enzyme. Based on the proximity between the HypA and metal binding sites in HypB, possible role of HypA/HypB interaction for hydrogenase maturation is discussed. Results HypA and HypB from A. fulgidus form 1:1 heterodimer in solution The interaction between A. fulgidus HypA (AfHypA) and HypB (AfHypB) was demonstrated by in vitro pull-down. AfHypB was mixed with cell lysate of E. coli overexpressing GST-tagged AfHypA and loaded onto the glutathione resin. After extensive washing with phosphate buffered saline, AfHypB was co-eluted with GST- AfHypA (lane 1, figure 1A). In contrast, no elution of AfHypB was detected in the resin and the GST controls (lane 3 and 4, figure 1A). The identity of AfHypB in the pull-down assay was confirmed by mass spectroscopy analysis of trypsinized fragments. The result suggests that AfHypA interacts with AfHypB. A. fulgidus HypB only forms stable dimer in the presence of both GMPPNP and nickel Previous study showed that HypB forms dimer in the presence of GTP or Ni. Here, we measured the molecular weight of AfHypB preloaded with GMPPNP and/or Ni (Figure 1B) by size exclusion chromatography with light scattering (SEC/LS). AfHypB preloaded with GDP was eluted as a single peak with a molecular weight of 25 kDa, which is consistent with the theoretical molecular weight of monomeric AfHypB (24.8 kDa). AfHypB preloaded with Ni was eluted as two peaks with molecular weights of 42 kDa and 25 kDa, and that preloaded with GMPPNP only was eluted as a broadened peak with molecular weights of ,36 kDa, indicating the presence of both monomeric and dimeric AfHypB in the samples (Figure 1B). In contrast, AfHypB preloaded with both GMPPNP and Ni was eluted as a single major peak with a molecular weight of 51 kDa, suggesting AfHypB forms a stable dimer in the presence of both ligands. A. fulgidus HypA form complex with HypB monomer preloaded with GDP but not with HypB homodimer preloaded with GMPPNP and Ni Next, we addressed whether the oliogmeric state of HypB affects its interaction with HypA. AfHypA was mixed with AfHypB preloaded with GDP (monomeric form of AfHypB) or AfHypB preloaded with GMPPNP and Ni (dimeric form of AfHypB), and the molecular weights of the protein samples were measured by SEC/LS. When AfHypA was mixed with the monomeric form of AfHypB (preloaded with GDP), the two proteins was co-eluted at 10.5 ml (Figure 1E, lane 1) with a molecular weight of 37 kDa (Figure 1C), which is consistent with the theoretical molecular weight of 36.8 kDa for 1:1 dimer of AfHypA-AfHypB. When AfHypA was mixed with the dimeric form of AfHypB (preloaded with GMPPNP and Ni), two elution peaks were observed. The first peak at 10.3 mL with an observed molecular weight of 51 kDa accounted for AfHypB dimer, which identity was confirmed by SDS-PAGE (Figure 1E, lane 2). The second peak at 12.5 mL with an observed molecular weight of 13 kDa accounts for AfHypA (Figure 1E, lane 3). These results suggest that AfHypA can only interact with the monomeric GDP-bound form of AfHypB, but cannot interact with the dimeric form of AfHypB. The unstructured N-terminal residues of A. fulgidus HypB are required for HypA/HypB interaction In an attempt to map the HypA binding site on HypB, we performed scanning mutagenesis on HypB. Based on the crystal HypA/HypB Interaction and Hydrogenase Maturation structure of HypB from A. fulgidus (PDB: 2WSM), surface charged residues (Asp, Glu, Lys and Arg) on HypB were grouped into 12 clusters according to their proximity in its 3-dimentional structure. Twelve HypB variants were created by substituting residues in each cluster with alanine (figure 2A). In the crystal structure of AfHypB, residues 1–9 at the N-terminus and residues 215–221 at the C-terminus are disordered. This N-terminal region is also disordered in the crystal structures of M. jannaschii HypB [27]. To understand the role of these disordered residues, two additional truncation variants of HypB were created: an N-terminal truncated HypB variant (DN) with residues 1–9 deletion and a C-terminal truncated HypB variant (DC) with residues 215–221 deletion. To test the interaction between these AfHypB variants with AfHypA, in vitro pull-down assay using GST-AfHypA as bait was performed. All AfHypB variants were expressed as soluble proteins found in soluble fraction of crude lysate. Variants of AfHypB were mixed with GST-AfHypA, and loaded onto glutathione resin. After extensive washing, the bound proteins were eluted with 20 mM GSH and analyzed by 12.5% SDS- PAGE (figure 2B). The C1–C12 and the DC variants were co- eluted with GST-AfHypA, suggesting that these surface charged residues and the C-terminal unstructured residues are unnecessary for HypA/HypB interaction. On the other hand, the DN variant of HypB failed to co-elute with GST-HypA, showing that truncation of the unstructured N-terminal residues of HypB abolished interaction with HypA. Two conserved hydrophobic residues on A. fulgidus HypB are required to interact with HypA We have shown that the truncation of residues 1–9 of AfHypB abolished the interaction between HypA and HypB. Sequence alignment suggests that there are two conserved hydrophobic residues, Tyr-4 and Leu-6, in this region (Figure 2C). To test if these two residues are involved in HypA interaction, two AfHypB variants, Y4A and L6A, were constructed by site-directed mutagenesis. An E5A variant was also constructed to test the role of the non-conserved residue Glu-5. All three variants were expressed as soluble proteins found in soluble fraction of crude cell lysate. In vitro pull-down assay was performed using GST-AfHypA as bait. Our results showed that wild-type AfHypB and the E5A variant were co-eluted with AfHypA (Figure 2D, lane 3 and 5), suggesting that substitution with alanine at Glu-5 did not affect the HypA and HypB interaction. On the other hand, GST-AfHypA did not pull down the Y4A and L6A variants of AfHypB (Figure 2D, lane 4 and 6), suggesting that the two conserved non- polar residues, Tyr-4 and Leu-6, of AfHypB are required for interaction with HypA. Two conserved hydrophobic residues on E. coli HypB are required to interact with HypA E. coli HypB (EcHypB) has an extra N-terminal domain in addition to the GTPase domain comparing with AfHypB. Residues 1–9 of AfHypB correspond to residues 75–83 in E. coli HypB (EcHypB), which is the N-terminus of the GTPase domain of HypB (Figure 3A). The conserved non-polar residues, Tyr-4 and Leu-6, of AfHypB correspond to Leu-78 and Val-80 of EcHypB (Figure 3B). To test if these residues are involved in HypA/HypB interaction, EcHypB variants, L78A, E79A and V80A, were constructed by site-directed mutagenesis. These variants of EcHypB were expressed with an N-terminal His- SUMO (HS) tag and their interaction with EcHypA was tested by in vitro GST-pull-down assay (Figure 3C). EcHypB and its variants were expressed as soluble proteins found in the soluble fraction of PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e32592