Binding of ligand or monoclonal antibody 4B1 induces discrete structural changes in the lactose permease of Escherichia coli

Biochemistry
S FrillingosH R Kaback

Abstract

By using Cys-scanning mutagenesis with site-directed sulfhydryl modification in situ [Frillingos, S., & Kaback, H. R. (1996) Biochemistry 35, 3950-3956], conformational changes induced by binding of ligand or monoclonal antibody (mAb) 4B1 in the lactose permease of Escherichia coli were studied. Out of 31 single-Cys replacement mutants in helices I, V, VII, VIII, X, or XI, 4B1 binding alters the reactivity of Val238-->Cys (helix VII), Val331-->Cys (helix X), or single-Cys355 (helix XI) permease with N-ethylmaleimide (NEM) in right-side-out membrane vesicles. In addition, site-directed fluorescence spectroscopy shows that mAb 4B1 binding causes position 331 (helix X) in the permease to experience a more hydrophobic environment. In contrast, ligand binding elicits more widespread changes, as evidenced by enhancement of the NEM reactivity of Ala244-->Cys, Thr248-->Cys (helix VII), Thr265-->Cys (helix VIII), Val315-->Cys (helix X), Gln359-->Cys, or Met362-->Cys (helix XI) permease, none of which are altered by 4B1 binding. Furthermore, no effect of 4B1 is observed on the reactivity of Cys148 (helix V), Val264-->Cys, Gly268-->Cys, or Asn272-->Cys (helix VIII), positions which probably make direct contact with substrate. With respect...Continue Reading

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Citations

Jul 1, 1997·Protein Science : a Publication of the Protein Society·J SunH R Kaback
Aug 11, 2010·Proceedings of the National Academy of Sciences of the United States of America·Mikhail BogdanovWilliam Dowhan
Sep 13, 2012·The Journal of Biological Chemistry·Mikhail Bogdanov, William Dowhan
Feb 12, 1998·Proceedings of the National Academy of Sciences of the United States of America·Y J SeokA Peterkofsky
Dec 18, 2007·Journal of Molecular Biology·Alexej KedrovDaniel J Muller
Sep 29, 2011·Biochimica Et Biophysica Acta·William Dowhan, Mikhail Bogdanov
Aug 22, 2001·Biochimica Et Biophysica Acta·E Nachliel, M Gutman
Apr 3, 2019·The Protein Journal·William DowhanMikhail Bogdanov
Oct 8, 1998·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·S FrillingosH R Kaback

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