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W I Weis

Publications and source records attributed to W I Weis.

At least 19 recordsLinked to original sources
PubMed2001

Structural basis for selective recognition of oligosaccharides by DC-SIGN and DC-SIGNR.

Dendritic cell specific intracellular adhesion molecule-3 (ICAM-3) grabbing nonintegrin (DC-SIGN), a C-type lectin present on the surface of dendritic cells, mediates the initial interaction of dendritic cells with T cells by binding to ICAM-3. DC-SIGN and DC-SIGNR, a related receptor found on the endothelium of liver sinusoids, placental capillaries, and lymph nodes, bind to oligosaccharides that are present on the envelope of human immunodeficiency virus (HIV), an interaction that strongly promotes viral infection of T cells. Crystal structures of carbohydrate-recognition domains of DC-SIGN and of DC-SIGNR bound to oligosaccharide, in combination with binding studies, reveal that these receptors selectively recognize endogenous high-mannose oligosaccharides and may represent a new avenue for developing HIV prophylactics.

Acetylglucosamine↗
PubMed2001

Molecular mechanisms of beta-catenin recognition by adenomatous polyposis coli revealed by the structure of an APC-beta-catenin complex.

The adenomatous polyposis coli (APC) tumor suppressor protein plays a critical role in regulating cellular levels of the oncogene product beta-catenin. APC binds to beta-catenin through a series of homologous 15 and 20 amino acid repeats. We have determined the crystal structure of a 15 amino acid beta-catenin binding repeat from APC bound to the armadillo repeat region of beta-catenin. Although it lacks significant sequence homology, the N-terminal half of the repeat binds in a manner similar to portions of E-cadherin and XTcf3, but the remaining interactions are unique to APC. We discuss the implications of this new structure for the design of therapeutics, and present evidence from structural, biochemical and sequence data, which suggest that the 20 amino acid repeats can adopt two modes of binding to beta-catenin.

Adenomatous Polyposis Coli↗
PubMed2001

Crystal structure and biophysical properties of a complex between the N-terminal SNARE region of SNAP25 and syntaxin 1a.

SNARE proteins are required for intracellular membrane fusion. In the neuron, the plasma membrane SNAREs syntaxin 1a and SNAP25 bind to VAMP2 found on neurotransmitter-containing vesicles. These three proteins contain "SNARE regions" that mediate their association into stable tetrameric coiled-coil structures. Syntaxin 1a contributes one such region, designated H3, and SNAP25 contributes two SNARE regions to the fusogenic complex with VAMP2. Syntaxin 1a H3 (syn1aH3) and SNAP25 can form a stable assembly, which can then be bound by VAMP2 to form the full SNARE complex. Here we show that syn1aH3 can also form a stable but kinetically trapped complex with the N-terminal SNARE region of SNAP25 (S25N). The crystal structure of this complex reveals an extended parallel four-helix bundle similar to that of the core SNARE and the syn1aH3-SNAP25 complexes. The inherent ability of syn1aH3 and S25N to associate stably in vitro implies that the intracellular fusion machinery must prevent formation of, or remove, any non-productive complexes. Comparison with the syn1aH3-SNAP25 complex suggests that the linkage of the N- and C-terminal SNAP25 SNARE regions is kinetically advantageous in preventing formation of the non-productive syn1aH3-S25N complex. We also demonstrate that the syn1aH3-S25N complex can be disassembled by alpha-SNAP and N-ethylmaleimide-sensitive factor.

Animals↗
PubMed2001

The structure of the beta-catenin/E-cadherin complex and the molecular basis of diverse ligand recognition by beta-catenin.

