Dr Christian Siebold
| Research Area: | Protein Science and Structural Biology |
|---|---|
| Technology Exchange: | Crystallography, Microscopy (Confocal) and Protein interaction |
| Keywords: | Development, Hedgehog Signalling, Morphogens, Cell surface receptors, Membrane proteins and X-ray crystallography |
| Web Links: |
We are interested in the molecular mechanisms of hedgehog signalling. A handful of secreted growth factors and morphogens are responsible for almost all developmental decisions during embryogenesis and, among these, Hedgehog (Hh) family proteins are ubiquitous players in tissue growth, patterning and morphogenesis. Dysregulation of Hh signalling has pathological consequences, triggering developmental defects, neurodegenerative diseases and cancer. Hh-signalling is an intensely active field of research, with recent results underlining the clinical potential of modulating this pathway. Multiple cell surface receptors (Ptc, Smo, CDO, Hip, Gas1, etc.) transduce and/or regulate Hh signals (see Figure).
Our group aims to unravel the molecular mechanisms underlying Hh ligand reception and gradient formation and, ultimately, to understand how the Hh signal is transduced through the cell membrane. We are focusing on the structural and biophysical characterisation of secreted and full-length membrane-bound versions of Hh receptors and Hh ligand-receptor complexes. To achieve this, we are using a combination of X-ray crystallography, biophysical and cell culture-based methods.
| Name | Department | Institution | Country |
|---|---|---|---|
| Roger Patient | MRC Molecular Haematology Unit, WIMM, University of Oxford | UK | |
| Philip Ingham | Institute of Molecular and Cell Biology | Singapore |
2011. Modular mechanism of Wnt signaling inhibition by Wnt inhibitory factor 1. Nat Struct Mol Biol, 18 (8), pp. 886-893. Read abstract | Read more
Wnt morphogens control embryonic development and homeostasis in adult tissues. In vertebrates the N-terminal WIF domain (WIF-1(WD)) of Wnt inhibitory factor 1 (WIF-1) binds Wnt ligands. Our crystal structure of WIF-1(WD) reveals a previously unidentified binding site for phospholipid; two acyl chains extend deep into the domain, and the head group is exposed to the surface. Biophysical and cellular assays indicate that there is a WIF-1(WD) Wnt-binding surface proximal to the lipid head group but also implicate the five epidermal growth factor (EGF)-like domains (EGFs I-V) in Wnt binding. The six-domain WIF-1 crystal structure shows that EGFs I-V are wrapped back, interfacing with WIF-1(WD) at EGF III. EGFs II-V contain a heparan sulfate proteoglycan (HSPG)-binding site, consistent with conserved positively charged residues on EGF IV. This combination of HSPG- and Wnt-binding properties suggests a modular model for the localization of WIF-1 and for signal inhibition within morphogen gradients. Hide abstract
2011. Proteoglycan-specific molecular switch for RPTPσ clustering and neuronal extension. Science, 332 (6028), pp. 484-488. Read abstract | Read more
Heparan and chondroitin sulfate proteoglycans (HSPGs and CSPGs, respectively) regulate numerous cell surface signaling events, with typically opposite effects on cell function. CSPGs inhibit nerve regeneration through receptor protein tyrosine phosphatase sigma (RPTPσ). Here we report that RPTPσ acts bimodally in sensory neuron extension, mediating CSPG inhibition and HSPG growth promotion. Crystallographic analyses of a shared HSPG-CSPG binding site reveal a conformational plasticity that can accommodate diverse glycosaminoglycans with comparable affinities. Heparan sulfate and analogs induced RPTPσ ectodomain oligomerization in solution, which was inhibited by chondroitin sulfate. RPTPσ and HSPGs colocalize in puncta on sensory neurons in culture, whereas CSPGs occupy the extracellular matrix. These results lead to a model where proteoglycans can exert opposing effects on neuronal extension by competing to control the oligomerization of a common receptor. Hide abstract
2011. A dual binding mode for RhoGTPases in plexin signalling PLoS Biology, 9 (8),
2010. Structural basis of semaphorin-plexin signalling. Nature, 467 (7319), pp. 1118-1122. Read abstract | Read more
