Role of the irreplaceable residues in the LacY alternating access mechanism

Abstract

Few side chains in the galactoside/H + symporter LacY (lactose permease of Escherichia coli) are irreplaceable for an alternating access mechanism in which sugar binding induces closing of the cytoplasmic cavity and reciprocal opening of a periplasmic cavity. In this study, each irreplaceable residue was mutated individually, and galactoside-induced opening or closing of periplasmic or cytoplasmic cavities was probed by site-directed alkylation. Mutation of Glu126 (helix IV) or Arg144 (helix V), which are essential for sugar binding, completely blocks sugar-induced periplasmic opening as expected. Remarkably, however, replacement of Glu269 (helix VIII), His322 (helix X), or Tyr236 (helix VII) causes spontaneous opening of the periplasmic cavity in the absence of sugar and decreased closing of the cytoplasmic cavity in the presence of galactoside. In contrast, mutation of Arg302 (helix IX) or Glu325 (helix X) has no such effect, and sugar binding induces normal opening and closing of periplasmic and cytoplasmic cavities. Possibly, Glu269, His322, and Tyr236 act in concert to coordinate opening and closing of the cavities by binding water, which also acts as a cofactor in H + translocation. Mutation of the triad results in loss of the bound water, which destabilizes LacY, and the cavities open and close in an uncoordinated manner. Thus, the triad mutants exhibit poor affinity for sugar, and galactoside/H + symport is abolished as well.membrane protein | membranes | protein dynamics | transport T he lactose permease of Escherichia coli (LacY), a member of the major facilitator superfamily of membrane transport proteins, catalyzes the coupled translocation of a galactoside and an H + (i.e., galactoside/H + symport). Thus, LacY transduces free energy stored in an H + electrochemical gradient (Δμ H +) into a galactoside concentration gradient. As transport is obligatorily coupled, LacY also transduces free energy stored in a sugar concentration gradient into Δμ H +, the polarity of which depends on the direction of the sugar gradient (reviewed in ref. 1). LacY has been solubilized from the membrane, purified to homogeneity in a highly functional state (2), and is structurally (3, 4), as well as functionally (5), a monomer.X-ray crystal structures of LacY (6-9) and various independent biochemical and spectroscopic findings (10-17) provide converging evidence for an alternating access mechanism. By this means, sugar binding induces coordinated opening and closing of periplasmic and cytoplasmic cavities, respectively, thereby allowing accessibility of sugar-and H + -binding sites to either side of the membrane (i.e., the alternating access model; reviewed in refs. 18, 19). A similar model has been proposed for a number of other transporters (e.g., refs. 20-24). It is also likely that the alternating access model for LacY involves formation of an occluded intermediate (25,26), which is consistent with the highly dynamic nature of the protein (15, 27-31).Cys-scanning and site-directed mutagenesis of each residue in LacY de...

Function, Structure, and Evolution of the Major Facilitator Superfamily: The LacY Manifesto

“…Although earlier studies suggested by implication that Tyr236, Glu269, and His322 may be involved in H + transport, it is now apparent that these side chains are clearly ligands to the galactopyranoside ring [19]. Although mutation of these residues causes the periplasmic side of LacY to open spontaneously [136], there is no direct evidence that they are involved in H + translocation, although it is conceivable that one or more may play a dual role in the transport mechanism. It is also notable that every residue in LacY has been subjected to site-directed mutagenesis, and, with the exception of the irreplaceable residues described, each of which has a known function, there are no other side chains in the protein which are absolutely required for lactose/H + symport.…”

Structure of sugar-bound LacY

“…1 and 2), are clearly ligands to the galactopyranoside. Although mutation of these residues causes the periplasmic side of LacY to open spontaneously (53), there is no direct evidence that they are involved in H + translocation, although it is conceivable that they may play a dual role in the transport mechanism.…”

Evolutionary mix-and-match with MFS transporters II

“…Although His322 is clearly involved in affinity for galactosides, it has been suggested that it is also involved in H + translocation (54). Moreover, the notion has been put forward that structural water coordinated within a triad between His322, Tyr236, and Glu269 (two additional irreplaceable residues) may act as a cofactor for binding and galactoside/H + symport by forming a hydronium ion intermediate during turnover (54,55). In this regard, an analogous situation is apparent in PepT Gk where Glu35, which corresponds to His322 in LacY, is not a direct substrate ligand.…”