As a component of adherens junctions and the Wnt signaling pathway, beta-catenin binds cadherins, Tcf family transcription factors, and the tumor suppressor APC. We have determined the crystal structures of both unphosphorylated and phosphorylated E-cadherin cytoplasmic domain complexed with the arm repeat region of beta-catenin. The interaction spans all 12 arm repeats, and features quasi-independent binding regions that include helices which interact with both ends of the arm repeat domain and an extended stretch of 14 residues which closely resembles a portion of XTcf-3. Phosphorylation of E-cadherin results in interactions with a hydrophobic patch of beta-catenin that mimics the binding of an amphipathic XTcf-3 helix. APC contains sequences homologous to the phosphorylated region of cadherin, and is likely to bind similarly.

Adenomatous Polyposis Coli Protein↗
PubMed2001

A novel snare N-terminal domain revealed by the crystal structure of Sec22b.

Intra-cellular membrane fusion is facilitated by the association of SNAREs from opposite membranes into stable alpha-helical bundles. Many SNAREs, in addition to their alpha-helical regions, contain N-terminal domains that likely have essential regulatory functions. To better understand this regulation, we have determined the 2.4-A crystal structure of the 130-amino acid N-terminal domain of mouse Sec22b (mSec22b), a SNARE involved in endoplasmic reticulum/Golgi membrane trafficking. The domain consists of a mixed alpha-helical/beta-sheet fold that resembles a circular permutation of the actin/poly-proline binding protein, profilin, and the GAF/PAS family of regulatory modules. The structure is distinct from the previously characterized N-terminal domain of syntaxin 1A, and, unlike syntaxin 1A, the N-terminal domain of mSec22b has no effect on the rate of SNARE assembly in vitro. An analysis of surface conserved residues reveals a potential protein interaction site. Key residues in this site are distinct in two mammalian Sec22 variants that lack SNARE domains. Finally, sequence analysis indicates that a similar domain is likely present in the endosomal/lysosomal SNARE VAMP7.

Amino Acid Sequence↗
PubMed2001

beta-catenin: molecular plasticity and drug design.

The protein beta-catenin is an essential component of intercellular junctions and the Wnt growth factor signaling pathway. In many cancers, mutation of Wnt pathway components leads to activation of oncogenes by the beta-catenin-Tcf transcription factor complex. This complex is therefore an attractive target for anti-cancer drugs, but any such compound must selectively interfere with the beta-catenin-Tcf complex without disrupting other essential interactions of beta-catenin. Recent structural and biochemical studies have probed the molecular basis of ligand interaction by beta-catenin, and highlighted the possibilities and challenges of designing inhibitors of the beta-catenin-Tcf complex.

Animals↗
PubMed2000

The cadherin cytoplasmic domain is unstructured in the absence of beta-catenin. A possible mechanism for regulating cadherin turnover.

Cadherins are single pass transmembrane proteins that mediate Ca(2+)-dependent homophilic cell-cell adhesion by linking the cytoskeletons of adjacent cells. In adherens junctions, the cytoplasmic domain of cadherins bind to beta-catenin, which in turn binds to the actin-associated protein alpha-catenin. The physical properties of the E-cadherin cytoplasmic domain and its interactions with beta-catenin have been investigated. Proteolytic sensitivity, tryptophan fluorescence, circular dichroism, and (1)H NMR measurements indicate that murine E-cadherin cytoplasmic domain is unstructured. Upon binding to beta-catenin, the domain becomes resistant to proteolysis, suggesting that it structures upon binding. Cadherin-beta-catenin complex stability is modestly dependent on ionic strength, indicating that, contrary to previous proposals, the interaction is not dominated by electrostatics. Comparison of 18 cadherin sequences indicates that their cytoplasmic domains are unlikely to be structured in isolation. This analysis also reveals the presence of PEST sequences, motifs associated with ubiquitin/proteosome degradation, that overlap the previously identified beta-catenin-binding site. It is proposed that binding of cadherins to beta-catenin prevents recognition of degradation signals that are exposed in the unstructured cadherin cytoplasmic domain, favoring a cell surface population of catenin-bound cadherins capable of participating in cell adhesion.

