Engineered interdomain disulfide in the periplasmic receptor for sulfate transport reduces flexibility. Site-directed mutagenesis and ligand-binding studies.
Using recombinant DNA techniques, an Escherichia coli periplasmic sulfate receptor or sulfate-binding protein involved in active transport has been overexpressed and characterized. This protein is essentially identical in size, sequence, antigenicity, and ligand affinity and specificity to the sulfate receptor from Salmonella typhimurium whose crystal structure has been refined at 2 A resolution. The dehydrated sulfate is bound in the deep cleft between the two lobes of the bilobate protein. Using the structure of the S. typhimurium as a guide, three site-directed mutants (Ser129Cys, Gly46Cys, and Ser129Cys/Gly46Cys) have been made. In the Cys129/Cys46 mutant the disulfide has been successfully introduced across the opening of the ligand-binding site cleft of the E. coli sulfate-binding protein. The dissociation of sulfate from the double mutant protein is very slow under oxidizing conditions and increases more than 200-fold when reducing agent is added. This effect is attributed to a loss of interdomain structural flexibility in the presence of the disulfide, and underscores the importance of protein conformational change in binding protein function.
Jacobson
B L
BL
Department of Biochemistry and Structural Biology, Baylor College of Medicine, Houston, Texas 77030.
He
J J
JJ
Vermersch
P S
PS
Lemon
D D
DD
Quiocho
F A
FA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D033902:Periplasmic Binding Proteins; D015202:Protein Engineering; D012486:Salmonella typhimurium / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013431:Sulfates / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D033902:Periplasmic Binding Proteins; D013431:Sulfates; C489040:sbp protein, E coli; C044773:sulfate-binding protein, bacteria