The Trimeric Periplasmic Chaperone Skp of Escherichia coli Forms 1:1 Complexes with Outer Membrane Proteins via Hydrophobic and Electostatic Interactions

Abstract

The interactions of outer membrane proteins (OMPs) with the periplasmic chaperone Skp from Escherichia coli are not well understood. We have examined the binding of Skp to various OMPs of different origin, size, and function. These were OmpA, OmpG, and YaeT (Omp85) from Escherichia coli, the translocator domain of the autotransporter NalP from Neisseria meningitides, FomA from Fusobacterium nucleatum, and the voltagedependent anion-selective channel, human isoform 1 (hVDAC1) from mitochondria. Binding of Skp was observed for bacterial OMPs, but neither for hVDAC1 nor for soluble bovine serum albumin. The Skp trimer formed 1:1 complexes, OMP·Skp3, with bacterial OMPs, independent of their size or origin. The dissociation constants of these OMP·Skp3 complexes were all in the nanomolar range, indicating that they are stable. Complexes of Skp3 with YaeT displayed the smallest dissociation constants, complexes with NalP the largest. OMP binding to Skp3 was pH-dependent and not observed when either Skp or OMPs were neutralized at very basic or very acidic pH. When the ionic strength was increased, the free energies of binding of Skp to OmpA or OmpG were reduced. Electrostatic interactions were therefore necessary for formation and tability of OMP·Skp3 complexes. Light-scattering and circular dichroism experiments demonstratedthat Skp3 remained a stable trimer from pH 3 to pH 11. In the OmpA·Skp3 complex, Skp efficiently shielded tryptophan residues of the transmembrane strands of OmpA against fluorescence quenching by aqueous acrylamide. Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, bound to OmpA·Skp3 complexes at low stoichiometries. crylamide quenching of fluorescence indicated that in this ternary complex, the tryptophan residues of the transmembrane domain of OmpAwere located closer to the surface than in binary OmpA·Skp3 complexes. This may explain previous observations that folding of Skp-bound OmpA into lipid bilayers is facilitated in presence of LPS.