Interaction of E.coli RBP signal peptides with components of translocation system

Abstract

Signal peptides of secretory proteins interact with various membrane and cytosolic components during the course of translocation. The primary aim of this study is to elucidate the interaction of signal peptides of ribose binding protein (RBP) with Escherichia coli (E.coli) signal recognition particle (SRP) composed of Ffh protein and 4.5S RNA, SecA and lipid bilayer. The signal peptides, as the nascent chain emerging from ribosome, are thought to bind the methionine-rich domain of Ffh. The structure of a peptide corresponding to one of the proposed methionine-rich segment of Ffh was determined using CD and NMR. This peptide was found to have a very unusual strong propensity to form alpha-helix and assumes an appreciable alpha-helix conformation even in water. The interactions of methionie-rich segment of the M-domain and M-domain as a whole with signal peptides were checked. It was reported that the only signal peptides of precursor proteins which are translocated inhibit the GTPase activity of E.coli SRP (Miller et al., 1994. Nature 367, 657-659). But, the signal peptides of revertant precursor RBPs, which show recovered capability of secretion, had no effect on the activity as is the case of nonfunctional mutant. Although functional signal peptides were previously reported to stimulate the ATPase activity of SecA in the presence of lipid vesicles, revertant and mutant signal peptides of RBP were found to inhibit it under various conditions. Comparison of the effect of revertant signal peptides with that of the wild type was possible only with the corresponding precursor protein due to low solubility of the wild-type peptide. The enhancement of SecA ATPase activity and reduction of ANS binding to SecA induced by the wild type precursor RBP were much more pronounced than by either mutant or revertant precursor RBPs. Revertant signal peptides assume predominantly α-helical conformations in the presence of anionic phospholipid vesicles and restrict the acyl chain...