The Conformation of a Signal Peptide Bound by Escherichia coli Preprotein Translocase SecA

Abstract

To understand the structural nature of signal sequence recognition by the preprotein translocase SecA, we have characterized the interactions of a signal peptide corresponding to a LamB signal sequence (modified to enhance aqueous solubility) with SecA by NMR methods. One-dimensional NMR studies showed that the signal peptide binds SecA with a moderately fast exchange rate (K d ϳ 10 ؊5 M). The line-broadening effects observed from one-dimensional and two-dimensional NMR spectra indicated that the binding mode does not equally immobilize all segments of this peptide. The positively charged arginine residues of the n-region and the hydrophobic residues of the h-region had less mobility than the polar residues of the c-region in the SecA-bound state, suggesting that this peptide has both electrostatic and hydrophobic interactions with the binding pocket of SecA. Transferred nuclear Overhauser experiments revealed that the h-region and part of the c-region of the signal peptide form an ␣-helical conformation upon binding to SecA. One side of the hydrophobic core of the helical h-region appeared to be more strongly bound in the binding pocket, whereas the extreme C terminus of the peptide was not intimately involved. These results argue that the positive charges at the n-region and the hydrophobic helical h-region are the selective features for recognition of signal sequences by SecA and that the signal peptide-binding site on SecA is not fully buried within its structure.

Identification of Two Interaction Sites in SecY that Are Important for the Functional Interaction with SecA

“…This possibility is considered unlikely for the following reasons: The TMS4c peptides have a net negative charge, whereas that of signal peptides is usually positive due to the characteristic positively charged residues in the N terminus. It has recently been shown that these positively charged residues are directly involved in signal sequence binding to SecA, 31 which is in marked contrast to the negatively charged residue (Glu176) that is the main determinant for binding of TMS4c to SecA. Furthermore, if the interaction with TMS4c would represent SecA binding to the mature region of a pre-protein, many more peptides are expected to bind SecA.…”

The Long α-Helix of SecA Is Important for the ATPase Coupling of Translocation

“…The crystal structure of the monomeric form of Bacillus subtilis SecA revealed an open groove similar to the substrate-binding grooves of molecular chaperones (10). In addition, SecA has another and shallower groove located more closely to NFB1 and HSD (6, 10) with possible complementarity to a signal peptide (35). It is conceivable that such substrate-binding cavities are used for the second mode of preprotein binding used for the actual movement of the preprotein.…”

Use of synthetic signal sequences to explore the protein export machinery

“…92 The observed trNOEs showed that the bound signal peptide adopts an a-helical structure in the h-and c-regions, and differential line broadening argued that one side of the helix in the h-region is more strongly bound to SecA ( Figure 5). Additionally, the positive n-region and the hydrophobic h-region of the signal peptide were intimately involved in binding.…”