and heterologous CoFeSPMt or exogenous cobalamin catalyzed by MeTrCd were reported [17,18]. As shown in Table 2, both the turnover rates (kcat ) and efficiency (kcat/Km) for the methyl transfer reaction using CoFeSPcd as substrate were much better than the reaction using CoFeSPMt as substrate. According to the crystal structure of CoFeSPMt/MeTrMt complex, the molecular juggling conformational change between the two proteins played important roles in the methyl transfer reactions [29]. So, considering that CoFeSPMt and CoFeSPCd only shared a 38% homology, the mismatched residues in the interaction region of CoFeSPMt and MeTrCd might be the main reason for the lower methyl transfer activity in the

3.6. Pre-steady state and steady state kinetics Stopped-flow was applied to examine the kinetics of methyl transfer reaction between CH3-THF and CoFeSPCd catalyzed by MeTrCd. The presteady state kinetics was performed at pH 5.1 under saturating substrate conditions. The reaction was monitored at 390 nm for the

Table 1 Summary of the redox potentials of CoFeSPs. Enzymesa

[4Fe-4S]2+/1+ (mV)

Co2+/1+ (mV)

Ref.

CoFeSPCd CoFeSPMt CoFeSPCt

−439 (pH 7.0) −502 (pH 7.8) −523 (pH 7.6)

−423 (pH 7.0) −486 (pH 7.8) −504 (pH 7.6)

This work [20] [30]

a

All the potentials are converted to the NHE scale.

Fig. 6. The rate constants between CH3-THF (fixed at 60 μM) and CoFeSPCd (changed from 5 to 70 μM) catalyzed by MeTrCd (1 μM) were fit to the Michaelis-Menten equation, yielding values of kcat = 43.2 ± 2.6 s−1 and kcat/Km = 5.47 ± 0.5 μM−1 s−1 at 25 °C in 0.1 M MES at pH 5.1 and at an ionic strength of 0.1.

32

Y. Wei et al. / Journal of Inorganic Biochemistry 170 (2017) 26–33

Table 2 Kinetic parameters of methyl transfer reaction catalyzed by MeTrCd. Reaction

kcat (s−1)

Km (μM)

kcat/Km (s−1 μM−1)

pH

Co+ FeSPMt + CH3-THFa Co+ Cbl− + CH3-THFb Co+ FeSPCd + CH3-THF

2.6 77.6 43.2

17.8 58 7.9

0.15 1.33 5.47

5.1 [18] 5.1 [18] 5.1

a b

Initials: Co+ FeSP, corrinoid iron-sulfur protein. Initials: Co+ Cbl−, cobalamin.

loop in the middle part of MeTrCd (Helix 128–136). This interaction forms the primary interaction between MeTrCd and CoFeSPCd, and could make the MeTrCd get close to the B12-binding domain of CoFeSPCd, which ensures that the methyl transfer reaction can be carried out. Based on the molecular docking, the molecular juggling between CoFeSPCd and MeTrCd maintains a stable interaction conformation for the methyl transfer reaction, which is similar to the reported crystal structure of MeTrMt/CoFeSPMt complex [29]. 4. Conclusions

