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Daniel J Leahy

Publications and source records attributed to Daniel J Leahy.

17 recordsLinked to original sources
PubMed2006

Mutational analysis of Norrin-Frizzled4 recognition.

Norrin and Frizzled4 (Fz4) function as a ligand-receptor pair to control vascular development in the retina and inner ear. In mice and humans, mutations in either of the corresponding genes lead to defects in vascular development. The present work is aimed at defining the sequence determinants of binding specificity between Norrin and the Fz4 amino-terminal ligand-binding domain (the "cysteine-rich domain" (CRD)). The principal conclusions are as follows: 1) Norrin binds to the Fz4 CRD and does not detectably bind to the 14 other mammalian Frizzled and secreted Frizzled-related protein CRDs; 2) Norrin and Xenopus Wnt8 recognize largely overlapping regions of the Fz4 CRD; 3) surface determinants on the Fz4 and Fz8 CRDs that allow Norrin to distinguish between these two CRDs reside within several small regions on one face of the CRD; 4) Norrin function depends critically on three pairs of cysteines that form the highly conserved trio of disulfide bonds shared among all cystine knot proteins, but the remaining two putative disulfide bonds are less important; 5) Norrin-CRD binding depends on a largely contiguous group of amino acids in the extended beta-sheet domain of Norrin that are predicted to face away from the interface between the two monomers in the Norrin homodimer; 6) Norrin-CRD binding is strongly modulated by interactions involving charged amino acid side chains; and 7) Norrin-CRD binding is enhanced approximately 10-fold by the addition of heparin. These observations are discussed in the context of Frizzled signaling and the structure and function of other cystine knot proteins.

Amino Acid Sequence↗
PubMed2006

Structure of a heparin-dependent complex of Hedgehog and Ihog.

Hedgehog (Hh) signaling molecules mediate key tissue-patterning events during animal development, and inappropriate activation of Hh signaling in adults has been associated with human cancers. Recently, a conserved family of type I integral membrane proteins required for normal response to the Hh signal was discovered. One member of this family, Ihog (interference hedgehog), functions upstream or at the level of Patched (Ptc), but how Ihog participates in Hh signaling remains unclear. Here, we show that heparin binding induces Ihog dimerization and is required to mediate high-affinity interactions between Ihog and Hh. We also present crystal structures of a Hh-binding fragment of Ihog, both alone and complexed with Hh. Heparin is not well ordered in these structures, but a basic cleft in the first FNIII domain of Ihog (IhogFn1) is shown by mutagenesis to mediate heparin binding. These results establish that Hh directly binds Ihog and provide the first demonstration of a specific role for heparin in Hh responsiveness.

Animals↗
PubMed2006

Epidermal growth factor receptor activation in glioblastoma through novel missense mutations in the extracellular domain.

BACKGROUND: Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy. METHODS AND FINDINGS: Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR) kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132) of glioblastomas and 12.5% (1/8) of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors. CONCLUSIONS: Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma.

Animals↗
PubMed2005

The extracellular region of ErbB4 adopts a tethered conformation in the absence of ligand.

The human ErbB family of receptor tyrosine kinases comprises the epidermal growth factor receptor (EGFR/ErbB1/HER1), ErbB2 (HER2/Neu), ErbB3 (HER3), and ErbB4 (HER4). ErbBs play fundamental roles in cell growth and differentiation events in embryonic and adult tissues, and inappropriate ErbB activity has been implicated in several human cancers. We report here the 2.4 A crystal structure of the extracellular region of human ErbB4 in the absence of ligand and show that it adopts a tethered conformation similar to inactive forms of ErbB1 and ErbB3. This structure completes the gallery of unliganded ErbB receptors and demonstrates that all human ligand-binding ErbBs adopt the autoinhibited conformation. We also show that the binding of neuregulin-1beta to ErbB4 and ErbB3 and the binding of betacellulin to both ErbB4 and ErbB1 does not decrease at low pH, unlike the binding of epidermal growth factor and transforming growth factor-alpha to ErbB1. These results indicate an important role for ligand in determining pH-dependent binding and may explain different responses observed when the same ErbB receptor is stimulated by different ligands.

