Transient conformers of LacY are trapped by nanobodies

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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Engineered occluded apo-intermediate of LacY

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1816267115 ◽

2018 ◽

Vol 115 (50) ◽

pp. 12716-12721 ◽

Cited By ~ 1

Author(s):

Irina Smirnova ◽

Vladimir Kasho ◽

H. Ronald Kaback

Keyword(s):

Binding Site ◽

Lactose Permease ◽

Binding Studies ◽

X Ray Crystallography ◽

Sugar Binding ◽

Transport Cycle ◽

Open Structures ◽

Unrestricted Access ◽

Tight Association ◽

Alternating Access

The lactose permease of Escherichia coli (LacY) utilizes an alternating access symport mechanism with multiple conformational intermediates, but only inward (cytoplasmic)- or outward (periplasmic)-open structures have been characterized by X-ray crystallography. It is demonstrated here with sugar-binding studies that cross-linking paired-Cys replacements across the closed cytoplasmic cavity stabilize an occluded conformer with an inaccessible sugar-binding site. In addition, a nanobody (Nb) that stabilizes a periplasmic-open conformer with an easily accessible sugar-binding site in WT LacY fails to cause the cytoplasmic cross-linked mutants to become accessible to galactoside, showing that the periplasmic cavity is closed. These results are consistent with tight association of the periplasmic ends in two pairs of helices containing clusters of small residues in the packing interface between N- and C-terminal six-helix bundles of the symporter. However, after reduction of the disulfide bond, the Nb markedly increases the rate of galactoside binding, indicating unrestricted access to the Nb epitope and the galactoside-binding site from the periplasm. The findings indicate that the cross-linked cytoplasmic double-Cys mutants resemble an occluded apo-intermediate in the transport cycle.

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Crystal Structure of a ligand-bound LacY–Nanobody Complex

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1801774115 ◽

2018 ◽

Vol 115 (35) ◽

pp. 8769-8774 ◽

Cited By ~ 12

Author(s):

Hemant Kumar ◽

Janet S. Finer-Moore ◽

Xiaoxu Jiang ◽

Irina Smirnova ◽

Vladimir Kasho ◽

...

Keyword(s):

Crystal Structure ◽

Lactose Permease ◽

Transmembrane Helices ◽

Helical Bundle ◽

Flexible Loop ◽

Sugar Binding ◽

Open Conformation ◽

Substrate Analog ◽

Multiple Conformations ◽

Alternating Access

The lactose permease of Escherichia coli (LacY), a dynamic polytopic membrane transport 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. Camelid nanobodies were made against a double-mutant Gly46 → Trp/Gly262 → Trp (LacYWW) that produces an outward-open conformation, as opposed to the cytoplasmic open-state crystal structure of WT LacY. Nanobody 9047 (Nb9047) stabilizes WT LacY in a periplasmic-open conformation. Here, we describe the X-ray crystal structure of a complex between LacYWW, the high-affinity substrate analog 4-nitrophenyl-α-d-galactoside (NPG), and Nb9047 at 3-Å resolution. The present crystal structure demonstrates that Nb9047 binds to the periplasmic face of LacY, primarily to the C-terminal six-helical bundle, while a flexible loop of the Nb forms a bridge between the N- and C-terminal halves of LacY across the periplasmic vestibule. The bound Nb partially covers the vestibule, yet does not affect the on-rates or off-rates for the substrate binding to LacYWW, which implicates dynamic flexibility of the Nb–LacYWW complex. Nb9047-binding neither changes the overall structure of LacYWW with bound NPG, nor the positions of side chains comprising the galactoside-binding site. The current NPG-bound structure exhibits a more occluded periplasmic vestibule than seen in a previous structure of a (different Nb) apo-LacYWW/Nb9039 complex that we argue is caused by sugar-binding, with major differences located at the periplasmic ends of transmembrane helices in the N-terminal half of LacY.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1615414113 ◽

2016 ◽

Vol 113 (44) ◽

pp. 12420-12425 ◽

Cited By ~ 28

Author(s):

Xin Jiang ◽

Irina Smirnova ◽

Vladimir Kasho ◽

Jianping Wu ◽

Kunio Hirata ◽

...

Keyword(s):

Crystal Structure ◽

Binding Site ◽

Free Access ◽

Lactose Permease ◽

Sugar Binding ◽

Open Conformation ◽

Trp Mutant ◽

Multiple Conformations ◽

Polytopic Membrane Protein ◽

Alternating Access

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.

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The lactose permease of Escherichia coli : overall structure, the sugar-binding site and the alternating access model for transport

FEBS Letters ◽

10.1016/s0014-5793(03)01087-1 ◽

2003 ◽

Vol 555 (1) ◽

pp. 96-101 ◽

Cited By ~ 64

Author(s):

Jeff Abramson ◽

Irina Smirnova ◽

Vladimir Kasho ◽

Gillian Verner ◽

So Iwata ◽

...

