Mutational Alterations in the Homotetrameric Chaperone SecB That Implicate the Structure as Dimer of Dimers

Eva M Murén,Dominic Suciu,Traci B Topping,Carol A Kumamoto,Linda L Randall

doi:10.1074/jbc.274.27.19397

Eva M Murén, Dominic Suciu + Show 3 more

Open Access

https://doi.org/10.1074/jbc.274.27.19397

Copy DOI

Abstract

Variant forms of SecB with substitutions of aminoacyl residues in the region from 74 to 80 were analyzed with respect to their ability to bind a physiological ligand, precursor galactose-binding protein, and to their oligomeric states. SecBL75Q and SecBE77K are tetramers with affinity for ligand indistinguishable from that of the wild-type SecB, and thus the export defect exhibited by strains producing these variants must result from an effect on interactions between SecB and other components. SecBF74I is tetrameric but binds ligand with a lower affinity. Substitutions at positions 76, 78, and 80 cause a shift in the equilibrium so that the SecB tetramer dissociates into dimers. We conclude that the tetramer is a dimer of dimers and that the residues Cys76, Val78, and Gln80 must be involved either directly or indirectly in forming the interface between dimers. These variant species are defective in binding ligand; however, because their oligomeric state is altered no conclusion can be drawn concerning the direct role of these residues in ligand binding.

Highlights

SecB is a chaperone from Escherichia coli involved in the facilitation of export of proteins from the cytoplasm to the outer membrane, which lies beyond the cytoplasmic membrane, and to the periplasm, the aqueous space between the two membranes
SecB, which functions as a homotetramer [16, 17], is the product of the secB gene that encodes a polypeptide of 155 amino acids [18]
The equilibrium constant is such that even at 20 nM, which is well below the concentration in vivo, estimated to be 4 ␮M [2, 17], neither dimer nor monomer was detected when a solution was analyzed by size exclusion chromatography

Summary

EXPERIMENTAL PROCEDURES

Protein Purification—Mature galactose-binding protein and precursor galactose-binding protein were purified as described [4, 5]. Samples of 200 ␮l (for soluble lysates this volume contained the equivalent of 1.5 ϫ 109 cells) were injected, separation was carried out at 5 °C at 1 ml/min, and absorbance was monitored at 280 nm. Soluble lysates were injected onto the TSK G3000SW as described above and fractions of 1 ml were collected and processed for polyacrylamide gel electrophoresis followed by immunoblotting using an antibody to SecB. Interaction of SecB and SecB Variant Proteins with Precursor Galactose-binding Protein in Lysates—Soluble lysates were prepared (as above except that the growth temperature was 35 °C) from the following strains: CK1953 [10], which carries the secB::Tn5 mutation and is secBnull; BL21(DE3) pJW25 [6], which carries the wild-type secB on the chromosome as well as on the plasmid; and CK2212 with two plasmids both containing either secBC76Y or secBV78F. Immunoblotting was performed as described [13] using antisera to SecB and to galactose-binding protein and the chromogenic dye 4-chloro-1naphthol for detection

RESULTS

Kd n

DISCUSSION