An Asymmetric Conformational Change in LacY

作者：Smirnova Irina; Kasho Vladimir; Jiang Xiaoxu; Kaback H Ronald^*

来源：Biochemistry, 2017, 56(13): 1943-1950.

DOI：10.1021/acs.biochem.7b00134

Galactoside/H+ syinport by the lactose permease of Escherichia coli (Lacy) involves reciprocal opening and closing of periplasmic and cytoplasmic cavities so that sugar- and H+- binding sites become alternatively accessible to either side of the membrane. After reconstitution into proteoliposomes, LacY with the periplasmic cavity sealed by cross-linking paired-Cys residues does not bind sugar from the periplasmic side. However, reduction of the bond restores opening of the periplasmic cavity and galactoside binding. Furthermcire, nanobodies that stabilize the double-Cys mutant in a periplasmic-open con formation and allow free access of galactoside to the binding site do so only after reduction of the S-S bond: In contrast, when cross-linked LacY is solubilized in.detergent, galactoside binding is observed, indicating- that the cytoplasmic cavity is patent. Sugar binding from the cytoplasmic side exhibits nonlinear stopped-flow kinetics, and analysis reveals a two-step process in which a conformational change precedes binding. Because the cytoplasmic cavity is spontaneously closing and opening in the symporter with a sealed periplasmic cavity, it is apparent that an asymmetrical conformational transition controls access of sugar to the binding site.

• 出版日期2017-4-4