The Cys154→Gly Mutation in LacY Causes Constitutive Opening of the Hydrophilic Periplasmic Pathway

Abstract

The lactose permease of Escherichia coli (LacY) is a highly dynamic membrane transport protein, while the Cys154→Gly mutant is crippled conformationally. The mutant binds sugar with high affinity, but catalyzes very little translocation across the membrane. In order to further investigate the defect in the mutant, fluorescent maleimides were used to examine the accessibility/reactivity of single-Cys LacY in right-side-out membrane vesicles. As shown previously, sugar binding induces an increase in reactivity of single-Cys replacements in the tightly packed periplasmic domain of wildtype LacY, while decreased reactivity is observed on the cytoplasmic side. Thus, the predominant population of wild-type LacY in the membrane is in an inward-facing conformation in the absence of sugar, sugar binding induces opening of a hydrophilic pathway on the periplasmic side, and the sugar-binding site is alternatively accessible to either side of the membrane. In striking contrast, the accessibility/reactivity of periplasmic Cys replacements in the Cys154→Gly background is very high in the absence of sugar, and sugar binding has little or no effect. The observations indicate that an open hydrophilic pathway is present on the periplasmic side of the Cys154→Gly mutant and that this pathway is unaffected by ligand binding, a conclusion consistent with findings obtained from single-molecule fluorescence and double electron-electron resonance.

Role of the irreplaceable residues in the LacY alternating access mechanism

“…RSO membrane vesicles with a given singleCys mutant and tagged with 10-His residues at the C terminus were incubated with tetramethylrhodamine-5-maleimide (TMRM) in the absence or presence of p-nitrophenyl-α-D-galactopyranoside (αNPG). The membranes were then solubilized, and LacY was purified and subjected to SDS/PAGE (14,16,17). The gels were scanned for fluorescence and silver-stained for protein.…”

“…Labeling experiments were carried out following a modified procedure described previously (14,26). RSO membrane vesicles [0.1 mg total protein in 50 μL 100 mM KPi (pH 7.5)/10 mM MgSO 4 ] containing a specified single-Cys replacement without or with an additional mutation in an irreplaceable residue were incubated with 40 μM TMRM in the absence or presence of 10 mM αNPG at 0°C.…”

Trp replacements for tightly interacting Gly–Gly pairs in LacY stabilize an outward-facing conformation

“…Therefore, opening of the periplasmic cavity in the Gly→Trp mutants was examined by two approaches: site-directed alkylation of a Cys replacement for Asn245 and pre-steady state kinetics of sugar binding with protein solubilized in DDM or reconstituted into proteoliposomes. With respect to site-directed alkylation, it is well documented that position 245 is one of several positions on the periplasmic side of LacY at which Cys replacements exhibit very low reactivity/accessibility in the absence of bound sugar and a dramatic increase in reactivity/accessibility on sugar binding (11)(12)(13)(14)(15)59). Because a six-to 10-fold increase in reactivity/accessibility of Cys replacements on the sealed periplasmic side is mirrored by a six-to 10-fold decrease in reactivity/accessibility of Cys replacements on the open cytoplasmic side of LacY (14), an alternating access mechanism is likely central to the transport process.…”

“…In this conformation, the periplasmic side is tightly sealed and the sugar-binding site is inaccessible from the periplasm. However, a wealth of biophysical and spectroscopic data, which include site-directed alkylation (11)(12)(13)(14)(15)(16), single-molecule fluorescence resonance energy transfer (FRET) (17), double electron-electron resonance (18), site-directed cross-linking (19) and Trp fluorescence studies (20)(21)(22), provide converging evidence that the sugar-and H + -binding sites in LacY are alternatively accessible from either side of the membrane (the alternating access model, reviewed in refs. 6 and 23).…”

Electrophysiological characterization of LacY

“…In the absence of substrate, this mutant is in an inward-facing conformation, and paralyzed in an open conformation on the periplasmic side, but able to close on the cytoplasmic side on lactose binding (6)(7)(8)39). E325A LacY exhibits no active transport whatsoever; however, in contrast to C154G LacY, mutant E325A catalyzes exchange and counterflow at least as well as wild-type LacY, indicating that this mutant is permanently protonated (i.e., H ϩ dissociation is blocked in E325A LacY but binding is normal) (24,25,28).…”