AfHypA and AfHypB form 1∶1 heterodimeric complex.

<p>(A) Cell lysate containing GST-tagged AfHypA were loaded on to glutathione resin with (lane 1) or without (lane 2) AfHypB. After extensive washing, GST-AfHypA and AfHypB were co-eluted with 20 mM glutathione (lane 1). In contrast, no co-elution was observed in the resin (lane 3) and the GST (lane 4) controls. (B) Purified AfHypB dimer preloaded with various ligands were subjected to SEC/LS analysis. Refractive index (continuous lines) and molecular weight (dotted lines) were plotted in the chromatogram. AfHypB preloaded with both GMPPNP and Ni (red) was eluted as a major peak with observed molecular weigh 51 kDa. AfHypB preloaded with GMPPNP (green) showed a broadened peak with observed molecular weight ∼36 kDa. AfHypB preloaded with Ni (brown) showed two eluted peaks with observed molecular weight 42 kDa and 25 kDa. AfHypB preloaded with GDP (blue) was eluted as a monomer with observed molecular weight of 25 kDa. (C) Mixture of equimolar of AfHypA and AfHypB preloaded with GDP (green) was analyzed by SEC/LS. AfHypA and AfHypB were eluted as a complex of 37 kDa. Individual elution profiles of AfHypA (red) and AfHypB preloaded with GDP (blue) were included for comparison. The corresponding observed molecular weight of each peak was labeled in the chromatogram. (D) Similarly, mixture of equimolar of AfHypA and AfHypB preloaded with GMPPNP and Ni (green) was analyzed by SEC/LS. AfHypA and AfHypB were eluted separately in two peaks of molecular weight 51 kDa and 13 kDa. Individual elution profiles of AfHypA (red) and AfHypB preloaded with GMPPNP and Ni (blue) were plotted for comparison. (E) The eluted peaks of interest were analyzed by SDS-PAGE. AfHypB preloaded with GDP were co-eluted with AfHypA (lane 1), in contrast, AfHypB dimer preloaded with GMPPNP and Ni was eluted separately (lane 2 and 3).</p

The unstructured N-terminal residues of AfHypB is required for HypA/HypB interaction.

<p>(A) Design of variants to map the HypA binding site on HypB. Twelve clustered-charge-to-alanine variants designated C1–C12, were created according to the close proximity of the charge residues in the crystal structure of AfHypB (PDB code: 2WSM). Residues 1–9 and 214–221 were unstructured in AfHypB. Two truncation variants, ΔN and ΔC, were created to investigate the role of these unstructured residues. (B) Interaction of AfHypB variants with AfHypA was tested by <i>in vitro</i> pull-down assay. Cell lysate containing GST-AfHypA and HypB variants was loaded on to glutathione-resin. Bound proteins were eluted by 20 mM glutathione after extensive washing. All the HypB surface charge mutants and C-terminal mutant were co-eluted with HypA suggesting that interaction was not disrupted by the mutations. N-terminal truncated HypB mutant was incapable to be co-eluted with HypA indicating that the first nine amino acid residues of AfHypB is required to interact with HypA. (D) Residues 4 and 6 (numbering according to the sequence of AfHypB) are conserved among HypB. The sequence logo representation was generated by the program WEBLOGO <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032592#pone.0032592-Schneider1" target="_blank">[35]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032592#pone.0032592-Crooks1" target="_blank">[36]</a> using 62 non-redundant (with <90% identity) sequences from the NCBI non-redundant database. Aliphatic hydrophobic residues are always found at these two positions. (E) Interaction of AfHypB variants Y4A, E5A and L6A with AfHypA was tested by <i>in vitro</i> pull-down assay. The variant E5A (lane 3) retained its interaction with AfHypA like the wild-type AfHypB (lane 1). Y4A (lane 2) and L6A (lane 4) variants were incapable to interact with AfHypA indicating that both residues are required to interact with HypA.</p

Two conserved hydrophobic residues located at the N-terminus of the GTPase domain of HypB are required for HypA/HypB interaction.

<p>(A) Both <i>A. fulgidus</i> HypB (AfHypB) and <i>E. coli</i> 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.</p

Close proximity of the HypA and metal binding sites of HypB suggests possible metal transfer between HypA and HypB.

<p>(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 <i>M. jannaschii</i> 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 <i>M. jannaschii</i> 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.</p

Binding of GTP to HypB causes conformational changes leading to HypB dimerization.

