Conserved SecA Signal Peptide-Binding Site Revealed by Engineered Protein Chimeras and Förster Resonance Energy Transfer

Abstract

Signal peptides are critical for the initiation of protein transport in bacteria by virtue of their recognition by the SecA ATPase motor protein followed by their transfer to the lateral gate region of the SecYEG protein-conducting channel complex. In this study, we have constructed and validated the use of signal peptide-attached SecA chimeras for conducting structural and functional studies on the initial step of SecA signal peptide interaction. We utilized this system to map the location and orientation of the bound alkaline phosphatase and KRRLamB signal peptides to a peptide-binding groove adjacent to the two-helix finger subdomain of SecA. These results support the existence of a single conserved SecA signal peptide-binding site that positions the signal peptide parallel to the two-helix finger subdomain of SecA, and they are also consistent with the proposed role of this subdomain in the transfer of the bound signal peptide from SecA into the protein-conducting channel of SecYEG protein. In addition, our work highlights the utility of this system to conveniently engineer and study the interaction of SecA with any signal peptide of interest as well as its potential use for X-ray crystallographic studies given issues with exogenous signal peptide solubility.

Alignment of the protein substrate hairpin along the SecA two-helix finger primes protein transport in Escherichia coli

“…The determined distances have a larger error than that obtained experimentally for the efficiencies alone to account for the error introduced by the uncertainty in dye position as determined from steady-state anisotropy values (21,30,31). Using this information, we could position the signal peptide and early mature region of PhoA within the SecA-SecYEG complex.…”

“…1). We also demonstrated that the lambda receptor signal peptide, KRRLamB, bound to SecA in the same location and orientation as its PhoA counterpart, indicative of a common binding site and orientation (21).…”

“…To determine the location of the signal peptide and early mature region of the protein substrate within the purified SecYEGbound SecA complex, we used our genetically engineered SecAPhoA chimeras and FRET mapping approach as reported previously (21). We have demonstrated that the SecA and PhoA signal peptide portions of the chimera were functional in vivo and in vitro, and that the attached PhoA signal peptide bound specifically to SecA at the previously mapped binding site (20).…”

“…SecA also bound SecYEG with They include (N-terminal to C-terminal on SecA): the nucleotide-binding domain-1 (NBD-1) (blue), the preprotein cross-linking domain (PPXD) (gold), the nucleotide-binding domain-2 (NBD-2) (light blue), the central helix subdomain (CH) (green), the helical wing domain (HWD) (dark green), the two helix-finger subdomain (THF) (cyan), and the carboxyl-terminal linker (CTL). The CTL is depicted in red and serves as a model of PhoA signal peptide bound to B. subtilis SecA based on the FRET mapping study of Zhang et al (21).…”

“…Previously, we used Förster resonance energy transfer (FRET)-based methods to map the location of the SecA signal peptidebinding site using chimeras containing SecA and the alkaline phosphatase (PhoA) or lambda receptor variant (KRRLamB) signal peptides (20,21). We found that the signal peptide interacts Significance Specialized protein complexes transport and integrate peptides into membranes in all living cells.…”