An alternate mode of oligomerization for E. coli SecA

Abstract

SecA is the ATPase of preprotein translocase. SecA is a dimer in solution and changes in its oligomeric state may function in preprotein translocation. The SecA-N68 construct, in which the C-terminal helical domains of SecA are deleted, was used to investigate the mechanism of SecA oligomerization. SecA-N68 is in equilibrium between monomers, dimers, and tetramers. Subunit interactions in the SecA-N68 tetramer are mediated entirely by unstructured regions at its N- and C-termini: when the termini are deleted to yield SecA-N68∆NC, the construct is completely monomeric. This monomeric construct yielded crystals diffracting to 2.6 Å that were used to solve the structure of SecA-N68, including the “preprotein crosslinking domain” (PPXD) that was missing from previous E. coli SecA structures. The SecA-N68 structure was combined with small angle X-ray scattering (SAXS) data to construct a model of the SecA-N68 tetramer that is consistent with the essential roles of the extreme N- and C-termini in oligomerization. This mode of oligomerization, which depends on binding of the extreme N-terminus to the DEAD motor domains, NBD1 and NBD2, was used to model a novel parallel and flexible SecA solution dimer that agrees well with SAXS data.

Structure of the substrate-engaged SecA-SecY protein translocation machine

“…3i and j). The β-strand in PPXD is caused by the presence of substrate, as it is not seen in previous structures, including those of E. coli and M. tuberculosis SecA 13,35,36 . Similarly, the β-strand in the substrate itself is also induced.…”

Direct visualization of the E. coli Sec translocase engaging precursor proteins in lipid bilayers

“…What is the origin of the complex trimodal height distribution exhibited by translocases engaging pOmpA during hydrolysis? SecA 2 has been reported in at least three different orientations, and SecA protomers are thought to act independently (with little cooperativity) and to slide or to rotate about the interface through electrostatic and/or hydrophobic interactions (4,7,(22)(23)(24)(25)(26)(27). Hence, the complex height distribution could be related to different orientational states of the SecA dimer interface.…”

“…This suggests a high likelihood of dimers, based on the dissociation constant of ≤1 M (1,(16)(17)(18)(19); however, the presence of binding partners can directly affect the oligomeric state (18,20,21). Crystal structures of SecA from different species show two prominent orientations of dimer organization (parallel or antiparallel), though others have been identified (7,(22)(23)(24)(25)(26)(27). It has been argued that the two protomers within the SecA dimer (SecA 2 ) can undergo relative motion without dissociating, enabled by hydrophobic and/or electrostatic interactions along the dimer interface that allow SecA protomers to slide and to rotate (4).…”