Figure 3. Two conserved hydrophobic residues located at the N- terminus of the GTPase domain of HypB are required for HypA/ HypB interaction. (A) Both A. fulgidus HypB (AfHypB) and E. coli HypB (EcHypB) have a GTPase domain. The unstructured N-terminal residues of AfHypB (residues 1–9) correspond to residues 75–83 of EcHypB located at the N-terminus of the GTPase domain. (B) Sequence alignment of HypB from five selected species was shown. The two conserved hydrophobic residues were shaded. (C) Interaction between variants of EcHypB and EcHypA were tested by GST pull-down assay. EcHypB was expressed with an N-terminal His-SUMO (HS) tag. Cell lysate expressing HS-EcHypB or its variants was mixed with that expressing GST-EcHypA, and was loaded onto glutathione-resin. After extensive washing, wild-type HS-EcHypB and GST-EcHypA was co-eluted with 20 mM GSH (lane 3). While E79A variant of EcHypB retained its interaction with HypA to be co-eluted with GST-EcHypA (lane 5), no co-elution was observed for the L78A and V80A variants (lane 4 and 6). No co-elution was observed for the negative control experiments (lane 1 and 2), suggesting the interaction between EcHypA and EcHypB is specific. doi:10.1371/journal.pone.0032592.g003 and Val-80, of EcHypB are required for interaction with HypA. This observation is consistent with the observation in the pull down experiment of AfHypA and AfHypB. HypA/HypB interaction is required for hydrogenase maturation in E. coli To test if HypA/HypB interaction is required for its biological function, in vivo hydrogenase reactivation experiment was carried HypA/HypB Interaction and Hydrogenase Maturation out. Hydrogenase activity of E. coli DhypB strain (F-, D(araD- 2 araB)567, DlacZ4787(::rrnB-3), l , DhypB731::kan, rph-1, D(rhaD- rhaB)568, hsdR514) expressing HypB and HypB variants were compared to the activity of the parental strain (F-, D(araD- araB)567, DlacZ4787(::rrnB-3), l2, rph-1, D(rhaD-rhaB)568, hsdR514) (Figure 4). DhypB strain with transformed pBAD-EcHypB showed a relative activity of 73621% compared to the parental strain, suggesting that HypB can complement the loss of HypB gene. Transformation of the E79A mutant of EcHypB showed a relative activity of 63613%, suggesting that the mutation of E79A did not affect the hydrogenase maturation significantly. On the other hand, transformation of the L78A and V80A mutants resulted in only 1063% and 564% relative activity, respectively. These values were comparable to 763% for the empty vector, suggesting that the mutations of L78A and V80A abolished in vivo hydrogenase maturation. Our results suggest that the L78A and V80A mutations disrupted HypA and HypB interaction and abolished in vivo maturation of hydrogenase in E. coli. Discussion In this study, we addressed the question of how HypB interacts with HypA, and if such interaction plays a role in hydrogenase maturation. We demonstrated that A. fulgidus HypA form a 1:1 heterodimer with the monomeric GDP-bound form of HypB, but does not interact with the dimeric form of HypB preloaded with GMPPNP and Ni. We showed that residues located at the N- terminus of the GTPase domain of HypB are required for interaction with HypA. Single residue substitution of two conserved hydrophobic residues (Y4 and L6 in A. fulgidus HypB, L78 and V80 in E. coli HypB) in this region disrupted HypA/ HypB interaction and abolished hydrogenase maturation in E. coli. We have extended this finding to Helicobacter pylori HypA/HypB interaction, and showed that the substitutions of N-terminal residues of H. pylori HypB also break the interaction with HypA Figure 4. Mutations that disrupted HypA/HypB interactions also abolished restoration of hydrogenase activity in vivo. The DhypB strain was transformed with the empty vector control (pBAD), wild-type pBAD-EcHypB and its mutants (L78A, E79A or V80A). The hydrogenase activity of the cell lysate was compared to that of the parental strain. Expression of wild-type HypB recovered 73621% hydrogenase activity. Noteworthy, for the mutations (L78A and V80A) that disrupted HypA/HypB interactions, the hydrogenase activity was significantly reduced, and was similar to that for the empty vector control. The p-values were obtained by Dunnett’s Multiple Comparison Test to compare each setup with DhypB strain-WT. doi:10.1371/journal.pone.0032592.g004 PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e32592