Amino Acid Sequence↗
PubMed2000

Self-association of the H3 region of syntaxin 1A. Implications for intermediates in SNARE complex assembly.

Intracellular membrane fusion requires SNARE proteins found on the vesicle and target membranes. SNAREs associate by formation of a parallel four-helix bundle, and it has been suggested that formation of this complex promotes membrane fusion. The membrane proximal region of the cytoplasmic domain of the SNARE syntaxin 1A, designated H3, contributes one of the four helices to the SNARE complex. In the crystal structure of syntaxin 1A H3, four molecules associate as a homotetramer composed of two pairs of parallel helices that are anti-parallel to each other. The H3 oligomer observed in the crystals is also found in solution, as assessed by gel filtration and chemical cross-linking studies. The crystal structure reveals that the highly conserved Phe-216 packs against conserved Gln-226 residues present on the anti-parallel pair of helices. Modeling indicates that Phe-216 prevents parallel tetramer formation. Mutation of Phe-216 to Leu appears to allow formation of parallel tetramers, whereas mutation to Ala destabilizes the protein. These results indicate that Phe-216 has a role in preventing formation of stable parallel helical bundles, thus favoring the interaction of the H3 region of syntaxin 1a with other proteins involved in membrane fusion.

Amino Acid Sequence↗
PubMed2000

Mechanism of pH-dependent N-acetylgalactosamine binding by a functional mimic of the hepatocyte asialoglycoprotein receptor.

Efficient release of ligands from the Ca(2+)-dependent carbohydrate-recognition domain (CRD) of the hepatic asialoglycoprotein receptor at endosomal pH requires a small set of conserved amino acids that includes a critical histidine residue. When these residues are incorporated at corresponding positions in an homologous galactose-binding derivative of serum mannose-binding protein, the pH dependence of ligand binding becomes more like that of the receptor. The modified CRD displays 40-fold preferential binding to N-acetylgalactosamine compared with galactose, making it a good functional mimic of the asialoglycoprotein receptor. In the crystal structure of the modified CRD bound to N-acetylgalactosamine, the histidine (His(202)) contacts the 2-acetamido methyl group and also participates in a network of interactions involving Asp(212), Arg(216), and Tyr(218) that positions a water molecule in a hydrogen bond with the sugar amide group. These interactions appear to produce the preference for N-acetylgalactosamine over galactose and are also likely to influence the pK(a) of His(202). Protonation of His(202) would disrupt its interaction with an asparagine that serves as a ligand for Ca(2+) and sugar. The structure of the modified CRD without sugar displays several different conformations that may represent structures of intermediates in the release of Ca(2+) and sugar ligands caused by protonation of His(202).

Acetylgalactosamine↗
PubMed2000

Structure of a C-type carbohydrate recognition domain from the macrophage mannose receptor.

The mannose receptor of macrophages and liver endothelium mediates clearance of pathogenic organisms and potentially harmful glycoconjugates. The extracellular portion of the receptor includes eight C-type carbohydrate recognition domains (CRDs), of which one, CRD-4, shows detectable binding to monosaccharide ligands. We have determined the crystal structure of CRD-4. Although the basic C-type lectin fold is preserved, a loop extends away from the core of the domain to form a domain-swapped dimer in the crystal. Of the two Ca(2+) sites, only the principal site known to mediate carbohydrate binding in other C-type lectins is occupied. This site is altered in a way that makes sugar binding impossible in the mode observed in other C-type lectins. The structure is likely to represent an endosomal form of the domain formed when Ca(2+) is lost from the auxiliary calcium site. The structure suggests a mechanism for endosomal ligand release in which the auxiliary calcium site serves as a pH sensor. Acid pH-induced removal of this Ca(2+) results in conformational rearrangements of the receptor, rendering it unable to bind carbohydrate ligands.

Amino Acid Sequence↗
PubMed2000

Structural basis of the Axin-adenomatous polyposis coli interaction.