Cell-cell signalling of semaphorin ligands through interaction with plexin receptors is important for the homeostasis and morphogenesis of many tissues and is widely studied for its role in neural connectivity, cancer, cell migration and immune responses. SEMA4D and Sema6A exemplify two diverse vertebrate, membrane-spanning semaphorin classes (4 and 6) that are capable of direct signalling through members of the two largest plexin classes, B and A, respectively. In the absence of any structural information on the plexin ectodomain or its interaction with semaphorins the extracellular specificity and mechanism controlling plexin signalling has remained unresolved. Here we present crystal structures of cognate complexes of the semaphorin-binding regions of plexins B1 and A2 with semaphorin ectodomains (human PLXNB1(1-2)-SEMA4D(ecto) and murine PlxnA2(1-4)-Sema6A(ecto)), plus unliganded structures of PlxnA2(1-4) and Sema6A(ecto). These structures, together with biophysical and cellular assays of wild-type and mutant proteins, reveal that semaphorin dimers independently bind two plexin molecules and that signalling is critically dependent on the avidity of the resulting bivalent 2:2 complex (monomeric semaphorin binds plexin but fails to trigger signalling). In combination, our data favour a cell-cell signalling mechanism involving semaphorin-stabilized plexin dimerization, possibly followed by clustering, which is consistent with previous functional data. Furthermore, the shared generic architecture of the complexes, formed through conserved contacts of the amino-terminal seven-bladed β-propeller (sema) domains of both semaphorin and plexin, suggests that a common mode of interaction triggers all semaphorin-plexin based signalling, while distinct insertions within or between blades of the sema domains determine binding specificity. Hide abstract
2010. Interactions between Hedgehog proteins and their binding partners come into view GENE DEV, 24 (18), pp. 2001-2012. | Read more
2009. Structural insights into hedgehog ligand sequestration by the human hedgehog-interacting protein HHIP. Nat Struct Mol Biol, 16 (7), pp. 698-703. Read abstract | Read more
Hedgehog (Hh) morphogens have fundamental roles in development, whereas dysregulation of Hh signaling leads to disease. Multiple cell-surface receptors are responsible for transducing and/or regulating Hh signals. Among these, the Hedgehog-interacting protein (Hhip) is a highly conserved, vertebrate-specific inhibitor of Hh signaling. We have solved a series of crystal structures for the human HHIP ectodomain and Desert hedgehog (DHH) in isolation, as well as HHIP in complex with DHH (HHIP-DHH) and Sonic hedgehog (Shh) (HHIP-Shh), with and without Ca2+. The interaction determinants, confirmed by biophysical studies and mutagenesis, reveal previously uncharacterized and distinct functions for the Hh Zn2+ and Ca2+ binding sites--functions that may be common to all vertebrate Hh proteins. Zn2+ makes a key contribution to the Hh-HHIP interface, whereas Ca2+ is likely to prevent electrostatic repulsion between the two proteins, suggesting an important modulatory role. This interplay of several metal binding sites suggests a tuneable mechanism for regulation of Hh signaling. Hide abstract
2008. Biochemical and structural studies of ASPP proteins reveal differential binding to p53, p63, and p73. Structure, 16 (2), pp. 259-268. Read abstract | Read more
ASPP1 and ASPP2 are activators of p53-dependent apoptosis, whereas iASPP is an inhibitor of p53. Binding assays showed differential binding for C-terminal domains of iASPP and ASPP2 to the core domains of p53 family members p53, p63, and p73. We also determined a high-resolution crystal structure for the C terminus of iASPP, comprised of four ankyrin repeats and an SH3 domain. The crystal lattice revealed an interaction between eight sequential residues in one iASPP molecule and the p53-binding site of a neighboring molecule. ITC confirmed that a peptide corresponding to the crystallographic interaction shows specific binding to iASPP. The contributions of ankyrin repeat residues, in addition to those of the SH3 domain, generate distinctive architecture at the p53-binding site suitable for inhibition by small molecules. These results suggest that the binding properties of iASPP render it a target for antitumor therapeutics and provide a peptide-based template for compound design. Hide abstract
2007. Structure of a tyrosine phosphatase adhesive interaction reveals a spacer-clamp mechanism. Science, 317 (5842), pp. 1217-1220. Read abstract | Read more
Cell-cell contacts are fundamental to multicellular organisms and are subject to exquisite levels of control. Human RPTPmu is a type IIB receptor protein tyrosine phosphatase that both forms an adhesive contact itself and is involved in regulating adhesion by dephosphorylating components of cadherin-catenin complexes. Here we describe a 3.1 angstrom crystal structure of the RPTPmu ectodomain that forms a homophilic trans (antiparallel) dimer with an extended and rigid architecture, matching the dimensions of adherens junctions. Cell surface expression of deletion constructs induces intercellular spacings that correlate with the ectodomain length. These data suggest that the RPTPmu ectodomain acts as a distance gauge and plays a key regulatory function, locking the phosphatase to its appropriate functional location. Hide abstract
2007. Mutations in alpha-tubulin cause abnormal neuronal migration in mice and lissencephaly in humans. Cell, 128 (1), pp. 45-57. Read abstract | Read more
