Selective Photoaffinity Labeling Identifies the Signal Peptide Binding Domain on SecA

Abstract

SecA, an ATPase crucial to the Sec-dependent translocation machinery in Escherichia coli, recognizes and directly binds the N-terminal signal peptide of an exported preprotein. This interaction plays a central role in the targeting and transport of preproteins via the SecYEG channel. Here we identify the Signal Peptide Binding Groove (SPBG) on SecA addressing a key issue regarding the SecA-preprotein interaction. We employ a synthetic signal peptide containing the photoreactive benzoylphenylalanine to efficiently and specifically label SecA containing a unique Factor Xa site. Comparison of the photolabeled fragment from the subsequent proteolysis of several SecAs, which vary only in the location of the Factor Xa site, reveals one 53-residue segment in common with the entire series. The covalently modified SecA segment produced is the same in aqueous solution and in lipid vesicles. This spans amino acids 269 to 322 of the E. coli protein, which is distinct from a previously proposed signal peptide binding site, and contributes to a hydrophobic peptide binding groove evident in molecular models of SecA.

Use of synthetic signal sequences to explore the protein export machinery

“…Using fragments, deletional analysis, and chemical crosslinking of synthetic signal peptides, Economou's group concluded that the region at the base of the PPXD (so-called stem region) serves to bind the signal sequence. 69,89 Results of a FRET-based study with complementary crosslinking approaches also implicated the PPXD (specifically the third Using Signal Peptides to Explore Protein Export 315 helix) in signal sequence binding, 93,95 which is consistent with the early crosslinking studies from Mizushima's lab. 94 We have performed a sequence alignment of 550 SecA proteins, and the region proposed by Musial-Siwek et al 93 has a low conservation score (J. L. Maki and L. M. Gierasch, unpublished observations), raising doubt about whether it serves as a direct binding site for signal sequences.…”

“…69,89 Results of a FRET-based study with complementary crosslinking approaches also implicated the PPXD (specifically the third Using Signal Peptides to Explore Protein Export 315 helix) in signal sequence binding, 93,95 which is consistent with the early crosslinking studies from Mizushima's lab. 94 We have performed a sequence alignment of 550 SecA proteins, and the region proposed by Musial-Siwek et al 93 has a low conservation score (J. L. Maki and L. M. Gierasch, unpublished observations), raising doubt about whether it serves as a direct binding site for signal sequences. Taken together, it seems that the central region adjacent and perhaps including the PPXD is involved in initial signal sequence recognition, but the exact location of the signal sequencebinding site on cytoplasmic SecA remains elusive.…”

Chaperones and chaperone–substrate complexes: Dynamic playgrounds for NMR spectroscopists

“…The observed trNOEs showed that the bound signal peptide adopts an α-helical structure and differential line broadening results suggested that the initial signal sequence-binding pocket on SecA diplays both electrostatic and hydrophobic character [265]. Subsequently, the PBD was identified by NMR chemical shift perturbations and selective photoaffinity measurements as the site where preproteins bind on SecA [254,260,266] and it was shown that this domain is able to control conformation and ATP catalysis within the helicase motor [267]. The most accurate structural information to date was reported by the Kalodimos group, obtained from high-resolution NMR structure determination in combination with PRE measurements of a LamB signal sequence bound to the SecA protein [260].…”

“…To translocate polypeptides via the SecYEG-translocon across the bacterial inner membrane towards the periplasmic space, the substrate has to be delivered by the SecA chaperone [254,255]. Besides its molecular chaperone function, SecA serves as the initial recognition site for the signal sequence and as the motor domain of the SecYEG-channel, whereupon it uses the energy generated by ATP-hydrolysis [256].…”

Cryo-electron Microscopic Structure of SecA Protein Bound to the 70S Ribosome

“…The cleft between the PPXD and NBD2 is referred to as the clamp, and, depending on the position of the PPXD, it can be in an open (5), partially open (8), or closed form (10). The PPXD has been proposed to interact with preproteins (11)(12)(13)(14).…”