Adult↗
PubMed2005

The crystal structures of EAP domains from Staphylococcus aureus reveal an unexpected homology to bacterial superantigens.

The Eap (extracellular adherence protein) of Staphylococcus aureus functions as a secreted virulence factor by mediating interactions between the bacterial cell surface and several extracellular host proteins. Eap proteins from different Staphylococcal strains consist of four to six tandem repeats of a structurally uncharacterized domain (EAP domain). We have determined the three-dimensional structures of three different EAP domains to 1.8, 2.2, and 1.35 A resolution, respectively. These structures reveal a core fold that is comprised of an alpha-helix lying diagonally across a five-stranded, mixed beta-sheet. Comparison of EAP domains with known structures reveals an unexpected homology with the C-terminal domain of bacterial superantigens. Examination of the structure of the superantigen SEC2 bound to the beta-chain of a T-cell receptor suggests a possible ligand-binding site within the EAP domain (Fields, B. A., Malchiodi, E. L., Li, H., Ysern, X., Stauffacher, C. V., Schlievert, P. M., Karjalainen, K., and Mariuzza, R. (1996) Nature 384, 188-192). These results provide the first structural characterization of EAP domains, relate EAP domains to a large class of bacterial toxins, and will guide the design of future experiments to analyze EAP domain structure/function relationships.

Amino Acid Sequence↗
PubMed2005

Structure and Notch receptor binding of the tandem WWE domain of Deltex.

Deltex is a cytosolic effector of Notch signaling thought to bind through its N-terminal domain to the Notch receptor. Here we report the structure of the Drosophila Deltex N-terminal domain, which contains two tandem WWE sequence repeats. The WWE repeats, which adopt a novel fold, are related by an approximate two-fold axis of rotation. Although the WWE repeats are structurally distinct, they interact extensively and form a deep cleft at their junction that appears well suited for ligand binding. The two repeats are thermodynamically coupled; this coupling is mediated in part by a conserved segment that is immediately C-terminal to the second WWE domain. We demonstrate that although the Deltex WWE tandem is monomeric in solution, it forms a heterodimer with the ankyrin domain of the Notch receptor. These results provide structural and functional insight into how Deltex modulates Notch signaling, and how WWE modules recognize targets for ubiquitination.

Amino Acid Sequence↗
PubMed2004

Crystal structure of Caenorhabditis elegans HER-1 and characterization of the interaction between HER-1 and TRA-2A.

HER-1 is a secreted protein that promotes male development in the nematode Caenorhabditis elegans. HER-1 inhibits the function of TRA-2A, a multipass integral membrane protein thought to serve as its receptor. We report here the 1.5-A crystal structure of HER-1. The structure was solved by the multiwavelength anomalous diffraction method by using selenomethionyl-substituted HER-1 produced in Chinese hamster ovary cells. The HER-1 structure consists of two all-helical domains and is not closely homologous to any known structure. Sites of amino acid substitutions known to impair HER-1 function were mapped on the HER-1 structure and classified according to the likely mechanism by which they affect HER-1 activity. A subset of these and other amino acid substitutions on the HER-1 surface were assayed for their ability to disrupt interactions between HER-1 and TRA-2A-expressing cells, and a localized region on the HER-1 surface important for mediating this interaction was identified.

Amino Acid Sequence↗
PubMed2004

Insights into ErbB signaling from the structure of the ErbB2-pertuzumab complex.

We have determined the 3.2 A X-ray crystal structure of the extracellular domain of the human epidermal growth factor receptor 2 (ErbB2 or HER2) in a complex with the antigen binding fragment of pertuzumab, an anti-ErbB2 monoclonal antibody also known as 2C4 or Omnitarg. Pertuzumab binds to ErbB2 near the center of domain II, sterically blocking a binding pocket necessary for receptor dimerization and signaling. The ErbB2-pertuzumab structure, combined with earlier mutagenesis data, defines the pertuzumab residues essential for ErbB2 interaction. To analyze the ErbB2 side of the interface, we have mutated a number of residues contacting pertuzumab and examined the effects of these mutations on pertuzumab binding and ErbB2-ErbB3 heterodimerization. We have also shown that conserved residues previously shown to be necessary for EGF receptor homodimerization may be dispensible for ErbB2-ErbB3 heterodimerization.

