Chaperone properties of the bacterial periplasmic substrate-binding proteins.

Gilbert Richarme,Teresa Dantas Caldas

doi:10.1074/jbc.272.25.15607

Abstract

Bacterial periplasmic substrate-binding proteins are initial receptors in the process of active transport across cell membranes and/or chemotaxis. Each of them binds a specific substrate (e.g. sugar, amino acid, or ion) with high affinity. For transport, each binding protein interacts with a cognate membrane complex consisting of two hydrophobic proteins and two subunits of a hydrophilic ATPase. For chemotaxis, binding proteins interact with specific membrane chemotaxis receptors. We report, herewith, that the oligopeptide-binding protein OppA of Escherichia coli, the maltose-binding protein MalE of E. coli, and the galactose-binding protein MglB of Salmonella typhimurium interact with unfolded and denatured proteins, such as the molecular chaperones that are involved in protein folding and protein renaturation after stress. These periplasmic substrate-binding proteins promote the functional folding of citrate synthase and alpha-glucosidase after urea denaturation. They prevent the aggregation of citrate synthase under heat shock conditions, and they form stable complexes with several unfolded proteins, such as reduced carboxymethyl alpha-lactalbumin and unfolded bovine pancreatic trypsin inhibitor. These chaperone-like functions are displayed by both the liganded and ligand-free forms of binding proteins, and they occur at binding protein concentrations that are 10-100-fold lower than their periplasmic concentration. These results suggest that bacterial periplasmic substrate-binding proteins, in addition to their function in transport and chemotaxis, might be implicated in protein folding and protection from stress in the periplasm.

Highlights

The periplasmic binding proteins of Gram-negative bacteria constitute a set of at least 30 proteins that are involved in the transport of, and chemotaxis toward, substrates
That the oligopeptide-binding protein OppA of Escherichia coli, the maltose-binding protein MalE of E. coli, and the galactose-binding protein MglB of Salmonella typhimurium interact with unfolded and denatured proteins, such as the molecular chaperones that are involved in protein folding and protein renaturation after stress
Periplasmic Binding Proteins Increase the Amount of Correctly Folded Citrate Synthase and ␣-Glucosidase—We first investigated whether OppA, MglB, and MalE act as molecular chaperones in the folding of proteins

Summary

EXPERIMENTAL PROCEDURES

Purification of MglB, MalE, OppA, DnaK, and DnaJ—MglB and MalE were purified as described previously [24]. The spheroplast supernatant from 10 g of bacteria was prepared as described by Higgins and Staros [25], loaded onto a DE52-Sephacel column (20-ml bed volume, Pharmacia Biotech Inc.) equilibrated in 20 mM Tris-HCl, pH 8, 1 mM dithiothreitol, and eluted with a linear gradient of 0 to 0.3 M NaCl in the same buffer. The active fractions were loaded onto a hydroxylapatite column (6-ml bed volume, Bio-Rad) in the same buffer, and the column was eluted with a linear gradient of 0 to 0.3 M sodium phosphate, pH 8, in the same buffer. DnaK and DnaJ were prepared, as described previously [27,28,29], from an overproducing

Chaperone Properties of Periplasmic Binding Proteins

RESULTS

DISCUSSION