MeTrCd/CoFeSPMt reaction system. Moreover, the kcat/Km for the methyl transfer to CoFeSPCd was also better than that of cobalamin. This result also proved that the molecular juggling between CoFeSPCd and MeTrCd was essential for the catalytic reaction. 3.7. Molecular docking To study the molecular juggling between CoFeSPCd and MeTrCd, molecular docking was performed. The dimensional structures of CoFeSPCd and MeTrCd were successfully built using homology modeling (SwissModel). Generated 3D structures were further set for validation on the basis of Ramachandran Plot and profile-3D analysis. The Ramachandran Plot studies showed most of the residues of the modeled proteins in most favored regions, while almost no residues were found in the disallowed region as represented (Figs. S9A, S9B and S10A). Meanwhile, profile-3D studies showed the Verfy Score of the modeled proteins were close to their Verify Expected High Score (GammaCd, 179.7 to 204.1; DeltaCd, 117.7 to 142.9; MeTrCd, 114.8 to 119.5) and almost all the invalid residues were located in the loops, which meant the models were reliable (Figs. S9C, 9D and S10B). Then, a systematic search for optimal interactions was performed by Z-DOCK as mentioned in the materials and methods section. The most reasonable poses for the MeTrCdCoFeSPCd complexes were finally chosen. As showed in Fig. 7, the assembly mode of MeTrCd/CoFeSPCd complex is similar to the reported crystal structure of MeTrMt/CoFeSPMt complex, in which the folate-binding site of MeTr is close to the corrinoid cofactor of CoFeSP [29]. The interaction between MeTrCd and CoFeSPCd mainly lies in two points. Firstly, a weak interaction lies between α helix in the N-terminal domain of CoFeSP gamma subunit (Helix 30–35) and a loop in the C-terminal of MeTrCd loop 180–190, which could stabilize the highly flexible [4Fe-4S] domain that is responsible for B12 activation. Secondly, a stronger interaction lies between the α helix in middle TIM barrel domain of CoFeSP gamma subunit (CoFeSPCd, Helix 81–86) and a

In this study, a key protein CoFeSPCd from the acetyl-coenzyme A synthesis pathway of the human pathogen Clostridium difficile was cloned, expressed and purified for the first time. An efficient recombinant expression system was established successfully. The structure and function of CoFeSPCd with complete metal center and methyl transfer activity, prepared by co-expression system and reconstituting the corrinoid cofactor in vitro, were investigated using homology structure modeling, spectroscopy, electrochemistry, enzyme kinetics and molecular docking. UV–Vis and EPR results indicated that CoFeSPCd has identical corrinoid cofactor and [4Fe-4S] cluster with both CoFeSPMt and CoFeSPCh, which was predicted via gene sequencing and a structure modeling analysis. DPV were also performed to characterize the two metal centers of CoFeSPCd for the first time. The results showed that [4Fe-4S] cluster had a more negative Em value than corrinoid cofactor, which is consistent with its role of electron transfer to the cobalt center reported in CoFeSPMt. Furthermore, the methyl transfer activity between CH3-THF and CoFeSPCd catalyzed by MeTrCd was determined. Both the turnover rates (kcat) and efficiency (kcat/Km) for the methyl transfer reaction between CH3-THF and CoFeSPCd were much better than the reactions using CoFeSPMt and cobalamin as substrates. This result indicated that the molecular juggling between MeTrCd and CoFeSPCd might be essential for the catalytic reaction. In the end, molecular docking was used to further study the mechanism of the methyl transfer between MeTrCd and CoFeSPs. The result showed that the hydrogen bond and salt bridge between MeTrCd and CoFeSPCd enhanced their interaction, which might be the reason for the increased catalytic activity. As we know, the human pathogen C. difficile infection (CDI) is one of the most important healthcare-associated infections. This study on CoFeSPCd, which is a key enzyme in the acetyl-coenzyme A synthesis pathway in anaerobic bacteria, deepens our understanding on the Wood-Ljungdahl pathway of C. difficile. The active metal center of CoFeSPCd and the interaction between CoFeSPCd and MeTrCd can become innovative targets for drug design to treat human CDI.

Fig. 7. Molecular docking of CoFeSPCd and MeTrCd complex. Ribbon representation of MeTrCd in pink, CoFeSPCd small subunits in orange, large subunit [4Fe-4S] domains in cyan, TIM barrel domains in green, and B12 domains in blue. B12 cofactors in sticks. [4Fe-4S] clusters in spheres: Fe in orange, S in yellow.