Keyword(s):

Escherichia Coli ◽

Binding Site ◽

Lactose Permease ◽

Sugar Binding ◽

Alternating Access ◽

Access Model

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Crystal structures of an E1–E2–ubiquitin thioester mimetic reveal molecular mechanisms of transthioesterification

Nature Communications ◽

10.1038/s41467-021-22598-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Lingmin Yuan ◽

Zongyang Lv ◽

Melanie J. Adams ◽

Shaun K. Olsen

Keyword(s):

Complex Formation ◽

Crystal Structures ◽

Molecular Mechanisms ◽

Conformational State ◽

Biochemical Data ◽

Conformational States ◽

Product Release ◽

Closed Conformation ◽

Open Conformation ◽

Conjugating Enzymes

AbstractE1 enzymes function as gatekeepers of ubiquitin (Ub) signaling by catalyzing activation and transfer of Ub to tens of cognate E2 conjugating enzymes in a process called E1–E2 transthioesterification. The molecular mechanisms of transthioesterification and the overall architecture of the E1–E2–Ub complex during catalysis are unknown. Here, we determine the structure of a covalently trapped E1–E2–ubiquitin thioester mimetic. Two distinct architectures of the complex are observed, one in which the Ub thioester (Ub(t)) contacts E1 in an open conformation and another in which Ub(t) instead contacts E2 in a drastically different, closed conformation. Altogether our structural and biochemical data suggest that these two conformational states represent snapshots of the E1–E2–Ub complex pre- and post-thioester transfer, and are consistent with a model in which catalysis is enhanced by a Ub(t)-mediated affinity switch that drives the reaction forward by promoting productive complex formation or product release depending on the conformational state.

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NMR Studies Of complexes of L. Casei Dihydrofolate Reductase with Antifolate Drugs: Multiple Conformations and Conformational Selection of Bound Rotational Isomers

Bioorganic Chemistry in Healthcare and Technology ◽

10.1007/978-1-4684-1354-0_9 ◽

1991 ◽

pp. 109-120

Author(s):

J. Feeney

Keyword(s):

Dihydrofolate Reductase ◽

Nmr Studies ◽

Conformational Selection ◽

Rotational Isomers ◽

Multiple Conformations ◽

Antifolate Drugs ◽

Selection Of

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New open conformation of SMYD3 implicates conformational selection and allostery

AIMS Biophysics ◽

10.3934/biophy.2017.1.1 ◽

2016 ◽

Vol 4 (1) ◽

pp. 1-18 ◽

Cited By ~ 4

Author(s):

Nicholas Spellmon ◽

◽

Xiaonan Sun ◽

Wen Xue ◽

Joshua Holcomb ◽

...

Keyword(s):

Conformational Selection ◽

Open Conformation

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Oversized galactosides as a probe for conformational dynamics in LacY

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1800706115 ◽

2018 ◽

Vol 115 (16) ◽

pp. 4146-4151 ◽

Cited By ~ 3

Author(s):

Irina Smirnova ◽

Vladimir Kasho ◽

Xiaoxu Jiang ◽

Hong-Ming Chen ◽

Stephen G. Withers ◽

...

Keyword(s):

Binding Site ◽

Conformational Dynamics ◽

Dissociation Rate ◽

Binding Kinetics ◽

Lactose Permease ◽

E Coli ◽

Open Conformation ◽

Dissociation Rate Constants ◽

Kinetics Of ◽

Micromolar Range

Binding kinetics of α-galactopyranoside homologs with fluorescent aglycones of different sizes and shapes were determined with the lactose permease (LacY) of Escherichia coli by FRET from Trp151 in the binding site of LacY to the fluorophores. Fast binding was observed with LacY stabilized in an outward-open conformation (kon = 4–20 μM−1·s−1), indicating unobstructed access to the binding site even for ligands that are much larger than lactose. Dissociation rate constants (koff) increase with the size of the aglycone so that Kd values also increase but remain in the micromolar range for each homolog. Phe27 (helix I) forms an apparent constriction in the pathway for sugar by protruding into the periplasmic cavity. However, replacement of Phe27 with a bulkier Trp does not create an obstacle in the pathway even for large ligands, since binding kinetics remain unchanged. High accessibility of the binding site is also observed in a LacY/nanobody complex with partially blocked periplasmic opening. Remarkably, E. coli expressing WT LacY catalyzes transport of α- or β-galactopyranosides with oversized aglycones such as bodipy or Aldol518, which may require an extra space within the occluded intermediate. The results confirm that LacY specificity is strictly directed toward the galactopyranoside ring and also clearly indicate that the opening on the periplasmic side is sufficiently wide to accommodate the large galactoside derivatives tested here. We conclude that the actual pathway for the substrate entering from the periplasmic side is wider than the pore diameter calculated in the periplasmic-open X-ray structures.

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