PubMed Health⌕ Search

Biomedical subjects

William I Weis

Publications and source records attributed to William I Weis.

At least 19 recordsLinked to original sources
PubMed2006

Multiple modes of binding enhance the affinity of DC-SIGN for high mannose N-linked glycans found on viral glycoproteins.

The dendritic cell surface receptor DC-SIGN and the closely related endothelial cell receptor DC-SIGNR specifically recognize high mannose N-linked carbohydrates on viral pathogens. Previous studies have shown that these receptors bind the outer trimannose branch Manalpha1-3[Manalpha1-6]Manalpha present in high mannose structures. Although the trimannoside binds to DC-SIGN or DC-SIGNR more strongly than mannose, additional affinity enhancements are observed in the presence of one or more Manalpha1-2Manalpha moieties on the nonreducing termini of oligomannose structures. The molecular basis of this enhancement has been investigated by determining crystal structures of DC-SIGN bound to a synthetic six-mannose fragment of a high mannose N-linked oligosaccharide, Manalpha1-2Manalpha1-3[Manalpha1-2Manalpha1-6]Manalpha1-6Man and to the disaccharide Manalpha1-2Man. The structures reveal mixtures of two binding modes in each case. Each mode features typical C-type lectin binding at the principal Ca2+-binding site by one mannose residue. In addition, other sugar residues form contacts unique to each binding mode. These results suggest that the affinity enhancement displayed toward oligosaccharides decorated with the Manalpha1-2Manalpha structure is due in part to multiple binding modes at the primary Ca2+ site, which provide both additional contacts and a statistical (entropic) enhancement of binding.

Carbohydrate Conformation↗
PubMed2006

Organization of the SH3-SH2 unit in active and inactive forms of the c-Abl tyrosine kinase.

The tyrosine kinase c-Abl is inactivated by interactions made by its SH3 and SH2 domains with the distal surface of the kinase domain. We present a crystal structure of a fragment of c-Abl which reveals that a critical N-terminal cap segment, not visualized in previous structures, buttresses the SH3-SH2 substructure in the autoinhibited state and locks it onto the distal surface of the kinase domain. Surprisingly, the N-terminal cap is phosphorylated on a serine residue that interacts with the connector between the SH3 and SH2 domains. Small-angle X-ray scattering (SAXS) analysis shows that a mutated form of c-Abl, in which the N-terminal cap and two other key contacts in the autoinhibited state are deleted, exists in an extended array of the SH3, SH2, and kinase domains. This alternative conformation of Abl is likely to prolong the active state of the kinase by preventing it from returning to the autoinhibited state.

Amino Acid Sequence↗
PubMed2005

Deconstructing the cadherin-catenin-actin complex.

Spatial and functional organization of cells in tissues is determined by cell-cell adhesion, thought to be initiated through trans-interactions between extracellular domains of the cadherin family of adhesion proteins, and strengthened by linkage to the actin cytoskeleton. Prevailing dogma is that cadherins are linked to the actin cytoskeleton through beta-catenin and alpha-catenin, although the quaternary complex has never been demonstrated. We test this hypothesis and find that alpha-catenin does not interact with actin filaments and the E-cadherin-beta-catenin complex simultaneously, even in the presence of the actin binding proteins vinculin and alpha-actinin, either in solution or on isolated cadherin-containing membranes. Direct analysis in polarized cells shows that mobilities of E-cadherin, beta-catenin, and alpha-catenin are similar, regardless of the dynamic state of actin assembly, whereas actin and several actin binding proteins have higher mobilities. These results suggest that the linkage between the cadherin-catenin complex and actin filaments is more dynamic than previously appreciated.

Actins↗
PubMed2005

Alpha-catenin is a molecular switch that binds E-cadherin-beta-catenin and regulates actin-filament assembly.

Epithelial cell-cell junctions, organized by adhesion proteins and the underlying actin cytoskeleton, are considered to be stable structures maintaining the structural integrity of tissues. Contrary to the idea that alpha-catenin links the adhesion protein E-cadherin through beta-catenin to the actin cytoskeleton, in the accompanying paper we report that alpha-catenin does not bind simultaneously to both E-cadherin-beta-catenin and actin filaments. Here we demonstrate that alpha-catenin exists as a monomer or a homodimer with different binding properties. Monomeric alpha-catenin binds more strongly to E-cadherin-beta-catenin, whereas the dimer preferentially binds actin filaments. Different molecular conformations are associated with these different binding states, indicating that alpha-catenin is an allosteric protein. Significantly, alpha-catenin directly regulates actin-filament organization by suppressing Arp2/3-mediated actin polymerization, likely by competing with the Arp2/3 complex for binding to actin filaments. These results indicate a new role for alpha-catenin in local regulation of actin assembly and organization at sites of cadherin-mediated cell-cell adhesion.