Y. Wei et al. / Journal of Inorganic Biochemistry 170 (2017) 26–33

Abbreviations CDI CoFeSP CH3-THF MeTr ACS CODH Ni–NTA MBP TEV IPTG ICP-OES EPR DPV SCE MPA NHE

Clostridium difficile infection corrinoid iron-sulfur protein CH3-H4folate methyl transferase Acetyl-CoA synthase carbon monoxide dehydrogenase nickel-nitrilotriacetic acid Maltose binding protein Tobacco Etch Virus isopropyl-g-D-thiogalactopyranoside inductively coupled plasma optical emission spectrometer electron paramagnetic resonance differential pulse voltammetry saturated calomel reference electrode 3-mercaptopropionic acid normal hydrogen electrode

Acknowledgment The authors would thank Prof. Jihu Su at University of Science and Technology of China for his support of EPR simulation. This work was supported partly by the National Natural Science Foundation of China (Nos. 31270869, 31670817, 21472027, 91013001), the PhD Program of the Education Ministry of China (20100071110011), Shanghai & Beijing Synchrotron Radiation Facility, and High Magnetic Field Laboratory, Chinese Academy of Sciences. References [1] M. Rupnik, M.H. Wilcox, D.N. Gerding, Clostridium difficile infection: new developments in epidemiology and pathogenesis, Nat. Rev. Microbiol. 7 (2009) 526–536. [2] V.G. Loo, L. Poirier, M.A. Miller, M. Oughton, M.D. Libman, S. Michaud, A.M. Bourgault, T. Nguyen, C. Frenette, M. Kelly, A. Vibien, P. Brassard, S. Fenn, K. Dewar, T.J. Hudson, R. Horn, P. Rene, Y. Monczak, A. Dascal, A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality, N. Engl. J. Med. 353 (2005) 2442–2449. [3] L.C. McDonald, G.E. Killgore, A. Thompson, R.C. Owens Jr., S.V. Kazakova, S.P. Sambol, S. Johnson, D.N. Gerding, An epidemic, toxin gene-variant strain of Clostridium difficile, N. Engl. J. Med. 353 (2005) 2433–2441. [4] O.M. Williams, R.C. Spencer, The management of Clostridium difficile infection, Br. Med. Bull. 91 (2009) 87–110. [5] G.J. Babcock, T.J. Broering, H.J. Hernandez, R.B. Mandell, K. Donahue, N. Boatright, A.M. Stack, I. Lowy, R. Graziano, D. Molrine, D.M. Ambrosino, W.D. Thomas Jr., Human monoclonal antibodies directed against toxins A and B prevent Clostridium difficile-induced mortality in hamsters, Infect. Immun. 74 (2006) 6339–6347. [6] L.V. McFarland, Evidence-based review of probiotics for antibiotic-associated diarrhea and Clostridium difficile infections, Anaerobe 15 (2009) 274–280. [7] D. Shah, M.D. Dang, R. Hasbun, H.L. Koo, Z.D. Jiang, H.L. DuPont, K.W. Garey, Clostridium difficile infection: update on emerging antibiotic treatment options and antibiotic resistance, Expert Rev. Anti-Infect. Ther. 8 (2010) 555–564. [8] N.S. Grewal, A. Salim, Clostridium difficile colitis: a call for aggressive management, Scand. J. Surg. 99 (2010) 90–92. [9] B. Faris, A. Blackmore, N. Haboubi, Review of medical and surgical management of Clostridium difficile infection, Tech. Coloproctol. 14 (2010) 97–105. [10] H.L. Koo, K.W. Garey, H.L. Dupont, Future novel therapeutic agents for Clostridium difficile infection, Expert Opin. Investig. Drugs 19 (2010) 825–836. [11] M. Sebaihia, B.W. Wren, P. Mullany, N.F. Fairweather, N. Minton, R. Stabler, N.R. Thomson, A.P. Roberts, A.M. Cerdeno-Tarraga, H. Wang, M.T. Holden, A. Wright, C. Churcher, M.A. Quail, S. Baker, N. Bason, K. Brooks, T. Chillingworth, A. Cronin, P. Davis, L. Dowd, A. Fraser, T. Feltwell, Z. Hance, S. Holroyd, K. Jagels, S. Moule, K. Mungall, C. Price, E. Rabbinowitsch, S. Sharp, M. Simmonds, K. Stevens, L. Unwin,

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