Outward-facing conformers of LacY stabilized by nanobodies

Смирнова, И. Н.; Kasho, Vladimir N.; Jiang, Xiaoxu; Pardon, Els; Steyaert, Jan; Kaback, H. Ronald

doi:10.1073/pnas.1422265112

Cited by 22 publications

(69 citation statements)

References 41 publications

(47 reference statements)

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“…Recently, we reported development of Nbs that specifically bind to the periplasmic side of LacY and trap outward-open conformation(s) (15). The Nbs were prepared against the double-Trp mutant of LacY G46W/G262W reconstituted into proteoliposomes, where the protein is oriented with periplasmic side out, as in the bacterial membrane, so that the periplasmic epitopes are exposed.…”

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confidence: 99%

Transient conformers of LacY are trapped by nanobodies

Смирнова

Kasho

Jiang

et al. 2015

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

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The lactose permease of Escherichia coli (LacY), a highly dynamic membrane protein, catalyzes symport of a galactopyranoside and an H+ by using an alternating access mechanism, and the transport cycle involves multiple conformational states. Single-domain camelid nanobodies (Nbs) developed against a LacY mutant immobilized in an outward (periplasmic)-open conformation bind to the flexible WT protein and stabilize the open-outward conformation(s). Here, we use site-directed, distance-dependent Trp quenching/unquenching of fluorescent probes inserted on opposite surfaces of LacY to assess the conformational states of the protein complexed with each of eight unique Nbs that bind exclusively to the periplasmic side and block transport, but increase the accessibility of the sugar-binding site. Nb binding involves conformational selection of LacY molecules with exposed binding epitopes. Each of eight Nbs induces quenching with three pairs of cytoplasmic Trp/fluorophore probes, indicating closing of cytoplasmic cavity. In reciprocal fashion, the same Nbs induce unquenching of fluorescence in three pairs of periplasmic probes due to opening of the periplasmic cavity. Because the extent of fluorescence change with various Nbs differs and the differences correlate with changes in the rate of sugar binding, it is also concluded that the Nbs stabilize several different outward-open conformations of LacY.

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confidence: 99%

Transient conformers of LacY are trapped by nanobodies

Смирнова

Kasho

Jiang

et al. 2015

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

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“…The Nbs, 9039 in particular, bind to LacY ww with 1:1 stoichiometry, which is apparent from the X-ray structure of the complex ( Fig. 1) as well as from biochemical assays (30,31). The interface is formed mainly by the variable regions of the Nb and periplasmic aspect of the apo LacY ww C-terminal domain ( Fig.…”

Section: Resultsmentioning

confidence: 92%

“…Discovery of Nb 9039 against G46W/G262W LacY mutant was published previously (30). Expression and purification of Nb 9039 is described in detail in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

“…Production of Nb 9039 and purification and reconstitution of LacY were carried out as described previously (30). The LacY-Nb 9039 complex was formed by mixing 20 to 30 μM LacY with 1.5 to 2 molar excess of Nb for 30 min at room temperature in 50 mM NaP i (pH 7.5) containing 0.02% DDM or, in the case of reconstituted LacY, in the absence of DDM.…”

Section: Methodsmentioning

confidence: 99%

“…Nbs are small in size and have a unique structure that allows flexible loops with complementarity-determining regions (CDRs) to insert into nooks and crannies. Nbs developed against the periplasmic-open conformation of LacY ww have been shown to block transport activity in WT LacY by stabilizing various outward-open conformations (30,31). Here we report an X-ray crystal structure of a LacY ww -Nb 9039 complex in the absence of sugar (apo LacY ww -Nb 9039).…”

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confidence: 93%

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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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Plant glucose transporter structure and function

Geiger

2020

Pflugers Arch - Eur J Physiol

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The carbohydrate D-glucose is the main source of energy in living organisms. In contrast to animals, as well as most fungi, bacteria, and archaea, plants are capable to synthesize a surplus of sugars characterizing them as autothrophic organisms. Thus, plants are de facto the source of all food on earth, either directly or indirectly via feed to livestock. Glucose is stored as polymeric glucan, in animals as glycogen and in plants as starch. Despite serving a general source for metabolic energy and energy storage, glucose is the main building block for cellulose synthesis and represents the metabolic starting point of carboxylate- and amino acid synthesis. Finally yet importantly, glucose functions as signalling molecule conveying the plant metabolic status for adjustment of growth, development, and survival. Therefore, cell-to-cell and long-distance transport of photoassimilates/sugars throughout the plant body require the fine-tuned activity of sugar transporters facilitating the transport across membranes. The functional plant counterparts of the animal sodium/glucose transporters (SGLTs) are represented by the proton-coupled sugar transport proteins (STPs) of the plant monosaccharide transporter(-like) family (MST). In the framework of this special issue on “Glucose Transporters in Health and Disease,” this review gives an overview of the function and structure of plant STPs in comparison to the respective knowledge obtained with the animal Na+-coupled glucose transporters (SGLTs).

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Outward-facing conformers of LacY stabilized by nanobodies

Cited by 22 publications

References 41 publications

Transient conformers of LacY are trapped by nanobodies

Transient conformers of LacY are trapped by nanobodies

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

Plant glucose transporter structure and function

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