Animals↗
PubMed2003

Netrin binds discrete subdomains of DCC and UNC5 and mediates interactions between DCC and heparin.

Netrins are secreted proteins that elicit both attractive and repulsive responses in migrating cells in the central and peripheral nervous systems. Netrins interact with members of two distinct families of transmembrane receptors, represented by DCC (deleted in colorectal cancer) and UNC5. A human netrin fragment (soluble netrin; sNetrin) was purified from an engineered Chinese hamster ovary cell line and used in a pull-down assay to map the interactions between netrin and its receptors. We find that sNetrin binds exclusively to the fifth fibronectin type III repeat of DCC and to each immunoglobulin repeat of UNC5. Both DCC and UNC5 bind to sNetrin with 1:1 stoichiometry in solution, and the minimal receptor fragments behave similarly to larger fragments in cross-linking experiments with purified sNetrin. We find no evidence for formation of a ternary complex between sNetrin and soluble forms of DCC and UNC5. We also find no evidence for an interaction between DCC and heparin and instead demonstrate that a loop on the fifth fibronectin type III repeat of DCC previously implicated in mediating interactions with heparin is important for sNetrin binding. Since netrin binds heparin, our results suggest that interactions between DCC and heparin are probably mediated by netrin.

Algorithms↗
PubMed2003

Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab.

HER2 (also known as Neu, ErbB2) is a member of the epidermal growth factor receptor (EGFR; also known as ErbB) family of receptor tyrosine kinases, which in humans includes HER1 (EGFR, ERBB1), HER2, HER3 (ERBB3) and HER4 (ERBB4). ErbB receptors are essential mediators of cell proliferation and differentiation in the developing embryo and in adult tissues, and their inappropriate activation is associated with the development and severity of many cancers. Overexpression of HER2 is found in 20-30% of human breast cancers, and correlates with more aggressive tumours and a poorer prognosis. Anticancer therapies targeting ErbB receptors have shown promise, and a monoclonal antibody against HER2, Herceptin (also known as trastuzumab), is currently in use as a treatment for breast cancer. Here we report crystal structures of the entire extracellular regions of rat HER2 at 2.4 A and human HER2 complexed with the Herceptin antigen-binding fragment (Fab) at 2.5 A. These structures reveal a fixed conformation for HER2 that resembles a ligand-activated state, and show HER2 poised to interact with other ErbB receptors in the absence of direct ligand binding. Herceptin binds to the juxtamembrane region of HER2, identifying this site as a target for anticancer therapies.

Animals↗
PubMed2003

EGF activates its receptor by removing interactions that autoinhibit ectodomain dimerization.

Epidermal growth factor (EGF) receptor is the prototype of the ErbB (HER) family receptor tyrosine kinases (RTKs), which regulate cell growth and differentiation and are implicated in many human cancers. EGF activates its receptor by inducing dimerization of the 621 amino acid EGF receptor extracellular region. We describe the 2.8 A resolution crystal structure of this entire extracellular region (sEGFR) in an unactivated state. The structure reveals an autoinhibited configuration, where the dimerization interface recently identified in activated sEGFR structures is completely occluded by intramolecular interactions. To activate the receptor, EGF binding must promote a large domain rearrangement that exposes this dimerization interface. This contrasts starkly with other RTK activation mechanisms and suggests new approaches for designing ErbB receptor antagonists.

Crystallography, X-Ray↗
PubMed2003

An open-and-shut case? Recent insights into the activation of EGF/ErbB receptors.

Recent crystallographic studies have provided significant new insight into how receptor tyrosine kinases from the EGF receptor or ErbB family are regulated by their growth factor ligands. EGF receptor dimerization is mediated by a unique dimerization arm, which becomes exposed only after a dramatic domain rearrangement is promoted by growth factor binding. ErbB2, a family member that has no ligand, has its dimerization arm constitutively exposed, and this explains several of its unique properties. We outline a mechanistic view of ErbB receptor homo- and heterodimerization, which suggests new approaches for interfering with these processes when they are implicated in human cancers.

