I03-Macromolecular Crystallography
I23-Long wavelength MX
I24-Microfocus Macromolecular Crystallography
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Tomas
Malinauskas
,
Gareth
Moore
,
Amalie F.
Rudolf
,
Holly
Eggington
,
Hayley L.
Belnoue-Davis
,
Kamel
El Omari
,
Samuel C.
Griffiths
,
Rachel E.
Woolley
,
Ramona
Duman
,
Armin
Wagner
,
Simon J.
Leedham
,
Clair
Baldock
,
Hilary L.
Ashe
,
Christian
Siebold
Diamond Proposal Number(s):
[28534, 14744, 19946]
Open Access
Abstract: Twisted gastrulation (TWSG1) is an evolutionarily conserved secreted glycoprotein which controls signaling by Bone Morphogenetic Proteins (BMPs). TWSG1 binds BMPs and their antagonist Chordin to control BMP signaling during embryonic development, kidney regeneration and cancer. We report crystal structures of TWSG1 alone and in complex with a BMP ligand, Growth Differentiation Factor 5. TWSG1 is composed of two distinct, disulfide-rich domains. The TWSG1 N-terminal domain occupies the BMP type 1 receptor binding site on BMPs, whereas the C-terminal domain binds to a Chordin family member. We show that TWSG1 inhibits BMP function in cellular signaling assays and mouse colon organoids. This inhibitory function is abolished in a TWSG1 mutant that cannot bind BMPs. The same mutation in the Drosophila TWSG1 ortholog Tsg fails to mediate BMP gradient formation required for dorsal-ventral axis patterning of the early embryo. Our studies reveal the evolutionarily conserved mechanism of BMP signaling inhibition by TWSG1.
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Jun 2024
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I23-Long wavelength MX
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Diamond Proposal Number(s):
[14744, 9946]
Open Access
Abstract: The receptor tyrosine kinase ROR2 mediates noncanonical WNT5A signaling to orchestrate tissue morphogenetic processes, and dysfunction of the pathway causes Robinow syndrome, Brachydactyly B and metastatic diseases. The domain(s) and mechanisms required for ROR2 function, however, remain unclear. We solved the crystal structure of the extracellular cysteine-rich (CRD) and Kringle (Kr) domains of ROR2 and found that, unlike other CRDs, the ROR2 CRD lacks the signature hydrophobic pocket that binds lipids/lipid-modified proteins, such as WNTs, suggesting a novel mechanism of ligand reception. Functionally, we showed that the ROR2 CRD, but not other domains, is required and minimally sufficient to promote WNT5A signaling, and Robinow mutations in the CRD and the adjacent Kr impair ROR2 secretion and function. Moreover, using function-activating and -perturbing antibodies against the Frizzled (FZ) family of WNT receptors, we demonstrate the involvement of FZ in WNT5A-ROR signaling. Thus, ROR2 acts via its CRD to potentiate the function of a receptor super-complex that includes FZ to transduce WNT5A signals.
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May 2024
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I24-Microfocus Macromolecular Crystallography
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Maia
Kinnebrew
,
Rachel E.
Woolley
,
T. Bertie
Ansell
,
Eamon F. X.
Byrne
,
Sara
Frigui
,
Giovanni
Luchetti
,
Ria
Sircar
,
Sigrid
Nachtergaele
,
Laurel
Mydock-Mcgrane
,
Kathiresan
Krishnan
,
Simon
Newstead
,
Mark S. P.
Sansom
,
Douglas F.
