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

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.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. SecB keeps these proteins in a nonnative conformation, which is competent for export, while delivering them to SecA, the next component in the export pathway. There is no specific sequence of amino acids that distinguishes a polypeptide as a ligand for SecB. The only requirement for binding is that the ligand be devoid of tertiary structure. The binding frames for three physiological ligands, maltose-binding protein, galactose-binding protein, and the oligopeptide-binding protein, have been determined (1, 2).1 In all three cases it was shown that the sequence in contact with SecB is large, minimally comprising 150 amino acids in a continuous stretch. There is no similarity in sequence among the binding frames nor is it obvious that a structural element provides recognition. The large area of contact between SecB and its ligands allows for interaction at multiple subsites with each site having low affinity yet together giving tight binding; the dissociation constant for ligands of SecB is in the range of 10 Ϫ8 M (3-5). Even though the affinity is high there are several studies that indicate that the complex of SecB and its ligand is in rapid equilibrium with the unbound ...

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

“…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 rebinding to SecB. 13,14 Precursor galactose-binding protein is an ideal ligand for these studies because the rate of folding can be modulated by temperature, as is true for the folding of all proteins, and also by calcium. The native structure of galactose-binding protein is stabilized by a calcium ion and the rate of folding of the polypeptide is drastically decreased in the absence of calcium.…”

“…The native structure of galactose-binding protein is stabilized by a calcium ion and the rate of folding of the polypeptide is drastically decreased in the absence of calcium. 14 In the experimental design used here the precursor, unfolded in 1 N GnHCl, was added to the SecB by rapid dilution into a solution held on ice so that the final concentration of the GnHCl was 0.17 N and calcium was chelated by 1 mM EGTA. The spectra were collected at 6°C.…”

“…20 Precursor galactose-binding protein was purified as described. 14 The protein concentrations were determined spectrophotometrically at 280 nm using extinction coefficients of 47,600 M −1 cm −1 for SecB tetramer and 37410 M −1 cm −1 for precursor galactose-binding protein.…”

Crystal structure of SecB from Escherichia coli

“…SecB is involved in two fundamentally different types of interactions: interactions with a diverse set of unfolded proteins, that are in rapid equilibrium with the free pool (Topping and Randall, 1997) and the unique interaction with SecA that makes the precursor enter the translocation pathway. SecB might also engage in functions completely unrelated to export (Panse et al, 2000) possibly acting as a general buffer for the unfolded state of polypeptides in the cytosol (Randall and Hardy, 2002).…”

The Sec system

“…Therefore, SecB will not be able to distinguish between cvtosolic and precursor proteins on the basis of the folding behaviour only: Moreover, even when a preprotcin escapcs the SecB binding at the ribosome, it will be bound in the cvtosol before it can fold into a native-like structure. Once SecB has bound an unfolded preprotein, stable %lding is prevented in the binding equilibriuna by repcatcd association and dissociation events [9,10]. Taken togethcr, the specificity of the SecB-preprotein interaction it1 ~'iz'o remains unclear.…”