Solution NMR Structure and X-ray Absorption Analysis of the C-Terminal Zinc-Binding Domain of the SecA ATPase^†

The solution NMR structure of a 22-residue Zn²⁺-binding domain (ZBD) from Esherichia coli preprotein translocase subunit SecA is presented. In conjunction with X-ray absorption analysis, the NMR structure shows that three cysteines and a histidine in the sequence CXCXSGX₈CH assume a tetrahedral arrangement around the Zn²⁺ atom, with an average Zn²⁺−S bond distance of 2.30 Å and a Zn²⁺−N bond distance of 2.03 Å. The NMR structure shows that ND1 of His20 binds to the Zn²⁺ atom. The ND1−Zn²⁺ bond is somewhat strained:  it makes an angle of approximately 17° with the plane of the ring, and it also shows a significant “in-plane” distortion of 13°. A comprehensive sequence alignment of the SecA-ZBD from many different organisms shows that, along with the four Zn²⁺ ligands, there is a serine residue (Ser12) that is completely conserved. The NMR structure indicates that the side chain of this serine residue forms a strong hydrogen bond with the thiolate of the third cysteine residue (Cys19); therefore, the conserved serine appears to have a critical role in the structure. SecB, an export-specific chaperone, is the only known binding partner for the SecA-ZBD. A phylogenetic analysis using 86 microbial genomes shows that 59 of the organisms carry SecA with a ZBD, but only 31 of these organisms also possess a gene for SecB, indicating that there may be uncharacterized binding partners for the SecA-ZBD.