Structural biology of morphogen signalling

Project Overview

Top: Crystal structures of the Hh morphogen signal transducer and GPCR Smoothened in different ...

My group aims to decipher the function of proteins and protein complexes that mediate signalling by morphogen-activated signalling cascades, such as the Hedgehog (Hh) and Bone Morphogenetic Protein (BMP) pathways using state-of-the-art structural (x-ray crystallography, cryo-electron microscopy, solution x-ray scattering), biophysical and cell-based (e.g. live cell super-resolution imaging) techniques. We aim to answer the following key questions: What are the determinants for morphogen interactions with their receptors and modulators at the signal-receiving cell? How is the extracellular morphogen signal communicated through the cell? What are the consequences of morphogen dysfunction in disease? To shed light on these questions, we aim to obtain molecular snapshots of these processes and combine atomic details with biochemical and cellular studies. Our work led to a significant number of crystal structures and new strategies to modulate these fundamental signalling pathways. e.g. the structural and functional analysis of the Hedgehog signal transducer, oncoprotein and G protein coupled receptor Smoothened (Nature 2016, Elife 2016 and 2013, Fig. 1 top panel), complexes of Hedgehog morphogens with their receptors (Nature Struc Mol Biol 2009, PNAS 2013), and the Repulsive Guidance Molecule (RGM) protein family with BMP morphogens (Science 2013, Nat Struc Mol Biol 2015, Fig. 1 bottom panel).

Training Opportunities

The student will join a very dynamic and lively team of DPhil students and postdocs and will gain experience in the whole spectrum of modern structural biology techniques with a focus on X-ray crystallography and cryo electron microscopy of human membrane proteins as well as soluble multi-subunit protein-protein complexes. Techniques range from molecular biology and protein expression, biochemical and biophysical analyses to X-ray crystallography, in silico structural analysis and super-resolution fluorescence and cryo electron microscopy applying cutting-edge technologies. The work will benefit from our expertise in glycoprotein complexes and large multi-spanning transmembrane receptors, established mammalian cell culture facilities, and the developments and ready access to the DIAMOND synchrotron site. Additionally, the student will have the opportunity to interface with a network of collaborations in Oxford, UK and international to relate his/her structural and molecular results to the biomedical and in vivo functional context.

Theme

Protein Science & Structural Biology and Cancer Biology

Admissions

Project reference number: 118

Funding and admissions information

Supervisors

Name Department Institution Country Email
Professor Christian Siebold Structural Biology Oxford University, Henry Wellcome Building of Genomic Medicine GBR christian.siebold@strubi.ox.ac.uk
Professor E. Yvonne Jones FRS FMedSci Structural Biology Oxford University, Henry Wellcome Building of Genomic Medicine GBR jones-pa@strubi.ox.ac.uk

Byrne EFX, Sircar R, Miller PS, Hedger G, Luchetti G, Nachtergaele S, Tully MD, Mydock-McGrane L, Covey DF, Rambo RP, Sansom MSP, Newstead S, Rohatgi R, Siebold C. 2016. Structural basis of Smoothened regulation by its extracellular domains. Nature, 535 (7613), pp. 517-522. Read abstract | Read more

Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzledclass G-protein-coupled receptors (GPCRs) composed of both a heptahelical transmembrane domain (TMD) and an extracellular cysteine-rich domain (CRD). How the large extracellular domains of GPCRs regulate signalling by the TMD is unknown. We present crystal structures of the Hh signal transducer and oncoprotein Smoothened, a GPCR that contains two distinct ligand-binding sites: one in its TMD and one in the CRD. The CRD is stacked a top the TMD, separated by an intervening wedge-like linker domain. Structure-guided mutations show that the interface between the CRD, linker domain and TMD stabilizes the inactive state of Smoothened. Unexpectedly, we find a cholesterol molecule bound to Smoothened in the CRD binding site. Mutations predicted to prevent cholesterol binding impair the ability of Smoothened to transmit native Hh signals. Binding of a clinically used antagonist, vismodegib, to the TMD induces a conformational change that is propagated to the CRD, resulting in loss of cholesterol from the CRD-linker domain-TMD interface. Our results clarify the structural mechanism by which the activity of a GPCR is controlled by ligand-regulated interactions between its extracellular and transmembrane domains. Hide abstract

Healey EG, Bishop B, Elegheert J, Bell CH, Padilla-Parra S, Siebold C. 2015. Repulsive guidance molecule is a structural bridge between neogenin and bone morphogenetic protein. Nat. Struct. Mol. Biol., 22 (6), pp. 458-65. Read abstract | Read more

