[28]. Taken together, our results showed that HypA/HypB interaction is required for hydrogenase maturation. How HypA/HypB interaction contributes to hydrogenase maturation is poorly understood. Previous studies showed that HypA and HypB are responsible for the delivery of nickel ion to the active site of [NiFe] hydrogenases [1,2,3,4,5,6,7]. It has been demonstrated that binding of GTP induces HypB dimerization [20,27,29]. As observed in the crystal structure of Methanococcus jannaschii HypB in complex with GTPcS, metal ions are chelated by three invariant residues Cys-95, His-96 and Cys-127 [27]. Mutagenesis studies have suggested that these invariant residues are essential to hydrogenase maturation [24,25]. Gasper et al. observed that GTP-dependent dimerization brought Cys-95 and Cys-96 from each chain of HypB together to allow both chains to bind a nickel ion at the interface (Figure 5A) [27]. GTP hydrolysis was shown to be essential for the maturation of hydrogenase [3,20,26]. It is possible that GTP hydrolysis will dissociate the HypB dimer, and release the nickel ion bound at the dimeric interface to the hydrogenase. This hypothesis is supported by the observation that mutations disrupting GTP-dependent dimerization of HypB also resulted in reduced hydrogenase activity [30,31]. In this study, we showed that the N-terminal residues of the GTPase domain of HypB is responsible for binding HypA. It is noteworthy that the N- terminus of the GTPase domain is located in close proximity to the invariant residues responsible for metal binding (Figure 5B). The close proximity of the HypA interaction site and the metal binding site of HypB may facilitate transfer of nickel ions between HypA and HypB. Here we suggest that the nickel ion is transferred from HypA to HypB via the formation of HypA/HypB complex, and is then delivered to the catalytic core of hydrogenase upon GTP hydrolysis. Another point of interest is when HypB forms dimer, N- terminus of helix 1 on each copy of HypB is pointing towards each other. We anticipate that dimerization of HypB may induce steric hinderance between the HypA binding sites and prevent HypA from interacting with the dimeric form of HypB (Figure 5B). HypA/HypB Interaction and Hydrogenase Maturation Taken together, HypA may function as a metal chaperone by binding to HypB. After loading of nickel and binding of GTP to HypB, HypB dimerizes and HypA dissociates from HypB. HypB dimer in complex with GTP and Ni then delivers its bound nickel to hydrogenase precursor upon GTP hydrolysis. Materials and Methods Cloning and mutagenesis Full length hypA and hypB genes were amplified by PCR from the A. fulgidus (ATCC: 49558) and E. coli (strain DH5a) genomic DNA, and cloned into the expression vector pRSET-His-SUMO (an in-house expression plasmid based on pRSET-A (Invitrogen) with an N-terminal His-SUMO tag), pRSET-GST (an in-house expression plasmid based on pRSET-A (Invitrogen) with an N-terminal GST fusion tag) and pBAD-A (Invitrogen). All mutants of HypB were constructed by QuikChange mutagenesis kit from Stratagene on constructs pRSET- His-SUMO-AfHypB, pRSET-His-SUMO-EcHypB and pBAD- EcHypB. Constructs created and the primers used are listed in table S1. All constructs were verified by DNA sequencing. Protein Expression All proteins were expressed by transforming the corresponding expression plasmids into E. coli BL21 (DE3) pLysS host. The bacterial cells were grown in LB medium at 37uC supplemented with 100 mg/mL ampicillin and 50 mg/mL chloramphenicol. Protein expression was induced by addition of 0.4 mM isopro- pyl-D-1-thiogalactopyranoside when OD600 reached 0.8. Cells were allowed to grow overnight at 25uC and then were harvested by centrifugation at 5,0006 g, 15 min, 4uC. Cell pellets were stored in 220uC freezer before use. Purification of AfHypA and AfHypB Cell pellet expressing His-SUMO-AfHypA or His-SUMO- AfHypB from 1 L culture was resuspended in 20 mL of lysis buffer Figure 5. Close proximity of the HypA and metal binding sites of HypB suggests possible metal transfer between HypA and HypB. (A) The N-terminal residues of HypB, responsible for HypA binding, were not resolved in the crystal structures of HypB (red dashed line). HypB contains an invariant metal binding site composed of two cysteine residues and one histidine residue (C95, H96 and C127 in M. jannaschii HypB, PDB code: 2HF8). The close proximity between the metal binding site at the dimeric interface and the HypA binding site at the N-terminus of the GTPase domain suggest that HypA/HypB interaction may facilitate transfer of nickel between the two proteins. (B) Binding of HypA to HypB may facilitate transfer of nickel between HypA and the metal binding sites (C95, H96 and C127 in M. jannaschii HypB) situated at the dimeric interface of HypB. This metal binding site at the dimeric interface was occupied by zinc (green). Dimerization of HypB may induce steric hinderance between the HypA binding sites and prevent HypA from interacting with the dimeric form of HypB. doi:10.1371/journal.pone.0032592.g005 PLoS ONE | www.plosone.org 6 February 2012 | Volume 7 | Issue 2 | e32592