I23-Long wavelength MX
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Amalie F.
Rudolf
,
Maia
Kinnebrew
,
Christiane
Kowatsch
,
T. Bertie
Ansell
,
Kamel
El Omari
,
Benjamin
Bishop
,
Els
Pardon
,
Rebekka A.
Schwab
,
Tomas
Malinauskas
,
Mingxing
Qian
,
Ramona
Duman
,
Douglas F.
Covey
,
Jan
Steyaert
,
Armin
Wagner
,
Mark S. P.
Sansom
,
Rajat
Rohatgi
,
Christian
Siebold
Abstract: Hedgehog (HH) ligands, classical morphogens that pattern embryonic tissues in all animals, are covalently coupled to two lipids—a palmitoyl group at the N terminus and a cholesteroyl group at the C terminus. While the palmitoyl group binds and inactivates Patched 1 (PTCH1), the main receptor for HH ligands, the function of the cholesterol modification has remained mysterious. Using structural and biochemical studies, along with reassessment of previous cryo-electron microscopy structures, we find that the C-terminal cholesterol attached to Sonic hedgehog (Shh) binds the first extracellular domain of PTCH1 and promotes its inactivation, thus triggering HH signaling. Molecular dynamics simulations show that this interaction leads to the closure of a tunnel through PTCH1 that serves as the putative conduit for sterol transport. Thus, Shh inactivates PTCH1 by grasping its extracellular domain with two lipidic pincers, the N-terminal palmitate and the C-terminal cholesterol, which are both inserted into the PTCH1 protein core.
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Oct 2019
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I24-Microfocus Macromolecular Crystallography
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Shuo
Chen
,
Jiale
Wu
,
Shan
Zhong
,
Yuntong
Li
,
Ping
Zhang
,
Jingyi
Ma
,
Jingshan
Ren
,
Yun
Tan
,
Yunhao
Wang
,
Kin Fai
Au
,
Christian
Siebold
,
Gareth L.
Bond
,
Zhu
Chen
,
Min
Lu
,
E. Yvonne
Jones
,
Xin
Lu
Open Access
Abstract: The most frequently mutated protein in human cancer is p53, a transcription factor (TF) that regulates myriad genes instrumental in diverse cellular outcomes including growth arrest and cell death. Cell context-dependent p53 modulation is critical for this life-or-death balance, yet remains incompletely understood. Here we identify sequence signatures enriched in genomic p53-binding sites modulated by the transcription cofactor iASPP. Moreover, our p53–iASPP crystal structure reveals that iASPP displaces the p53 L1 loop—which mediates sequence-specific interactions with the signature-corresponding base—without perturbing other DNA-recognizing modules of the p53 DNA-binding domain. A TF commonly uses multiple structural modules to recognize its cognate DNA, and thus this mechanism of a cofactor fine-tuning TF–DNA interactions through targeting a particular module is likely widespread. Previously, all tumor suppressors and oncoproteins that associate with the p53 DNA-binding domain—except the oncogenic E6 from human papillomaviruses (HPVs)—structurally cluster at the DNA-binding site of p53, complicating drug design. By contrast, iASPP inhibits p53 through a distinct surface overlapping the E6 footprint, opening prospects for p53-targeting precision medicine to improve cancer therapy.
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Aug 2019
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14744, 10627]
Open Access
Abstract: Semaphorin ligands and their plexin receptors are one of the major cell guidance factors that trigger localised changes in the cytoskeleton. Binding of semaphorin homodimer to plexin brings two plexins in close proximity which is a prerequisite for plexin signalling. This model appears to be too simplistic to explain the complexity and functional versatility of these molecules. Here, we determine crystal structures for all members of Drosophila class 1 and 2 semaphorins. Unlike previously reported semaphorin structures, Sema1a, Sema2a and Sema2b show stabilisation of sema domain dimer formation via a disulfide bond. Unexpectedly, our structural and biophysical data show Sema1b is a monomer suggesting that semaphorin function may not be restricted to dimers. We demonstrate that semaphorins can form heterodimers with members of the same semaphorin class. This heterodimerization provides a potential mechanism for cross-talk between different plexins and co-receptors to allow fine-tuning of cell signalling.
