Chaperone SecB from Escherichia coli Mediates Kinetic Partitioning via a Dynamic Equilibrium with Its Ligands

Traci B Topping,Linda L Randall

doi:10.1074/jbc.272.31.19314

Abstract

We have shown that the complexes between SecB, a chaperone from Escherichia coli, and two physiological ligands, galactose-binding protein and maltose-binding protein, are in rapid, dynamic equilibrium between the bound and free states. Binding to SecB is readily reversible, and each time the ligand is released it undergoes a kinetic partitioning between folding to its native state and re-binding to SecB. Binding requires that the polypeptide be devoid of tertiary structure; once the protein has folded, it is no longer a ligand. Conditions were established in which folding of the polypeptides was sufficiently slow so that at each cycle of dissociation rebinding was favored over folding and a kinetically stable complex between SecB and each polypeptide ligand was observed. Evidence that the ligand is continually released to the bulk solution and rebound was obtained by altering the conditions to increase the rate of folding of each ligand so that folding of the ligand was faster than reassociation with SecB thereby allowing the system to partition to free SecB and folded polypeptide ligand. We conclude that complexes between the chaperone SecB and ligands are in dynamic, rapid equilibrium with the free states. This mode of binding is simpler than that documented for chaperones that function to facilitate folding such as the Hsp70s and Hsp60s, where hydrolysis of ATP is coupled to the binding and release of ligands. This difference may reflect the fact that SecB does not mediate folding but is specialized to facilitate protein export. Without a requirement for exogenous energy it efficiently performs its sole duty: to keep proteins in a nonnative conformation and thus competent for export.

Highlights

SecB is a cytosolic chaperone from Escherichia coli that binds a subset of polypeptides destined to be exported to the periplasmic space or to the outer membrane
In this model the ligand is in dynamic, rapid equilibrium between the free and bound states, and the proportion of the polypeptides that are properly localized is a function of the rate constant of association relative to the rate constant of folding
SecB is classified as a protein chaperone based on its ability to selectively bind polypeptides that are in a nonnative state, but it is distinguished from other members of the family by at least one distinctive property

Summary

EXPERIMENTAL PROCEDURES

Materials—Ultra-pure guanidinium chloride (GdmHCl) was from ICN Biochemicals, Inc. EGTA was from Fluka; calcium chloride and potassium acetate were from J. The pellet that contained membrane and precursor galactose-binding protein in inclusion bodies was suspended at an equivalent cell density of 3 ϫ 1010 cells/ml in 10 mM Tris-Cl, pH 7.6, 3% (w/v) Triton X-100, and 5 mM EDTA to solubilize the membrane proteins. Fractions containing the precursor galactose-binding protein as detected by SDS-gel electrophoresis were pooled and dialyzed against 1 M GdmHCl, 20 mM HEPES-KOH, pH 7.4. Analysis of Complexes between SecB and the Binding Proteins—A complex between SecB and galactose-binding protein was formed by diluting unfolded galactose-binding protein to a solution held on ice containing SecB to give final concentrations of 0.09 M GdmHCl, 10 mM HEPES-KOH, pH 7.0, 150 mM potassium acetate, 0.5 mM EGTA, 5.5 ␮M SecB tetramer, and 3.7 ␮M galactose-binding protein. One quarter of the protein in each fraction and the equivalent of 1/20 of the sample applied to the column were loaded on the gel unless indicated otherwise

RESULTS

SecB Binds Ligands in Dynamic Equilibrium

DISCUSSION