Mapping of the Binding Frame for the Chaperone SecB within a Natural Ligand, Galactose-binding Protein

Vijaya J Khisty,Gerhard R Munske,Linda L Randall

doi:10.1074/jbc.270.43.25920

Vijaya J Khisty, Gerhard R Munske + Show 1 more

Open Access

https://doi.org/10.1074/jbc.270.43.25920

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Abstract

The chaperone SecB selectively binds polypeptides that are in a non-native state; however, the details of the interaction between SecB and its ligands are unknown. As a step in elucidation of the molecular mechanism of binding, we have mapped the region of a physiologic ligand (galactose-binding protein) that is in contact with SecB. The binding frame comprises approximately 160 aminoacyl residues and is located in the central portion of the primary sequence. Comparison to the binding frame within maltose-binding protein, which is similarly long and positioned around the center of that polypeptide, reveals no similarity in sequence or in folding motif. The results are consistent with the proposal that the selectivity in binding exhibited by SecB is based on the simultaneous occupancy of multiple binding sites, each of which demonstrates low specificity, by flexible stretches of polypeptide that are only accessible in non-native proteins.

Highlights

Was identified that consisted of multiple contiguous sites positioned around the center of the primary sequence and covering ϳ170 residues (Topping and Randall, 1994)
As was shown previously for the interaction of SecB with another of its natural ligands, periplasmic maltose-binding protein, SecB forms a complex with galactose-binding protein only if the protein is presented to SecB in a non-native state
The defining characteristic of the binding is that it occurs with low specificity, but when the ligand is a long polypeptide, the affinity is high (dissociation constants are in the range of 1–100 nM (Randall, 1992))

Summary

EXPERIMENTAL PROCEDURES

Materials—Guanidinium chloride (ultrapure) was purchased from Schwarz/Mann, and HEPES and proteinase K were from Sigma. The concentrations of the proteins were determined by absorbance at 280 nm using extinction coefficients for the SecB monomer and galactose-binding protein of 11,900 and 37,700 MϪ1 cmϪ1, respectively. 1) Denatured galactose-binding protein in 1 M GdmCl, 150 mM potassium acetate, 10 mM HEPES, pH 7.6, was diluted into a solution containing SecB; or 2) denatured galactose-binding protein was diluted into a solution without SecB, and SecB was added within 10 s In both cases, the solutions were held on ice, and the final concentrations were as follows: 37 ␮M SecB tetramer, 18 ␮M galactose-binding protein, 50 mM GdmCl, 100 mM NaCl, 7.5 mM potassium acetate, 50 mM Tris-HCl, 1.5 mM HEPES, pH 7.0. After an additional incubation for 10 min on ice, phenylmethylsulfonyl fluoride

The abbreviations used are

RESULTS

DISCUSSION