Actin-Related Protein 2-3 Complex↗
PubMed2005

Thermodynamics of beta-catenin-ligand interactions: the roles of the N- and C-terminal tails in modulating binding affinity.

beta-Catenin is a structural component of adherens junctions, where it binds to the cytoplasmic domain of cadherin cell adhesion molecules. beta-Catenin is also a transcriptional coactivator in the Wnt signaling pathway, where it binds to Tcf/Lef family transcription factors. In the absence of a Wnt signal, nonjunctional beta-catenin is present in a multiprotein complex containing the proteins axin and adenomatous polyposis coli (APC), both of which bind directly to beta-catenin. The thermodynamics of beta-catenin binding to E-cadherin, Lef-1, APC, axin, and the transcriptional inhibitor ICAT have been determined by isothermal titration calorimetry. Most of the interactions showed large, unfavorable entropy changes, consistent with these ligands being natively unstructured in the absence of beta-catenin. Phosphorylation of serine residues present in a sequence motif common to cadherins and APC increased the affinity for beta-catenin 300-700-fold, and surface plasmon resonance measurements revealed that phosphorylation of E-cadherin both enhanced its on rate and decreased its off rate. The effects of the N- and C-terminal "tails" that flank the beta-catenin armadillo repeat domain on ligand binding have also been investigated using constructs lacking one or both tails. Contrary to earlier studies that employed less direct binding assays, the tails did not affect the affinity of beta-catenin for tight ligands such as E-cadherin, Lef-1, and phosphorylated APC. However, the beta-catenin C-terminal tail was found to decrease the affinity for the weaker ligands APC and axin, suggesting that this region may have a regulatory role in beta-catenin degradation.

Adenomatous Polyposis Coli Protein↗
PubMed2005

Crystal structure of the S.cerevisiae exocyst component Exo70p.

The exocyst is an evolutionarily conserved multiprotein complex required for the targeting and docking of post-Golgi vesicles to the plasma membrane. Through its interactions with a variety of proteins, including small GTPases, the exocyst is thought to integrate signals from the cell and signal that vesicles arriving at the plasma membrane are ready for fusion. Here we describe the three-dimensional crystal structure of one of the components of the exocyst, Exo70p, from Saccharomyces cerevisiae at 3.5A resolution. Exo70p binds the small GTPase Rho3p in a GTP-dependent manner with an equilibrium dissociation constant of approximately 70 microM. Exo70p is an extended rod approximately 155 angstroms in length composed principally of alpha helices, and is a novel fold. The structure provides a first view of the Exo70 protein family and provides a framework to study the molecular function of this exocyst component.

Amino Acid Sequence↗
PubMed2005

Beta-catenin directly displaces Groucho/TLE repressors from Tcf/Lef in Wnt-mediated transcription activation.

Wnt growth factors mediate cell fate determination during embryogenesis and in the renewal of tissues in the adult. Wnts act by stabilizing cellular levels of the transcriptional coactivator beta-catenin, which forms complexes with sequence-specific DNA-binding Tcf/Lef transcription factors. In the absence of nuclear beta-catenin, Tcf/Lefs act as transcriptional repressors by binding to Groucho/TLE proteins. The molecular basis of the switch from transcriptional repression to activation during Wnt signaling has not been clear, in particular whether factors other than beta-catenin are required to disrupt the interaction between Groucho/TLE and Tcf/Lef. Using highly purified proteins, we demonstrate that beta-catenin displaces Groucho/TLE from Tcf/Lef by binding to a previously unidentified second, low-affinity binding site on Lef-1 that includes sequences just N-terminal to the DNA-binding domain, and that overlaps the Groucho/TLE-binding site.

Animals↗
PubMed2005

Conformational changes of p97 during nucleotide hydrolysis determined by small-angle X-Ray scattering.

