Preprotein Conformational Dynamics Drive Bivalent Translocase Docking and Secretion

Abstract

Most bacterial secretory proteins destined beyond the plasma membrane are secreted post-translationally by the Sec translocase. In the first step of translocation, preproteins are targeted for binding to their 2-site receptor SecA, the peripheral ATPase subunit of the translocase. We now reveal that secretory preproteins use a dual-key mechanism to bridge the signal peptide and mature domain receptor sites and cooperatively enhance their affinities. Docking of targeting-competent mature domains requires that their extensive disorder is finely tuned. This is achieved through amino-terminal mature domain regions acting as conformational rheostats. By being linked to the rheostats, signal peptides regulate long-range preprotein disorder. Concomitant conformational changes in SecA sterically adapt its two receptor sites to optimally recognize hundreds of dissimilar preproteins. This novel intramolecular conformational crosstalk in the preprotein chains and the dynamic interaction with their receptor are mechanistically coupled to preprotein engagement in the translocase and essential for secretion.

Hierarchical protein targeting and secretion is controlled by an affinity switch in the type III secretion system of enteropathogenic Escherichia coli

“…Attesting to this, mutations on the SepL-binding sites on EscV severely compromised secretion of Tir ( Fig EV5G). Analogous promiscuity is seen in the universal Sec system where the single SecA receptor recognizes the 505 secreted proteins of E. coli K12 on two adjacent sites Sardis et al, 2017). SepL participates in multiple interactions (i.e., association with membranes, with EscV, with three different translocator chaperones, with its own two chaperones) and is flexible enough to become secreted through the translocase.…”

“…Attesting to this, mutations on the SepL‐binding sites on EscV severely compromised secretion of Tir (Fig EV5G). Analogous promiscuity is seen in the universal Sec system where the single SecA receptor recognizes the 505 secreted proteins of E. coli K12 on two adjacent sites (Chatzi et al , ; Sardis et al , ).…”

Cardiolipin is required in vivo for the stability of bacterial translocon and optimal membrane protein translocation and insertion

“…bacterial secretory proteins destined beyond the IM are secreted post-translationally by the SecYEG translocon 50 , E. coli alkaline phosphatase and OmpA are widely used model proteins, which are capable for both co-and post-translational modes in vivo and in vitro 51,52 . Measurement of alkaline phosphatase enzymatic activity in intact toluene permeabilized cells is a reliable assay of translocation of preproteins across IM and enzymatic activity is used very often to quantitate the extent of translocon-assisted translocation in vivo 53 .…”

The Preprotein Binding Domain of SecA Displays Intrinsic Rotational Dynamics

“…For residue-specific labeling via maleimide-modified dyes reacting with thiol groups of cys residues ( Figure S1C), we used the fully functional SecA(Cys À ) Sardis et al, 2017). The secA(cys À ) gene derivatives with specific cysteine pairs were generated.…”

“…We used the fully functional SecA(cys -) that has its 4 cysteinyl residues substituted Sardis et al, 2017) (positions 98, 885, 887 and 896) substituted by serine (C98S) or alanines (885, 887 and 896). Gene-mutations were introduced by following the QuickChange Site-Directed Mutagenesis protocol (Stratagene-Agilent); templates and primers are listed in Tables S3 and S4.…”