<p>(A) Surface representation of (i) apo-AfHypB (ii) GTPγS-bound MjHypB. A GTP molecule is modeled into the apo-AfHypB for comparison. In apo-AfHypB, the invariant Asp-66 occupies the γ-phosphate binding pocket. Therefore, it is not possible for HypB to accommodate a GTP molecule in such conformation. The surface of the invariant Asp is colored in orange. (B) Binding of GTPγS causes conformational changes in the switch I loop and helix-3. The movements of the conserved residues Asp-66, Asp-72, and Arg-75 are indicated by arrows. Apo-AfHypB and GTPγS-MjHypB are colored cyan and white respectively. (C) Binding of GTP causes swinging movement of helix-3 from the apo-conformation (cyan) to the GTP-bound state conformation (white). It causes Asp-72 to move by 4.9 Å to the dimeric interface. This allows Asp-72 to form a salt bridge with Lys-148 on the opposite chain (yellow). In apo-form conformation of HypB, the switch I loop blocks the site for Lys 148 insertion. All residues are numbered with reference to the sequence of AfHypB.</p

AfHypB can recognizes guanine nucleotide despite having a non-canonical NKxA G4-motif.

<p>(A) In MjHypB, the aspartate residue of the NKxD motif serves as a hydrogen acceptor that form specific hydrogen bonds with the guanine N1 and N2 atoms. In AfHypB (green), the aspartate residue in the canonical NKxD G4-motif is replaced by an alanine residue (Ala-165). The role of a hydrogen acceptor is fulfilled by a nearby Asp-194 on the G5 loop that can form hydrogen bond to the N1 atom of the guanine base. (B–C) AfHypB specifically binds GDP and GTP but not ADP. Binding of MANT-GDP (grey dashed line) to AfHypB resulted in an increase in fluorescence at wavelength 400–500 nm with excitation at 290 nm (black solid lines). Addition of excess GDP or GTP (grey solid lines) resulted in significant decreases of fluorescence, indicating both GDP and GTP could competitively displace MANT-GDP from AfHypB. (D) On the other hand, addition of excess ADP (grey solid line) resulted in no significant changes in fluorescence, indicating that AfHypB binds specifically to guanine nucleotide but not to adenine nucleotide.</p

Mutation of the invariant lysine residue of <i>E. coli</i> HypB blocked <i>in vivo</i> hydrogenase maturation.

<p>The Δ<i>hypB</i> strain was transformed with the plasmids encoding wild-type EcHypB(WT), the K224A mutant, or the empty vector control pBAD. The hydrogenase activity of the lysates of these Δ<i>hypB</i> strains was compared relative to that of the wild type E. coli strain.</p

AfHypB undergoes GTP-dependent dimerization.

<p>Molecular weight of AfHypB in the presence of guanine nucleotides are determined by analytical size exclusion chromatography coupled with static light scattering. Both apo AfHypB (24.8 kDa) and GDP-bound AfHypB (23.6 kDa) remain as monomer (24.7 kDa). In contrast, AfHypB has an increased apparent molecular weight (41.0 kDa) in the presence of GMPPNP.</p

Substitutions of K148A disrupted GTP-dependent dimerization of AfHypB, but has no significant effect on guanine nucleotide binding and GTPase activity.

<p>(A) Molecular weight of AfHypB K148A variant in the presence of 5 mM GDP (blue), GMPPNP (green) or without nucleotides (red) were determined by SEC/LS. (B) The dissociation constants of AfHypB and the K148A variants for binding MANT-GDP and MANT-GMPPNP. (C) GTP hydrolysis rate in the presence of AfHypB or K148A mutant were determined as free phosphate release per hour (left). 0.2 mM of purified protein was mixed with 2 mM GTP. The GTP hydrolysis rates of wild-type and K148A AfHypB were found to be 0.041±0.006 hr⁻¹ and 0.051±0.008 hr⁻¹. Error bars indicate the standard deviation of hydrolysis rate over three independent experiments. The same experiment was repeated with protein pre-incubated with equimolar of GDP (right). The GTP hydrolysis rates of wild-type and K148A AfHypB pre-incubated with equimolar of GDP were found to be 0.039±0.008 hr⁻¹ and 0.049±0.008 hr⁻¹. Pre-incubation of GDP did not show significant effect to GTP hydrolysis activity of AfHypB or the K148A variant.</p

The ΔΔG_u^ele values of Asp and Glu of L30e* at 298 K and 333 K.

<p>The ΔΔG_u^ele values of Asp and Glu of L30e* at 298 K and 333 K.</p