HypA/HypB Interaction and Hydrogenase Maturation 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). doi:10.1371/journal.pone.0032592.g001 crude lysate. Lysate containing HS-EcHypB variants and the lysate containing GST-EcHypA were mixed and loaded on to GSH-resin for pull-down assay. Our results showed that wild-type HS-EcHypB was co-eluted with GST-EcHypA (Figure 3C, lane 3). The identity of HS-EcHypB was confirmed by mass spectroscopy analysis of trypsinized fragments. Like wild-type EcHypB, the E79A variant retained its interaction with GST- EcHypA. On the other hand, GST-EcHypA did not pull down the L79A and V80A variants of EcHypB (Figure 3C, lane 4 and 6), suggesting that the two conserved hydrophobic residues, Leu-78 Figure 2. The unstructured N-terminal residues of AfHypB is required for HypA/HypB interaction. (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, DN and DC, were created to investigate the role of these unstructured residues. (B) Interaction of AfHypB variants with AfHypA was tested by in vitro 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 [35,36] 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 in vitro 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. doi:10.1371/journal.pone.0032592.g002 PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e32592