scholarly journals Engineered occluded apo-intermediate of LacY

2018 ◽  
Vol 115 (50) ◽  
pp. 12716-12721 ◽  
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.

scholarly journals Transient conformers of LacY are trapped by nanobodies

2015 ◽  
Vol 112 (45) ◽  
pp. 13839-13844 ◽  
Author(s):  
Irina Smirnova ◽  
Vladimir Kasho ◽  
Xiaoxu Jiang ◽  
Els Pardon ◽  
Jan Steyaert ◽  
...  
Keyword(s):  
Lactose Permease ◽  
Conformational States ◽  
Dynamic Membrane ◽  
Conformational Selection ◽  
Sugar Binding ◽  
Open Conformation ◽  
Transport Cycle ◽  
Fluorescence Change ◽  
Alternating Access ◽  
Selection Of

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.

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

2016 ◽  
Vol 113 (44) ◽  
pp. 12420-12425 ◽  
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.

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

FEBS Letters ◽  
2003 ◽  
Vol 555 (1) ◽  
pp. 96-101 ◽  
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

scholarly journals Crystal Structure of a ligand-bound LacY–Nanobody Complex

2018 ◽  
Vol 115 (35) ◽  
pp. 8769-8774 ◽  
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.

scholarly journals 1a/1b Subtype Profiling of Nonnucleoside Polymerase Inhibitors of Hepatitis C Virus

2010 ◽  
Vol 84 (6) ◽  
pp. 2923-2934 ◽  
Author(s):  
Origène Nyanguile ◽  
Benoit Devogelaere ◽  
Leen Vijgen ◽  
Walter Van den Broeck ◽  
Frederik Pauwels ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Binding Site ◽  
Reverse Genetics ◽  
Bound Water ◽  
Clinical Isolates ◽  
Binding Studies ◽  
Hydrophobic Contact ◽  
X Ray Crystallography ◽  
Polymerase Inhibitors

ABSTRACT The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is an unusually attractive target for drug discovery since it contains five distinct drugable sites. The success of novel antiviral therapies will require nonnucleoside inhibitors to be active in at least patients infected with HCV of subtypes 1a and 1b. Therefore, the genotypic assessment of these agents against clinical isolates derived from genotype 1-infected patients is an important prerequisite for the selection of suitable candidates for clinical development. Here we report the 1a/1b subtype profiling of polymerase inhibitors that bind at each of the four known nonnucleoside binding sites. We show that inhibition of all of the clinical isolates tested is maintained, except for inhibitors that bind at the palm-1 binding site. Subtype coverage varies across chemotypes within this class of inhibitors, and inhibition of genotype 1a improves when hydrophobic contact with the polymerase is increased. We investigated if the polymorphism of the palm-1 binding site is the sole cause of the reduced susceptibility of subtype 1a to inhibition by 1,5-benzodiazepines by using reverse genetics, X-ray crystallography, and surface plasmon resonance studies. We showed Y415F to be a key determinant in conferring resistance on subtype 1a, with this effect being mediated through an inhibitor- and enzyme-bound water molecule. Binding studies revealed that the mechanism of subtype 1a resistance is faster dissociation of the inhibitor from the enzyme.

Eudicot plant-specific sphingolipids determine host selectivity of microbial NLP cytolysins

Science ◽  
2017 ◽  
Vol 358 (6369) ◽  
pp. 1431-1434 ◽  
Author(s):  
Tea Lenarčič ◽  
Isabell Albert ◽  
Hannah Böhm ◽  
Vesna Hodnik ◽  
Katja Pirc ◽  
...  
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Pathogenic Bacteria ◽  
Head Group ◽  
Microbial Infection ◽  
Binding Studies ◽  
X Ray ◽  
Plant Pathogenic Bacteria ◽  
X Ray Crystallography ◽  
Host Selectivity

Necrosis and ethylene-inducing peptide 1–like (NLP) proteins constitute a superfamily of proteins produced by plant pathogenic bacteria, fungi, and oomycetes. Many NLPs are cytotoxins that facilitate microbial infection of eudicot, but not of monocot plants. Here, we report glycosylinositol phosphorylceramide (GIPC) sphingolipids as NLP toxin receptors. Plant mutants with altered GIPC composition were more resistant to NLP toxins. Binding studies and x-ray crystallography showed that NLPs form complexes with terminal monomeric hexose moieties of GIPCs that result in conformational changes within the toxin. Insensitivity to NLP cytolysins of monocot plants may be explained by the length of the GIPC head group and the architecture of the NLP sugar-binding site. We unveil early steps in NLP cytolysin action that determine plant clade-specific toxin selectivity.