Valosin-containing protein (VCP)/p97 is an AAA family ATPase that has been implicated in the removal of misfolded proteins from the endoplasmic reticulum and in membrane fusion. p97 forms a homohexamer whose protomers consist of an N-terminal (N) domain responsible for binding to effector proteins, followed by two AAA ATPase domains, D1 and D2. Small-angle X-ray scattering (SAXS) measurements of p97 in the presence of AMP-PNP (ATP state), ADP-AlF(x) (hydrolysis transition state), ADP, or no nucleotide reveal major changes in the positions of the N domains with respect to the hexameric ring during the ATP hydrolysis cycle. Nucleotide binding and hydrolysis experiments indicate that D2 inhibits nucleotide exchange by D1. The data suggest that the conversion of the chemical energy of ATP hydrolysis into mechanical work on substrates involves transmission of conformational changes generated by D2 through D1 to move N.

Adenosine Triphosphatases↗
PubMed2004

Structure of the armadillo repeat domain of plakophilin 1.

The p120ctn subfamily of armadillo domain proteins has roles in modulating intercellular adhesion by cadherin-containing junctions. We have determined the crystal structure of the arm repeat domain from plakophilin-1 (PKP1), a member of the p120ctn subfamily that is found in desmosomes. The structure reveals that the domain has nine instead of the expected ten arm repeats. A sequence predicted to be an arm repeat is instead a large insert which serves as a wedge that produces a significant bend in the overall domain structure. Structure-based sequence alignments indicate that the nine repeats and large insert are common to this subfamily of armadillo proteins. A prominent basic patch on the surface of the protein may serve as a binding site for partners of these proteins.

Amino Acid Sequence↗
PubMed2004

Extended neck regions stabilize tetramers of the receptors DC-SIGN and DC-SIGNR.

The human cell surface receptors DC-SIGN (dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin) and DC-SIGNR (DC-SIGN-related) bind to oligosaccharide ligands found on human tissues as well as on pathogens including viruses, bacteria, and parasites. The extracellular portion of each receptor contains a membrane-distal carbohydrate-recognition domain (CRD) and forms tetramers stabilized by an extended neck region consisting of 23 amino acid repeats. Cross-linking analysis of full-length receptors expressed in fibroblasts confirms the tetrameric state of the intact receptors. Hydrodynamic studies on truncated receptors demonstrate that the portion of the neck of each protein adjacent to the CRD is sufficient to mediate the formation of dimers, whereas regions near the N terminus are needed to stabilize the tetramers. Some of the intervening repeats are missing from polymorphic forms of DC-SIGNR. Two different crystal forms of truncated DC-SIGNR comprising two neck repeats and the CRD reveal that the CRDs are flexibly linked to the neck, which contains alpha-helical segments interspersed with non-helical regions. Differential scanning calorimetry measurements indicate that the neck and CRDs are independently folded domains. Based on the crystal structures and hydrodynamic data, models for the full extracellular domains of the receptors have been generated. The observed flexibility of the CRDs in the tetramer, combined with previous data on the specificity of these receptors, suggests an important role for oligomerization in the recognition of endogenous glycans, in particular those present on the surfaces of enveloped viruses recognized by these proteins.

Animals↗
PubMed2004

Mechanism of phosphorylation-dependent binding of APC to beta-catenin and its role in beta-catenin degradation.

The transcriptional coactivator beta-catenin mediates Wnt growth factor signaling. In the absence of a Wnt signal, casein kinase 1 (CK1) and glycogen synthase kinase-3beta (GSK-3beta) phosphorylate cytosolic beta-catenin, thereby flagging it for recognition and destruction by the ubiquitin/proteosome machinery. Phosphorylation occurs in a multiprotein complex that includes the kinases, beta-catenin, axin, and the Adenomatous Polyposis Coli (APC) protein. The role of APC in this process is poorly understood. CK1epsilon and GSK-3beta phosphorylate APC, which increases its affinity for beta-catenin. Crystal structures of phosphorylated and nonphosphorylated APC bound to beta-catenin reveal a phosphorylation-dependent binding motif generated by mutual priming of CK1 and GSK-3beta substrate sequences. Axin is shown to act as a scaffold for substrate phosphorylation by these kinases. Phosphorylated APC and axin bind to the same surface of, and compete directly for, beta-catenin. The structural and biochemical data suggest a novel model for how APC functions in beta-catenin degradation.

Adenomatous Polyposis Coli Protein↗
PubMed2004

Structural basis for distinct ligand-binding and targeting properties of the receptors DC-SIGN and DC-SIGNR.