Covey
,
Christian
Siebold
,
Rajat
Rohatgi
Diamond Proposal Number(s):
[14744, 19946]
Open Access
Abstract: Smoothened (SMO) transduces the Hedgehog (Hh) signal across the plasma membrane in response to accessible cholesterol. Cholesterol binds SMO at two sites: one in the extracellular cysteine-rich domain (CRD) and a second in the transmembrane domain (TMD). How these two sterol-binding sites mediate SMO activation in response to the ligand Sonic Hedgehog (SHH) remains unknown. We find that mutations in the CRD (but not the TMD) reduce the fold increase in SMO activity triggered by SHH. SHH also promotes the photocrosslinking of a sterol analog to the CRD in intact cells. In contrast, sterol binding to the TMD site boosts SMO activity regardless of SHH exposure. Mutational and computational analyses show that these sites are in allosteric communication despite being 45 angstroms apart. Hence, sterols function as both SHH-regulated orthosteric ligands at the CRD and allosteric ligands at the TMD to regulate SMO activity and Hh signaling.
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Jun 2022
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Samuel C.
Griffiths
,
Rebekka A.
Schwab
,
Kamel
El Omari
,
Benjamin
Bishop
,
Ellen J.
Iverson
,
Tomas
Malinauskas
,
Ramin
Dubey
,
Mingxing
Qian
,
Douglas F.
Covey
,
Robert J. C.
Gilbert
,
Rajat
Rohatgi
,
Christian
Siebold
Diamond Proposal Number(s):
[19946, 14744]
Open Access
Abstract: Hedgehog (HH) morphogen signalling, crucial for cell growth and tissue patterning in animals, is initiated by the binding of dually lipidated HH ligands to cell surface receptors. Hedgehog-Interacting Protein (HHIP), the only reported secreted inhibitor of Sonic Hedgehog (SHH) signalling, binds directly to SHH with high nanomolar affinity, sequestering SHH. Here, we report the structure of the HHIP N-terminal domain (HHIP-N) in complex with a glycosaminoglycan (GAG). HHIP-N displays a unique bipartite fold with a GAG-binding domain alongside a Cysteine Rich Domain (CRD). We show that HHIP-N is required to convey full HHIP inhibitory function, likely by interacting with the cholesterol moiety covalently linked to HH ligands, thereby preventing this SHH-attached cholesterol from binding to the HH receptor Patched (PTCH1). We also present the structure of the HHIP C-terminal domain in complex with the GAG heparin. Heparin can bind to both HHIP-N and HHIP-C, thereby inducing clustering at the cell surface and generating a high-avidity platform for SHH sequestration and inhibition. Our data suggest a multimodal mechanism, in which HHIP can bind two specific sites on the SHH morphogen, alongside multiple GAG interactions, to inhibit SHH signalling.
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Dec 2021
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Krios I-Titan Krios I at Diamond
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Claire E.
Coupland
,
Sebastian A.
Andrei
,
T. Bertie
Ansell
,
Loic
Carrique
,
Pramod
Kumar
,
Lea
Sefer
,
Rebekka A.
Schwab
,
Eamon F. X.
Byrne
,
Els
Pardon
,
Jan
Steyaert
,
Anthony I.
Magee
,
Thomas
Lanyon-Hogg
,
Mark S. P.
Sansom
,
Edward W.
Tate
,
Christian
Siebold
Diamond Proposal Number(s):
[20223]
Open Access
Abstract: The Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). We present the high-resolution cryo-EM structure of HHAT bound to substrate analog palmityl-coenzyme A and a SHH-mimetic megabody, revealing a heme group bound to HHAT that is essential for HHAT function. A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. This structure of a membrane-bound O-acyltransferase (MBOAT) superfamily member provides a blueprint for other protein-substrate MBOATs and a template for future drug discovery.
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Dec 2021
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
Krios IV-Titan Krios IV at Diamond
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Ross A.
Robinson
,
Samuel C.
Griffiths
,
Lieke L.
Van De Haar
,
Tomas
Malinauskas
,
Eljo Y.
Van Battum
,
Pavol
Zelina
,
Rebekka A.
Schwab
,
Dimple
Karia
,
Lina
Malinauskaite
,
Sara
Brignani
,
Marleen H.
Van Den Munkhof
,
Özge
Düdükcü
,
Anna A.
De Ruiter
,
Dianne M.a.
