Cysteine scanning mutagenesis of the N‐terminal 32 amino acid residues in the lactose permease of <i>Escherichia coli</i>

Sahin–Tóth, Miklós; Persson, Bengt L.; Schwieger, Jeremy; Kaback, H. Ronald

doi:10.1002/pro.5560030208

Cited by 43 publications

(43 citation statements)

References 42 publications

(47 reference statements)

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“…2A). Although Phe27 could act as a gate, this seems unlikely, as the F27C mutant exhibits normal transport activity (37,38). Moreover, the side chain of Phe27 is not present in the overall density map contoured at 1.0 σ, unlike surrounding residues with well-defined densities, indicating high mobility of the Phe27 side chain.…”

Section: Discussionmentioning

confidence: 99%

Crystal structure of a LacY–nanobody complex in a periplasmic-open conformation

Jiang

Смирнова

Kasho

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The lactose permease of Escherichia coli (LacY), a dynamic polytopic membrane protein, catalyzes galactoside-H + symport and operates by an alternating access mechanism that exhibits multiple conformations, the distribution of which is altered by sugar binding. We have developed single-domain camelid nanobodies (Nbs) against a mutant in an outward (periplasmic)-open conformation to stabilize this state of the protein. Here we describe an X-ray crystal structure of a complex between a double-Trp mutant (Gly46→Trp/Gly262→Trp) and an Nb in which free access to the sugar-binding site from the periplasmic cavity is observed. The structure confirms biochemical data indicating that the Nb binds stoichiometrically with nanomolar affinity to the periplasmic face of LacY primarily to the C-terminal six-helix bundle. The structure is novel because the pathway to the sugar-binding site is constricted and the central cavity containing the galactoside-binding site is empty. Although Phe27 narrows the periplasmic cavity, sugar is freely accessible to the binding site. Remarkably, the side chains directly involved in binding galactosides remain in the same position in the absence or presence of bound sugar.X-ray structure | bioenergetics | membrane transporter | fluorescence | kinetics T he lactose permease of Escherichia coli (LacY) catalyzes the coupled transport of a galactopyranoside and an H + (galactoside-H + symport) across the cytoplasmic membrane (reviewed in refs. 1-3). Therefore, in the presence of an H + electrochemical gradient (ΔμH+; interior negative and/or alkaline), galactopyranosides can be concentrated 50-to 100-fold over the external concentration (4, 5). However, due to the activity of β-galactosidase, no free lactose is found within the cell under physiological conditions. So why does E. coli need an active transport system for lactose? The answer lies in the kinetics, as the major effect of ΔμH+ is to decrease the K m for transport (6, 7). By increasing the kinetic efficiency of LacY, ΔμH+ enables the cell to assimilate lactose effectively even when the environmental concentration is low.Initial X-ray crystal structures of conformationally restricted LacY (8-10), as well as WT LacY (11) + and is the poster child for the major facilitator superfamily, the largest family of membrane transport proteins. A detailed mechanism has been postulated involving alternating access of sugar-and H + -binding sites to either side of the membrane that is driven by sugar binding and dissociation and independent of the H + electrochemical gradient, which acts kinetically. To characterize structural intermediates in the transport cycle, stable conformers are essential, and camelid single-domain nanobodies (Nbs) are particularly useful in this context. Described herein is a structure of a LacY-Nb complex in which access to the sugar-binding site from the periplasmic cavity is diffusion-limited.Author contributions: X.J., I.S., V.K., N.Y., and H.R.K. designed research; X.J., I.S., V.K., J.W., K.H., and M.K. performed research...