Animals↗
PubMed2003

Structure and stability of the ankyrin domain of the Drosophila Notch receptor.

The Notch receptor contains a conserved ankyrin repeat domain that is required for Notch-mediated signal transduction. The ankyrin domain of Drosophila Notch contains six ankyrin sequence repeats previously identified as closely matching the ankyrin repeat consensus sequence, and a putative seventh C-terminal sequence repeat that exhibits lower similarity to the consensus sequence. To better understand the role of the Notch ankyrin domain in Notch-mediated signaling and to examine how structure is distributed among the seven ankyrin sequence repeats, we have determined the crystal structure of this domain to 2.0 angstroms resolution. The seventh, C-terminal, ankyrin sequence repeat adopts a regular ankyrin fold, but the first, N-terminal ankyrin repeat, which contains a 15-residue insertion, appears to be largely disordered. The structure reveals a substantial interface between ankyrin polypeptides, showing a high degree of shape and charge complementarity, which may be related to homotypic interactions suggested from indirect studies. However, the Notch ankyrin domain remains largely monomeric in solution, demonstrating that this interface alone is not sufficient to promote tight association. Using the structure, we have classified reported mutations within the Notch ankyrin domain that are known to disrupt signaling into those that affect buried residues and those restricted to surface residues. We show that the buried substitutions greatly decrease protein stability, whereas the surface substitutions have only a marginal affect on stability. The surface substitutions are thus likely to interfere with Notch signaling by disrupting specific Notch-effector interactions and map the sites of these interactions.

Amino Acid Sequence↗
PubMed2002

Structure of the extracellular region of HER3 reveals an interdomain tether.

We have determined the 2.6 angstrom crystal structure of the entire extracellular region of human HER3 (ErbB3), a member of the epidermal growth factor receptor (EGFR) family. The structure consists of four domains with structural homology to domains found in the type I insulin-like growth factor receptor. The HER3 structure reveals a contact between domains II and IV that constrains the relative orientations of ligand-binding domains and provides a structural basis for understanding both multiple-affinity forms of EGFRs and conformational changes induced in the receptor by ligand binding during signaling. These results also suggest new therapeutic approaches to modulating the behavior of members of the EGFR family.

Amino Acid Sequence↗
PubMed2002

Characterization of neuropilin-1 structural features that confer binding to semaphorin 3A and vascular endothelial growth factor 165.

Neuropilin-1 (Npn-1) is a receptor for both semaphorin 3A (Sema3A) and vascular endothelial growth factor 165 (VEGF(165)). To understand the role Npn-1 plays as a receptor for these structurally and functionally unrelated ligands, we set out to identify structural features of Npn-1 that confer binding to Sema3A or VEGF(165). We constructed Npn-1 variants containing deletions within the "a" and "b" domains of Npn-1. More than 16 variants were expressed in COS-1 cells and tested for alkaline phosphatase-Sema3A as well as alkaline phosphatase-VEGF(165) binding. Our results indicate that each of the two Npn-1 CUB domains and the amino-terminal coagulation factor V/VIII domain (CF V/VIII) are essential for Sema3A binding, but only the amino-terminal Npn-1 CF V/VIII domain is required for binding to VEGF(165). Guided by the structure of the bovine spermadhesin CUB domain, point mutants targeting defined surfaces of the Npn-1 a1 CUB domain were generated and tested for Sema3A and VEGF(165) binding. One Npn-1 variant, Npn-1(2ABC), exhibits complete loss of Sema3A binding while retaining normal VEGF(165) binding. Moreover, co-immunoprecipitation experiments show that Npn-1(2ABC) can form a signaling complex with the VEGF(165) signaling receptor KDR/VEGFR-2. These results establish the identity of contact sites between Npn-1 and its semaphorin ligands, and they provide a foundation for understanding how Npn-1 functions as a receptor for distinct classes of ligands in vivo.

Amino Acid Sequence↗