Van Den Heuvel
,
Benjamin
Bishop
,
Jonathan
Elegheert
,
A. Radu
Aricescu
,
R. Jeroen
Pasterkamp
,
Christian
Siebold
Diamond Proposal Number(s):
[19946, 20223]
Open Access
Abstract: During cell migration or differentiation, cell surface receptors are simultaneously exposed to different ligands. However, it is often unclear how these extracellular signals are integrated. Neogenin (NEO1) acts as an attractive guidance receptor when the Netrin-1 (NET1) ligand binds, but it mediates repulsion via repulsive guidance molecule (RGM) ligands. Here, we show that signal integration occurs through the formation of a ternary NEO1-NET1-RGM complex, which triggers reciprocal silencing of downstream signaling. Our NEO1-NET1-RGM structures reveal a “trimer-of-trimers” super-assembly, which exists in the cell membrane. Super-assembly formation results in inhibition of RGMA-NEO1-mediated growth cone collapse and RGMA- or NET1-NEO1-mediated neuron migration, by preventing formation of signaling-compatible RGM-NEO1 complexes and NET1-induced NEO1 ectodomain clustering. These results illustrate how simultaneous binding of ligands with opposing functions, to a single receptor, does not lead to competition for binding, but to formation of a super-complex that diminishes their functional outputs.
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Mar 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946]
Abstract: A relatively small number of proteins have been suggested to act as morphogens—signalling molecules that spread within tissues to organize tissue repair and the specification of cell fate during development. Among them are Wnt proteins, which carry a palmitoleate moiety that is essential for signalling activity1,2,3. How a hydrophobic lipoprotein can spread in the aqueous extracellular space is unknown. Several mechanisms, such as those involving lipoprotein particles, exosomes or a specific chaperone, have been proposed to overcome this so-called Wnt solubility problem4,5,6. Here we provide evidence against these models and show that the Wnt lipid is shielded by the core domain of a subclass of glypicans defined by the Dally-like protein (Dlp). Structural analysis shows that, in the presence of palmitoleoylated peptides, these glypicans change conformation to create a hydrophobic space. Thus, glypicans of the Dlp family protect the lipid of Wnt proteins from the aqueous environment and serve as a reservoir from which Wnt proteins can be handed over to signalling receptors.
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Jul 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14744, 10627]
Open Access
Abstract: Repulsive guidance molecules (RGMs) are cell surface proteins that regulate the development and homeostasis of many tissues and organs, including the nervous, skeletal, and immune systems. They control fundamental biological processes, such as migration and differentiation by direct interaction with the Neogenin (NEO1) receptor and function as coreceptors for the bone morphogenetic protein (BMP)/growth differentiation factor (GDF) family. We determined crystal structures of all three human RGM family members in complex with GDF5, as well as the ternary NEO1–RGMB–GDF5 assembly. Surprisingly, we show that all three RGMs inhibit GDF5 signaling, which is in stark contrast to RGM-mediated enhancement of signaling observed for other BMPs, like BMP2. Despite their opposite effect on GDF5 signaling, RGMs occupy the BMP type 1 receptor binding site similar to the observed interactions in RGM–BMP2 complexes. In the NEO1–RGMB–GDF5 complex, RGMB physically bridges NEO1 and GDF5, suggesting cross-talk between the GDF5 and NEO1 signaling pathways. Our crystal structures, combined with structure-guided mutagenesis of RGMs and BMP ligands, binding studies, and cellular assays suggest that RGMs inhibit GDF5 signaling by competing with GDF5 type 1 receptors. While our crystal structure analysis and in vitro binding data initially pointed towards a simple competition mechanism between RGMs and type 1 receptors as a possible basis for RGM-mediated GDF5 inhibition, further experiments utilizing BMP2-mimicking GDF5 variants clearly indicate a more complex mechanism that explains how RGMs can act as a functionality-changing switch for two structurally and biochemically similar signaling molecules.
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Jun 2020
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I04-Macromolecular Crystallography
I23-Long wavelength MX
I24-Microfocus Macromolecular Crystallography
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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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