Aromatic Stacking in the Sugar Binding Site of the Lactose Permease†

Biochemistry ◽  
2003 ◽  
Vol 42 (6) ◽  
pp. 1377-1382 ◽  
Author(s):  
Lan Guan ◽  
Yonglin Hu ◽  
H. Ronald Kaback
Keyword(s):  
Binding Site ◽  
Lactose Permease ◽  
Aromatic Stacking ◽  
Sugar Binding

scholarly journals Function, Structure, and Evolution of the Major Facilitator Superfamily: The LacY Manifesto

2014 ◽  
Vol 2014 ◽  
pp. 1-20 ◽  
Author(s):  
M. Gregor Madej
Keyword(s):  
Structural Information ◽  
Large Body ◽  
Major Facilitator Superfamily ◽  
Lactose Permease ◽  
Test Bed ◽  
X Ray ◽  
X Ray Crystallography ◽  
Major Facilitator ◽  
Mfs Transporters ◽  
Alternating Access

The major facilitator superfamily (MFS) is a diverse group of secondary transporters with members found in all kingdoms of life. A paradigm for MFS is the lactose permease (LacY) of Escherichia coli, which couples the stoichiometric translocation of a galactopyranoside and an H+ across the cytoplasmic membrane. LacY has been the test bed for the development of many methods applied for the analysis of transport proteins. X-ray structures of an inward-facing conformation and the most recent structure of an almost occluded conformation confirm many conclusions from previous studies. Although structure models are critical, they are insufficient to explain the catalysis of transport. The clues to understanding transport are based on the principles of enzyme kinetics. Secondary transport is a dynamic process—static snapshots of X-ray crystallography describe it only partially. However, without structural information, the underlying chemistry is virtually impossible to conclude. A large body of biochemical/biophysical data derived from systematic studies of site-directed mutants in LacY suggests residues critically involved in the catalysis, and a working model for the symport mechanism that involves alternating access of the binding site is presented. The general concepts derived from the bacterial LacY are examined for their relevance to other MFS transporters.

X-ray crystallographic studies of protein–ligand interactions

2003 ◽  
Vol 31 (5) ◽  
pp. 973-979 ◽  
Author(s):  
R.A. Palmer ◽  
H. Niwa
Keyword(s):  
Binding Site ◽  
Protein Structures ◽  
Three Dimensions ◽  
Small Samples ◽  
Mistletoe Lectin ◽  
Surface Binding ◽  
X Ray ◽  
X Ray Crystallography ◽  
Sugar Binding ◽  
Wide Range

X-ray crystallography enables details of covalent and non-covalent interactions to be analysed quantitatively in three dimensions, thus providing the basis for the understanding of binding of ligands to proteins as well as modes of action such as cell-surface binding. This article is concerned with current methods employed for the X-ray analysis of protein structures complexed with ligands. It deals mainly with ‘what can be done’ in current research, rather than providing details of ‘how to do it’. In recent years significant advances have been made in a variety of techniques: growing protein crystals from very small samples by scanning a wide range of conditions; X-ray intensity data collection and measurement through the use of charge-coupled devices and high-intensity, versatile synchrotron sources; cryo-crystallography which both stabilizes the crystals and provides improved data; methods for analysing and interpreting the structures, dependent, at least in part, on both structural and sequence databases; and improvements in hardware and software. To illustrate the type of results achievable two examples involving protein–sugar interactions are discussed: (i) SNAII (the lectin Sambucus nigra agglutinin-II from elder) N-terminal sugar-binding site where terminal sugar units in a glycosylation chain from a symmetry-related molecule bind and (ii) MLI (mistletoe lectin I) C-terminal sugar-binding site with lactose.

Sign in / Sign up

Export Citation Format

Share Document