Both the dendritic cell receptor DC-SIGN and the closely related endothelial cell receptor DC-SIGNR bind human immunodeficiency virus and enhance infection. However, biochemical and structural comparison of these receptors now reveals that they have very different physiological functions. By screening an extensive glycan array, we demonstrated that DC-SIGN and DC-SIGNR have distinct ligand-binding properties. Our structural and mutagenesis data explain how both receptors bind high-mannose oligosaccharides on enveloped viruses and why only DC-SIGN binds blood group antigens, including those present on microorganisms. DC-SIGN mediates endocytosis, trafficking as a recycling receptor and releasing ligand at endosomal pH, whereas DC-SIGNR does not release ligand at low pH or mediate endocytosis. Thus, whereas DC-SIGN has dual ligand-binding properties and functions both in adhesion and in endocytosis of pathogens, DC-SIGNR binds a restricted set of ligands and has only the properties of an adhesion receptor.

Binding Sites↗
PubMed2003

Crystal structure of the CUB1-EGF-CUB2 region of mannose-binding protein associated serine protease-2.

Serum mannose-binding proteins (MBPs) are C-type lectins that recognize cell surface carbohydrate structures on pathogens, and trigger killing of these targets by activating the complement pathway. MBPs circulate as a complex with MBP-associated serine proteases (MASPs), which become activated upon engagement of a target cell surface. The minimal functional unit for complement activation is a MASP homodimer bound to two MBP trimeric subunits. MASPs have a modular structure consisting of an N-terminal CUB domain, a Ca(2+)-binding EGF-like domain, a second CUB domain, two complement control protein modules and a C-terminal serine protease domain. The CUB1-EGF-CUB2 region mediates homodimerization and binding to MBP. The crystal structure of the MASP-2 CUB1-EGF-CUB2 dimer reveals an elongated structure with a prominent concave surface that is proposed to be the MBP-binding site. A model of the full six-domain structure and its interaction with MBPs suggests mechanisms by which binding to a target cell transmits conformational changes from MBP to MASP that allow activation of its protease activity.

Amino Acid Sequence↗
PubMed2002

Three-dimensional structure of the amino-terminal domain of syntaxin 6, a SNAP-25 C homolog.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are required for intracellular membrane fusion, and are differentially localized throughout the cell. SNAREs on vesicle and target membranes contain "SNARE motifs" which interact to form a four-helix bundle that contributes to the fusion of two membranes. SNARE motif sequences fall into four classes, homologous to the neuronal proteins syntaxin 1a, VAMP 2, and the N- and C-terminal SNARE motifs of SNAP-25 (S25N and S25C), and it is thought that one member from each class interacts to form a SNARE complex. Many SNAREs also feature N-terminal domains believed to function in regulating SNARE complex assembly or other aspects of vesicle transport. Syntaxin 6 is a SNARE found primarily in endosomal transport vesicles and whose SNARE motif shows significant homology to both syntaxin 1a and S25C. The crystal structure of the syntaxin 6 N-terminal domain reveals strong structural similarity with the N-terminal domains of syntaxin family members syntaxin 1a, Sso1p, and Vam3p, despite a very low level of sequence similarity. The syntaxin 6 SNARE motif can substitute for S25C in in vitro binding experiments, supporting the classification of syntaxin 6 as an S25C family member. Secondary structure prediction of SNARE proteins shows that the N-terminal domains of many syntaxin, S25N, and S25C family members are likely to be similar to one another, but are distinct from those of VAMP family members, indicating that syntaxin, S25N, and S25C SNAREs may have shared a common ancestor.

Animals↗
PubMed2002

Biochemical and structural definition of the l-afadin- and actin-binding sites of alpha-catenin.

alpha-Catenin is an integral component of adherens junctions, where it links cadherins to the actin cytoskeleton. alpha-Catenin is also required for the colocalization of the nectin/afadin/ponsin adhesion system to adherens junctions, and it specifically associates with the nectin-binding protein afadin. A proteolytic fragment of alpha-catenin, residues 385-651, contains the afadin-binding site. The three-dimensional structure of this fragment comprises two side-by-side four-helix bundles, both of which are required for afadin binding. The alpha-catenin fragment 385-651 binds afadin more strongly than the full-length protein, suggesting that the full-length protein harbors a cryptic binding site for afadin. Comparison of the alpha-catenin 385-651 structure with the recently solved structure of the alpha-catenin M-fragment (Yang, J., Dokurno, P., Tonks, N. K., and Barford, D. (2001) EMBO J. 20, 3645-3656) reveals a surprising flexibility in the orientation of the two four-helix bundles. alpha-Catenin and the actin-binding protein vinculin share sequence and most likely structural similarity within their actin-binding domains. Despite this homology, actin binding requires additional sequences adjacent to this region.

Actins↗