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Aug 2019
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B21-High Throughput SAXS
I24-Microfocus Macromolecular Crystallography
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Eamon F. X.
Byrne
,
Ria
Sircar
,
Paul S.
Miller
,
George
Hedger
,
Giovanni
Luchetti
,
Sigrid
Nachtergaele
,
Mark D.
Tully
,
Laurel
Mydock-mcgrane
,
Douglas F.
Covey
,
Robert P.
Rambo
,
Mark S. P.
Sansom
,
Simon
Newstead
,
Rajat
Rohatgi
,
Christian
Siebold
Diamond Proposal Number(s):
[10627]
Open Access
Abstract: Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzled-class 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 atop 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.
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Jul 2016
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Jonathan
Elegheert
,
W.
Kakegawa
,
J.
Clay
,
N. F.
Shanks
,
E.
Behiels
,
K.
Matsuda
,
K.
Kohda
,
E.
Miura
,
M.
Rossmann
,
Nikolaos
Mitakidis
,
J.
Motohashi
,
Veronica T.
Chang
,
Christian
Siebold
,
Ingo H.
Greger
,
Terunaga
Nakagawa
,
M.
Yuzaki
,
A. Radu
Aricescu
Diamond Proposal Number(s):
[8423, 10627]
Abstract: Ionotropic glutamate receptor (iGluR) family members are integrated into supramolecular complexes that modulate their location and function at excitatory synapses. However, a lack of structural information beyond isolated receptors or fragments thereof currently limits the mechanistic understanding of physiological iGluR signaling. Here, we report structural and functional analyses of the prototypical molecular bridge linking postsynaptic iGluR δ2 (GluD2) and presynaptic β-neurexin 1 (β-NRX1) via Cbln1, a C1q-like synaptic organizer. We show how Cbln1 hexamers “anchor” GluD2 amino-terminal domain dimers to monomeric β-NRX1. This arrangement promotes synaptogenesis and is essential for d-serine–dependent GluD2 signaling in vivo, which underlies long-term depression of cerebellar parallel fiber–Purkinje cell (PF-PC) synapses and motor coordination in developing mice. These results lead to a model where protein and small-molecule ligands synergistically control synaptic iGluR function.
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Jul 2016
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Veronica T
Chang
,
Ricardo A
Fernandes
,
Kristina A
Ganzinger
,
Steven F
Lee
,
Christian
Siebold
,
James
Mccoll
,
Peter
Jönsson
,
Matthieu
Palayret
,
Karl
Harlos
,
Charlotte H
Coles
,
Edith
Jones
,
Yuan
Lui
,
Elizabeth
Huang
,
Robert J C
Gilbert
,
David
Klenerman
,
A Radu
Aricescu
,
Simon J
Davis
Abstract: It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell antigen receptor (TCR) triggering, but how such segregation occurs and whether it can initiate signaling is unclear. Using structural and biophysical analysis, we show that the extracellular region of CD45 is rigid and extends beyond the distance spanned by TCR-ligand complexes, implying that sites of TCR-ligand engagement would sterically exclude CD45. We also show that the formation of 'close contacts', new structures characterized by spontaneous CD45 and kinase segregation at the submicron-scale, initiates signaling even when TCR ligands are absent. Our work reveals the structural basis for, and the potent signaling effects of, local CD45 and kinase segregation. TCR ligands have the potential to heighten signaling simply by holding receptors in close contacts.
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Mar 2016
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[10627]
Open Access
Abstract: 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.
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May 2015
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I03-Macromolecular Crystallography
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Open Access
Abstract: 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
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Oct 2013
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I03-Macromolecular Crystallography
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Abstract: 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, heparinand chondroitin sulfate.
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Sep 2013
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8423]
Abstract: 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).
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Jul 2013
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