Fourier Transform Infrared Analysis of Purified Lactose Permease:  A Monodisperse Lactose Permease Preparation Is Stably Folded, α-Helical, and Highly Accessible to Deuterium Exchange

156

234

According to x-ray structure, the lactose permease (LacY) is a monomer organized into N-and C-terminal six-helix bundles that form a deep internal cavity open on the cytoplasmic side with a single sugar-binding site at the apex. The periplasmic side of the molecule is closed. During sugar/H ؉ symport, a cavity facing the periplasmic side is thought to open with closure of the inwardfacing cytoplasmic cavity so that the sugar-binding site is alternately accessible to either face of the membrane. Double electronelectron resonance (DEER) is used here to measure interhelical distance changes induced by sugar binding to LacY. Nitroxidelabeled paired-Cys replacements were constructed at the ends of transmembrane helices on the cytoplasmic or periplasmic sides of wild-type LacY and in the conformationally restricted mutant Cys-1543 Gly. Distances were then determined in the presence of galactosidic or nongalactosidic sugars. Strikingly, specific binding causes conformational rearrangement on both sides of the molecule. On the cytoplasmic side, each of six nitroxide-labeled pairs exhibits decreased interspin distances ranging from 4 to 21 Å. Conversely, on the periplasmic side, each of three spin-labeled pairs shows increased distances ranging from 4 to 14 Å. Thus, the inward-facing cytoplasmic cavity closes, and a cleft opens on the tightly packed periplasmic side. In the Cys-1543 Gly mutant, sugarinduced closing is observed on the cytoplasmic face, but little or no change occurs on periplasmic side. The DEER measurements in conjunction with molecular modeling based on the x-ray structure provide strong support for the alternative access model and reveal a structure for the outward-facing conformer of LacY.conformational change ͉ double electron-electron resonance ͉ lactose permease ͉ major facilitator superfamily ͉ membrane transport T he lactose permease of Escherichia coli (LacY), a member of the major facilitator superfamily (MFS), utilizes free energy stored in an electrochemical H ϩ gradient (⌬˜Hϩ) to drive active transport by coupling the downhill, stoichiometric translocation of H ϩ with ⌬˜Hϩ to the uphill accumulation of galactopyranosides. In the absence of ⌬˜Hϩ, LacY can also use free energy released from downhill translocation of galactosides in either direction to drive uphill translocation of H ϩ with generation of ⌬˜Hϩ, the polarity of which depends on the direction of the sugar gradient (1, 2). An x-ray structure of LacY (3) combined with a wealth of biochemical data (1, 2, 4, 5) has led to an alternating access model for sugar translocation across the membrane. By this means, the inward-facing cytoplasmic cavity closes with opening of an outward-facing cleft, thereby making the sugar-binding site alternatively accessible to either face of the membrane. A similar model has been proposed for the glycerol phosphate/phosphate antiporter GlpT, a related MFS protein (6). The alternating access model involves a global conformational change, which is consistent with the highly dynamic nature of LacY (2, 7-9).A...

supporting

confidence: 67%

Sugar binding induces an outward facing conformation of LacY

Смирнова

Kasho

Choe

et al. 2007

156

234

“…A similar model has been proposed for the glycerol phosphate/phosphate antiporter GlpT, a related MFS protein (11), and the ABC transporter Sav 1866 (12). The alternating access model involves a global conformational change, which is consistent with the highly dynamic nature of LacY (2,(13)(14)(15)(16).…”

supporting

confidence: 54%

Opening and closing of the periplasmic gate in lactose permease

Zhou¹,

Guan²,

Freites

et al. 2008

105

X-ray crystal structures of lactose permease (LacY) reveal pseudosymmetrically arranged N-and C-terminal six-transmembrane helix bundles surrounding a deep internal cavity open on the cytoplasmic side and completely closed on the periplasmic side. The residues essential for sugar recognition and H ؉ translocation are located at the apex of the cavity and are inaccessible from the outside. On the periplasmic side, helices I/II and VII from the N-and C-six helix bundles, respectively, participate in sealing the cavity from the outside. Three paired double-Cys mutants-Ile-40 3 Cys/Asn-245 3 Cys, Thr-45 3 Cys/Asn-245 3 Cys, and Ile-32 3 Cys/Asn-245 3 Cys-located in the interface between helices I/II and VII on the periplasmic side of LacY were constructed. After cross-linking with homobifunctional reagents less than Ϸ15 Å in length, all three mutants lose the ability to catalyze lactose transport. Strikingly, however, full or partial activity is observed when cross-linking is mediated by flexible reagents greater than Ϸ15 Å in length. The results provide direct support for the argument that transport via LacY involves opening and closing of a large periplasmic cavity.cross-linking ͉ membrane proteins ͉ protein dynamics ͉ transport ͉ structure/function T he lactose permease of Escherichia coli (LacY) (1), a member of the major facilitator superfamily (MFS), transduces free energy in an electrochemical H ϩ gradient (⌬¯Hϩ) into a concentration gradient of galactopyranosides by coupling the downhill, stoichiometric translocation of H ϩ to the uphill accumulation of galactopyranosides. LacY can also use free energy released from downhill translocation of sugar in either direction to drive uphill translocation of H ϩ with generation of ⌬¯Hϩ, the polarity of which depends on the direction of the sugar gradient (1, 2).X-ray crystal structures of LacY (3-5) combined with a wealth of biochemical and biophysical data (2, 6 -10) have led to an alternating access mechanism for sugar translocation across the membrane. By this means, the inward-facing cavity closes with opening of an outward-facing cavity, thereby allowing alternative accessibility of the sugar-binding site to either face of the membrane. A similar model has been proposed for the glycerol phosphate/phosphate antiporter GlpT, a related MFS protein (11), and the ABC transporter Sav 1866 (12). The alternating access model involves a global conformational change, which is consistent with the highly dynamic nature of LacY (2, 13-16).Although it may seem intuitively obvious that a pathway must open on the periplasmic side of LacY to allow access of sugar to the binding site, insight is cloudy in this respect because all x-ray structures of LacY (3-5) are in the same inward-facing conformation with an open cytoplasmic cavity and a tightly closed periplasmic side. Nonetheless, site-directed alkylation (see refs. 7 and 10), single-molecule fluorescence resonance energy transfer (8) and double electron-electron resonance studies (9) clearly indicate that an outward-facing c...

“…[20][21][22][23][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)(28)(29)(30)(31).…”

mentioning

confidence: 66%

Role of the irreplaceable residues in the LacY alternating access mechanism

Zhou¹,

Jiang²,

Kaback

2012

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...