Axin and the adenomatous polyposis coli (APC) tumor suppressor protein are components of the Wnt/Wingless growth factor signaling pathway. In the absence of Wnt signal, Axin and APC regulate cytoplasmic levels of the proto-oncogene beta-catenin through the formation of a large complex containing these three proteins, glycogen synthase kinase 3beta (GSK3beta) and several other proteins. Both Axin and APC are known to be critical for beta-catenin regulation, and truncations in APC that eliminate the Axin-binding site result in human cancers. A protease-resistant domain of Axin that contains the APC-binding site is a member of the regulators of G-protein signaling (RGS) superfamily. The crystal structures of this domain alone and in complex with an Axin-binding sequence from APC reveal that the Axin-APC interaction occurs at a conserved groove on a face of the protein that is distinct from the G-protein interface of classical RGS proteins. The molecular interactions observed in the Axin-APC complex provide a rationale for the evolutionary conservation seen in both proteins.

Adenomatous Polyposis Coli Protein↗
PubMed2000

Three-dimensional structure of the neuronal-Sec1-syntaxin 1a complex.

Syntaxin 1a and neuronal Sec1 (nSec1) form an evolutionarily conserved heterodimer that is essential for vesicle trafficking and membrane fusion. The crystal structure of the nSec1-syntaxin 1a complex, determined at 2.6 A resolution, reveals that major conformational rearrangements occur in syntaxin relative to both the core SNARE complex and isolated syntaxin. We identify regions of the two proteins that seem to determine the binding specificity of particular Sec1 proteins for syntaxin isoforms, which is likely to be important for the fidelity of membrane trafficking. The structure also indicates mechanisms that might couple the action of upstream effector proteins to conformational changes in syntaxin 1a and nSec1 that lead to core complex formation and membrane fusion.

Amino Acid Sequence↗
PubMed2000

Protein-protein interactions in intracellular membrane fusion.

The fusion of intracellular vesicles with their target membranes is an essential feature of the compartmental structure of eukaryotic cells. This process requires proteins that dictate the targeting of a vesicle to the correct cellular location, mediate bilayer fusion and, in some systems, regulate the precise time at which fusion occurs. Recent biophysical and structural studies of these proteins have begun to provide a foundation for understanding their functions at a molecular level.

Membrane Fusion↗
PubMed2000

Structure of the dimerization and beta-catenin-binding region of alpha-catenin.

In adherens junctions, alpha-catenin links the cadherin-beta-catenin complex to the actin-based cytoskeleton. alpha-catenin is a homodimer in solution, but forms a 1:1 heterodimer with beta-catenin. The crystal structure of the alpha-catenin dimerization domain, residues 82-279, shows that alpha-catenin dimerizes through formation of a four-helix bundle in which two antiparallel helices are contributed by each protomer. A slightly larger fragment, comprising residues 57-264, binds to beta-catenin. A chimera consisting of the alpha-catenin-binding region of beta-catenin linked to the amino terminus of alpha-catenin 57-264 behaves as a monomer in solution, as expected, since beta-catenin binding disrupts the alpha-catenin dimer. The crystal structure of this chimera reveals the interaction between alpha- and beta-catenin, and provides a basis for understanding adherens junction assembly.

Amino Acid Sequence↗
PubMed1999

Crystal structure of the amino-terminal domain of N-ethylmaleimide-sensitive fusion protein.

The cytosolic ATPase N-ethylmaleimide-sensitive fusion protein (NSF) disassembles complexes of membrane-bound proteins known as SNAREs, an activity essential for vesicular trafficking. The amino-terminal domain of NSF (NSF-N) is required for the interaction of NSF with the SNARE complex through the adaptor protein alpha-SNAP. The crystal structure of NSF-N reveals two subdomains linked by a single stretch of polypeptide. A polar interface between the two subdomains indicates that they can move with respect to one another during the catalytic cycle of NSF. Structure-based sequence alignments indicate that in addition to NSF orthologues, the p97 family of ATPases contain an amino-terminal domain of similar structure.

