Transient conformers of LacY are trapped by nanobodies

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

doi:10.1073/pnas.1519485112

Cited by 25 publications

(40 citation statements)

References 20 publications

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“…S2B). The involvement of residues from TM5 and TM11 in the cytoplasmic gate of other MFS transporters (32,(37)(38)(39)(40) is consistent with this proposed role.…”

Section: Discussionsupporting

confidence: 77%

Emulating proton-induced conformational changes in the vesicular monoamine transporter VMAT2 by mutagenesis

Yaffe

Vergara-Jaque

Forrest

et al. 2016

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

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Neurotransporters located in synaptic vesicles are essential for communication between nerve cells in a process mediated by neurotransmitters. Vesicular monoamine transporter (VMAT), a member of the largest superfamily of transporters, mediates transport of monoamines to synaptic vesicles and storage organelles in a process that involves exchange of two H + per substrate. VMAT transport is inhibited by the competitive inhibitor reserpine, a second-line agent to treat hypertension, and by the noncompetitive inhibitor tetrabenazine, presently in use for symptomatic treatment of hyperkinetic disorders. During the transport cycle, VMAT is expected to occupy at least three different conformations: cytoplasm-facing, occluded, and lumen-facing. The lumen-to cytoplasm-facing transition, facilitated by protonation of at least one of the essential membrane-embedded carboxyls, generates a binding site for reserpine. Here we have identified residues in the cytoplasmic gate and show that mutations that disrupt the interactions in this gate also shift the equilibrium toward the cytoplasm-facing conformation, emulating the effect of protonation. These experiments provide significant insight into the role of proton translocation in the conformational dynamics of a mammalian H + -coupled antiporter, and also identify key aspects of the mode of action and binding of two potent inhibitors of VMAT2: reserpine binds the cytoplasm-facing conformation, and tetrabenazine binds the lumen-facing conformation.neurotransmitter transporter | ion coupling | reserpine | tetrabenazine

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“…S2B). The involvement of residues from TM5 and TM11 in the cytoplasmic gate of other MFS transporters (32,(37)(38)(39)(40) is consistent with this proposed role.…”

Section: Discussionsupporting

confidence: 77%

Emulating proton-induced conformational changes in the vesicular monoamine transporter VMAT2 by mutagenesis

Yaffe

Vergara-Jaque

Forrest

et al. 2016

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%

“…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).…”

mentioning

confidence: 93%

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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“…However, sugar binding from the periplasmic side is rescued upon reduction of the S–S bond with TCEP (Figure 3D and Figure S2E). Furthermore, Nb9065, which stabilizes the periplasmic-open conformation of WT LacY, 16,23 has no effect on the cross-linked mutant (Figure 5B and Figures S1C and S2F). Remarkably, TCEP reduction completely restores the effect of the Nb on activity, and binding of NPG to the mutant–Nb9065 complex via an open periplasmic cavity is observed with a high k on value (Figures 4B and 5A) like that obtained with WT LacY upon Nb9065 binding (Figure S4F).…”

Section: Discussionmentioning

confidence: 99%

“…In addition, a number of camelid Nbs developed against the periplasmic-open mutant G46W/G262W LacY (Figure 1C) bind to the periplasmic aspect of WT LacY and stabilize outward-facing conformations with dramatically increased k on values indicating a highly accessible galactoside-binding site. 10,16,23 …”

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confidence: 99%

An Asymmetric Conformational Change in LacY

et al. 2017

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Galactoside/H+ symport by the lactose permease of Escherichia coli (LacY) involves reciprocal opening and closing of periplasmic and cytoplasmic cavities so that sugar- and H+-binding sites become alternatively accessible to either side of the membrane. After reconstitution into proteoliposomes, LacY with the periplasmic cavity sealed by cross-linking paired-Cys residues does not bind sugar from the periplasmic side. However, reduction of the S–S bond restores opening of the periplasmic cavity and galactoside binding. Furthermore, nanobodies that stabilize the double-Cys mutant in a periplasmic-open conformation and allow free access of galactoside to the binding site do so only after reduction of the S–S bond. In contrast, when cross-linked LacY is solubilized in detergent, galactoside binding is observed, indicating that the cytoplasmic cavity is patent. Sugar binding from the cytoplasmic side exhibits nonlinear stopped-flow kinetics, and analysis reveals a two-step process in which a conformational change precedes binding. Because the cytoplasmic cavity is spontaneously closing and opening in the symporter with a sealed periplasmic cavity, it is apparent that an asymmetrical conformational transition controls access of sugar to the binding site.

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Transient conformers of LacY are trapped by nanobodies

Cited by 25 publications

References 20 publications

Emulating proton-induced conformational changes in the vesicular monoamine transporter VMAT2 by mutagenesis

Emulating proton-induced conformational changes in the vesicular monoamine transporter VMAT2 by mutagenesis

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

An Asymmetric Conformational Change in LacY

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