The development of the mammalian brain is dependent on extensive neuronal migration. Mutations in mice and humans that affect neuronal migration result in abnormal lamination of brain structures with associated behavioral deficits. Here, we report the identification of a hyperactive N-ethyl-N-nitrosourea (ENU)-induced mouse mutant with abnormalities in the laminar architecture of the hippocampus and cortex, accompanied by impaired neuronal migration. We show that the causative mutation lies in the guanosine triphosphate (GTP) binding pocket of alpha-1 tubulin (Tuba1) and affects tubulin heterodimer formation. Phenotypic similarity with existing mouse models of lissencephaly led us to screen a cohort of patients with developmental brain anomalies. We identified two patients with de novo mutations in TUBA3, the human homolog of Tuba1. This study demonstrates the utility of ENU mutagenesis in the mouse as a means to discover the basis of human neurodevelopmental disorders. Hide abstract
2006. MHC class II proteins and disease: a structural perspective. Nat Rev Immunol, 6 (4), pp. 271-282. Read abstract | Read more
MHC class II molecules on the surface of antigen-presenting cells display a range of peptides for recognition by the T-cell receptors of CD4+ T helper cells. Therefore, MHC class II molecules are central to effective adaptive immune responses, but conversely, genetic and epidemiological data have implicated these molecules in the pathogenesis of autoimmune diseases. Indeed, the strength of the associations between particular MHC class II alleles and disease render them the main genetic risk factors for autoimmune disorders such as type 1 diabetes. Here, we discuss the insights that the crystal structures of MHC class II molecules provide into the molecular mechanisms by which sequence polymorphisms might contribute to disease susceptibility. Hide abstract
2005. High-resolution structure of the catalytic region of MICAL (molecule interacting with CasL), a multidomain flavoenzyme-signaling molecule. Proc Natl Acad Sci U S A, 102 (46), pp. 16836-16841. Read abstract | Read more
Semaphorins are extracellular cell guidance cues that govern cytoskeletal dynamics during neuronal and vascular development. MICAL (molecule interacting with CasL) is a multidomain cytosolic protein with a putative flavoprotein monooxygenase (MO) region required for semaphorin-plexin repulsive axon guidance. Here, we report the 1.45-A resolution crystal structure of the FAD-containing MO domain of mouse MICAL-1 (residues 1-489). The topology most closely resembles that of the NADPH-dependent flavoenzyme p-hydroxybenzoate hydroxylase (PHBH). Comparison of structures before and after reaction with NADPH reveals that, as in PHBH, the flavin ring can switch between two discrete positions. In contrast with other MOs, this conformational switch is coupled with the opening of a channel to the active site, suggestive of a protein substrate. In support of this hypothesis, distinctive structural features highlight putative protein-binding sites in suitable proximity to the active site entrance. The unusual juxtaposition of this N-terminal MO (hydroxylase) activity with the characteristics of a multiprotein-binding scaffold exhibited by the C-terminal portion of the MICALs represents a unique combination of functionality to mediate signaling. Hide abstract
2004. Crystal structure of HLA-DQ0602 that protects against type 1 diabetes and confers strong susceptibility to narcolepsy. Proc Natl Acad Sci U S A, 101 (7), pp. 1999-2004. Read abstract | Read more
The MHC class II molecule DQ0602 confers strong susceptibility to narcolepsy but dominant protection against type 1 diabetes. The crystal structure of DQ0602 reveals the molecular features underlying these contrasting genetic properties. Structural comparisons to homologous DQ molecules with differential disease associations highlight a previously unrecognized interplay between the volume of the P6 pocket and the specificity of the P9 pocket, which implies that presentation of an expanded peptide repertoire is critical for dominant protection against type 1 diabetes. In narcolepsy, the volume of the P4 pocket appears central to the susceptibility, suggesting that the presentation of a specific peptide population plays a major role. Hide abstract
Molecular mechanisms of the Hedgehog signalling pathway
The morphogen Hedgehog (Hh) is a secreted, extracellular protein which activates an essential cellular pathway in the embryogenesis of various organisms (Beachy et al ). Hence, as well as its crucial role in embryonic development, Hh signalling is key in adult stem cell regulation in various tissues and species highlighting its potential importance in the field of regenerative medicine. The transduction of Hh signalling in cells is mediated by the two multi-transmembrane proteins Patched ...