Adenosine Triphosphatases↗
PubMed1998

Ca2+-dependent structural changes in C-type mannose-binding proteins.

C-type animal lectins are a diverse family of proteins which mediate cell-surface carbohydrate-recognition events through a conserved carbohydrate-recognition domain (CRD). Most members of this family possess a carbohydrate-binding activity that depends strictly on the binding of Ca2+ at two sites, designated 1 and 2, in the CRD. The structural transitions associated with Ca2+ binding in C-type lectins have been investigated by determining high-resolution crystal structures of rat serum mannose-binding protein (MBP) bound to one Ho3+ in place of Ca2+, and the apo form of rat liver MBP. The removal of Ca2+ does not affect the core structure of the CRD, but dramatic conformational changes occur in the loops. The most significant structural change in the absence of Ca2+ is the isomerization of a cis-peptide bond preceding a conserved proline residue in Ca2+ site 2. This bond adopts the cis conformation in all Ca2+-bound structures, whereas both cis and trans conformations are observed in the absence of Ca2+. The pattern of structural changes in the three loops that interact with Ca2+ is dictated in large part by the conformation of the prolyl peptide bond. The highly conserved nature of Ca2+ site 2 suggests that the transitions observed in MBPs are general features of Ca2+ binding in C-type lectins.

Amino Acid Sequence↗
PubMed1998

Coupling of prolyl peptide bond isomerization and Ca2+ binding in a C-type mannose-binding protein.

A proline residue flanked by two polar residues is a highly conserved sequence motif in the Ca2+- and carbohydrate-binding site of C-type animal lectins. Crystal structures of several C-type lectins have shown that the two flanking residues are only observed to act as Ca2+ ligands when the peptide bond preceding the proline residue is in the cis conformation. In contrast, structures of the apo- and one-ion forms of mannose-binding proteins (MBPs) reveal that, when the Ca2+-binding site is empty, the peptide bond preceding the proline can adopt either the cis or trans conformation, and distinct structures in adjacent regions are associated with the two proline isomers. In this work, measurements of Ca2+-induced changes in intrinsic tryptophan fluorescence, and fluorescence energy transfer from tryptophan to Tb3+, reveal a slow conformational change in rat liver MBP (MBP-C) accompanying the binding of either Ca2+ or Tb3+. The Ca2+-induced increase in intrinsic tryptophan fluorescence shows biphasic kinetics: a burst phase with a rate constant greater than 1 s(-1) is followed by a slow phase with a single-exponential rate constant ranging from 0.01 to 0.05 s(-1) (36 degrees C) that depends on the concentration of Ca2+. Likewise, addition of EGTA to Ca2+-bound or Tb3+-bound MBP-C causes a decrease in intrinsic tryptophan fluorescence with biphasic kinetics consisting of a burst phase with a rate constant greater than 1 s(-1), followed by a slow phase with a single-exponential rate constant of 0.065 s(-1). In contrast, Tb3+ fluorescence produced by resonant energy transfer from MBP-C decreases in a single kinetic phase with a rate constant greater than 1 s(-1), implying that the slow change in tryptophan fluorescence monitors a conformational change that is not limited in rate by ion dissociation. The rate constants of the slow phases accompanying Ca2+ binding and release are strongly affected by temperature and are weakly accelerated by the prolyl isomerase cyclophilin. These data strongly suggest that the binding of either Ca2+ or Tb3+ to MBP-C is coupled to a conformational change that involves the cis-trans isomerization of a peptide bond. Fitting of the data to kinetic models indicates that, in the absence of Ca2+, the proline in approximately 80% of the molecules is in the trans conformation. The slow kinetics associated with cis-trans proline isomerization may be exploited by endocytic receptors to facilitate sorting of carbohydrate-bearing ligands from the receptor in the endosome.

Animals↗