Repulsive guidance molecules (RGMs) control crucial processes including cell motility, adhesion, immune-cell regulation and systemic iron metabolism. RGMs signal via the neogenin (NEO1) and the bone morphogenetic protein (BMP) pathways. Here, we report crystal structures of the N-terminal domains of all human RGM family members in complex with the BMP ligand BMP2, revealing a new protein fold and a conserved BMP-binding mode. Our structural and functional data suggest a pH-linked mechanism for RGM-activated BMP signaling and offer a rationale for RGM mutations causing juvenile hemochromatosis. We also determined the crystal structure of the ternary BMP2-RGM-NEO1 complex, which, along with solution scattering and live-cell super-resolution fluorescence microscopy, indicates BMP-induced clustering of the RGM-NEO1 complex. Our results show how RGM acts as the central hub that links BMP and NEO1 and physically connects these fundamental signaling pathways. Hide abstract

Bell CH, Healey E, van Erp S, Bishop B, Tang C, Gilbert RJ, Aricescu AR, Pasterkamp RJ, Siebold C. 2013. Structure of the repulsive guidance molecule (RGM)-neogenin signaling hub. Science, 341 (6141), pp. 77-80. Read abstract | Read more

Repulsive guidance molecule family members (RGMs) control fundamental and diverse cellular processes, including motility and adhesion, immune cell regulation, and systemic iron metabolism. However, it is not known how RGMs initiate signaling through their common cell-surface receptor, neogenin (NEO1). Here, we present crystal structures of the NEO1 RGM-binding region and its complex with human RGMB (also called dragon). The RGMB structure reveals a previously unknown protein fold and a functionally important autocatalytic cleavage mechanism and provides a framework to explain numerous disease-linked mutations in RGMs. In the complex, two RGMB ectodomains conformationally stabilize the juxtamembrane regions of two NEO1 receptors in a pH-dependent manner. We demonstrate that all RGM-NEO1 complexes share this architecture, which therefore represents the core of multiple signaling pathways. Hide abstract

Bishop B, Aricescu AR, Harlos K, O'Callaghan CA, Jones EY, Siebold C. 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

Nachtergaele S, Whalen DM, Mydock LK, Zhao Z, Malinauskas T, Krishnan K, Ingham PW, Covey DF, Siebold C, Rohatgi R. 2013. Structure and function of the Smoothened extracellular domain in vertebrate Hedgehog signaling. Elife, 2 pp. e01340. Read abstract | Read more

The Hedgehog (Hh) signal is transduced across the membrane by the heptahelical protein Smoothened (Smo), a developmental regulator, oncoprotein and drug target in oncology. We present the 2.3 Å crystal structure of the extracellular cysteine rich domain (CRD) of vertebrate Smo and show that it binds to oxysterols, endogenous lipids that activate Hh signaling. The oxysterol-binding groove in the Smo CRD is analogous to that used by Frizzled 8 to bind to the palmitoleyl group of Wnt ligands and to similar pockets used by other Frizzled-like CRDs to bind hydrophobic ligands. The CRD is required for signaling in response to native Hh ligands, showing that it is an important regulatory module for Smo activation. Indeed, targeting of the Smo CRD by oxysterol-inspired small molecules can block signaling by all known classes of Hh activators and by clinically relevant Smo mutants. DOI:http://dx.doi.org/10.7554/eLife.01340.001. Hide abstract

Whalen DM, Malinauskas T, Gilbert RJ, Siebold C. 2013. Structural insights into proteoglycan-shaped Hedgehog signaling. Proc. Natl. Acad. Sci. U.S.A., 110 (41), pp. 16420-5. Read abstract | Read more

Hedgehog (Hh) morphogens play fundamental roles during embryogenesis and adulthood, in health and disease. Multiple cell surface receptors regulate the Hh signaling pathway. Among these, the glycosaminoglycan (GAG) chains of proteoglycans shape Hh gradients and signal transduction. We have determined crystal structures of Sonic Hh complexes with two GAGs, heparin and chondroitin sulfate. The interaction determinants, confirmed by site-directed mutagenesis and binding studies, reveal a previously not identified Hh site for GAG binding, common to all Hh proteins. The majority of Hh residues forming this GAG-binding site have been previously implicated in developmental diseases. Crystal packing analysis, combined with analytical ultracentrifugation of Sonic Hh-GAG complexes, suggests a potential mechanism for GAG-dependent Hh multimerization. Taken together, these results provide a direct mechanistic explanation of the observed correlation between disease and impaired Hh gradient formation. Moreover, GAG binding partially overlaps with the site of Hh interactions with an array of protein partners including Patched, hedgehog interacting protein, and the interference hedgehog protein family, suggesting a unique mechanism of Hh signaling modulation. Hide abstract