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Section: Discussionmentioning

confidence: 99%

Crystal structure of a LacY–nanobody complex in a periplasmic-open conformation

Jiang

Смирнова

Kasho

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…However, these hydrophobic interactions not only make no contribution to specificity, but individual mutation of the surrounding side chains in LacY has little or no effect on the ability of LacY to catalyze lactose/H + symport (21,49). Thus, they are not involved in the symport mechanism.…”

Section: Discussionmentioning

confidence: 99%

Structure of LacY with an α-substituted galactoside: Connecting the binding site to the protonation site

Johri

Finer-Moore

Kaback

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The X-ray crystal structure of a conformationally constrained mutant of the Escherichia coli lactose permease (the LacY double-Trp mutant Gly-46→Trp/Gly-262→Trp) with bound p-nitrophenyl-α-D-galactopyranoside (α-NPG), a high-affinity lactose analog, is described. With the exception of Glu-126 (helix IV), side chains Trp-151 (helix V), Glu-269 (helix VIII), Arg-144 (helix V), His-322 (helix X), and Asn-272 (helix VIII) interact directly with the galactopyranosyl ring of α-NPG to provide specificity, as indicated by biochemical studies and shown directly by X-ray crystallography. In contrast, are within van der Waals distance of the benzyl moiety of the analog and thereby increase binding affinity nonspecifically. Thus, the specificity of LacY for sugar is determined solely by side-chain interactions with the galactopyranosyl ring, whereas affinity is increased by nonspecific hydrophobic interactions with the anomeric substituent.X-ray structure | membrane protein | transport | major facilitator superfamily | conformational change T he lactose permease of Escherichia coli (LacY) binds and catalyzes the coupled stoichiometric transport of D-galactose or β-D-galactopyranosides and H + (galactoside/H + symport) but does not interact with glucopyranosides. Biochemical studies (1-7) indicate that affinity and specificity are distinct properties determined by different interactions with LacY. Specificity is determined entirely by interactions with the galactopyranosyl ring, whereas affinity is better with α-than β-galactopyranosides (anomeric at C1) and can be increased dramatically by hydrophobic anomeric substituents with no effect on specificity.By using the free energy released from the energetically downhill movement of H + in response to the electrochemical H + gradient (ΔμH+), LacY catalyzes uphill (active) transport of galactosides against a concentration gradient. Because coupling between sugar and H + translocation is obligatory, in the absence of ΔμH+, LacY can also transduce the free energy released from the downhill transport of sugar to drive uphill H + transport with the generation of ΔμH+, the polarity of which depends upon the direction of the sugar gradient (reviewed in refs. 8-10).Rates of equilibrium exchange and counterflow (exchange of one substrate molecule for another labeled molecule from the other side of the membrane) are unaffected by imposition of Δμ̃H+. Therefore, it is apparent that alternating accessibility of sugar-and H + -binding sites to either side of the membrane is the result of galactoside binding and dissociation and not ΔμH+ (reviewed in refs. 8-10). Moreover, downhill lactose/H + symport from a high to a low lactose concentration in the absence of ΔμH+ exhibits a primary deuterium isotope effect that is not observed for ΔμH+-driven lactose/H + symport, equilibrium exchange, or counterflow (11, 12). Thus, it is likely that the rate-limiting step for downhill symport is deprotonation (13,14), whereas in the presence of ΔμH+, opening of a cavity on the other side of the membrane ...

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“…DNA fragments encoding given single-Cys mutants were isolated from plasmids in the library of single-Cys mutations [22][23][24][25][26] by restriction enzyme digestion and inserted into plasmid pT7-5 encoding Cys-less LacY with either the C154Vor C154G mutation and a biotin acceptor domain (BAD) from a Klebsiella pneumoniae oxaloacetate decarboxylase at the C terminus. 27 All constructs were sequenced for the full length of lacY genes.…”

Section: Plasmid Constructionmentioning

confidence: 99%

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

Nie

Sabetfard

Kaback

2008

Journal of Molecular Biology

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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.

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Cysteine scanning mutagenesis of the N‐terminal 32 amino acid residues in the lactose permease of Escherichia coli

Cited by 43 publications

References 42 publications

Crystal structure of a LacY–nanobody complex in a periplasmic-open conformation

Crystal structure of a LacY–nanobody complex in a periplasmic-open conformation

Structure of LacY with an α-substituted galactoside: Connecting the binding site to the protonation site

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

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