Helix dynamics in LacY: helices II/IV

Biochemical and biophysical studies based upon crystal structures of both a mutant and wild-type lactose permease from Escherichia coli (LacY) in an inward-facing conformation have led to a model for the symport mechanism in which both sugarand H+-binding sites are alternatively accessible to either side of the membrane. Previous findings indicate that the face of helix II with Asp68 is important for the conformational changes that occur during turnover. As shown here, replacement of Asp68 at the cytoplasmic end of helix II, particularly with Glu, abolishes active transport, but the mutants retain the ability to bind galactopyranoside. In the x-ray structure, Asp68 and Lys131 (helix IV) lie within ∼4.2 Å of each other. Although a double mutant with Cys replacements at both position 68 and 131 cross-links efficiently, single replacements for Lys131 exhibit very significant transport activity. Site-directed alkylation studies show that sugar binding by the Asp68 mutants causes closure of the cytoplasmic cavity, like wild-type LacY; but strikingly, the probability of opening the periplasmic pathway upon sugar binding is markedly reduced. Taken together with previous mutagenesis and cross-linking studies, the findings lead to a model in which replacement of Asp68 blocks a conformational transition involving helices II and IV that is important for opening the periplasmic cavity. Evidence is also presented suggesting that movements of helices II and IV are coupled functionally with movements in the pseudosymmetrically paired helices VIII and X.