Sites of Interaction of a Precursor Polypeptide on the Export Chaperone SecB Mapped by Site-directed Spin Labeling

Abstract

SummaryExport of protein into the periplasm of Escherichia coli via the general secretory system requires that the transported polypeptides be devoid of stably folded tertiary structure. Capture of the precursor polypeptides before they fold is achieved by the promiscuous binding to the chaperone SecB. SecB delivers its ligand to export sites through its specific binding to SecA, a peripheral component of the membrane translocon. At the translocon the ligand is passed from SecB to SecA and subsequently through the SecYEG channel. We have previously used site-directed spin labeling and electron paramagnetic resonance spectroscopy to establish a docking model between SecB and SecA. Here we report use of the same strategy to map the pathway of a physiologic ligand, the unfolded form of precursor galactose-binding protein, on SecB. Our set of SecB variants each containing a single cysteine, which was used in the previous study, has been expanded to forty-eight residues which cover 49% of the surface of SecB. The residues on SecB involved in contacts were identified as those that, upon addition of the unfolded polypeptide ligand, showed changes in spectral line shape consistent with restricted motion of the nitroxide. We conclude that the bound precursor makes contact with a large portion of the surface of the small chaperone. The sites on SecB that interact with the ligand are compared with the previously identified sites that interact with SecA and a model for transfer of the ligand is discussed.

SecB-like chaperone controls a toxin–antitoxin stress-responsive system in Mycobacterium tuberculosis

“…1A). This alignment shows that Rv1957 has several clusters of key residues critical for SecB oligomerization and interaction with substrates or with its specific partner SecA at the Sec translocon (19)(20)(21)(22)(23)(24)(25). Previous work Author contributions: P.G.…”

Orientation of SecA and SecB in Complex, Derived from Disulfide Cross-Linking

“…1). For example, the last 13 residues of SecB (residues 143 to 155) that participate in 23 of the 41 values of 40% lie in regions that are intrinsically disordered as shown by nuclear magnetic resonance (18) and EPR studies (3,10). The amino terminus of SecA, which participates in 16 of the most abundant cross-links, is likely to be flexible since in the three X-ray structures of SecA from Bacillus subtilis the N-terminal 15 residues have different conformations.…”

“…Studies using site-directed spin labeling and EPR spectroscopy show that in the complex between SecB and a precursor, the polypeptide is wrapped around the surface of SecB. It occupies a channel at the interface of the dimer of dimers, crosses over the ends or the flat sides of SecB, and binds in the channel on the opposite side (3,10). Either of the orientations of SecB illustrated in Fig.…”

Chaperone addiction of toxin–antitoxin systems

“…This suggests that SecB chaperones can efficiently be rerouted to accommodate short C-terminal ChAD extensions without losing their ability to functionally bind a wide range of presecretory substrates. The fact that SecB possesses a remarkably large substrate-binding surface that can accommodate very long fragments within polypeptide substrates (up to 150 amino acids in length) indeed suggests that discreet changes within this surface could specialized the chaperone towards the ChAD sequence of an antitoxin without detectably modifying its overall generic chaperone properties4144. More work is warranted to identify such binding area on SecB TA chaperones.…”