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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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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Krios I-Titan Krios I at Diamond
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Qinrui
Wang
,
Robin A.
Corey
,
George
Hedger
,
Prafulla
Aryal
,
Mariana
Grieben
,
Chady
Nasrallah
,
Agnese
Baronina
,
Ashley C. W.
Pike
,
Jiye
Shi
,
Elisabeth P.
Carpenter
,
Mark S. P.
Sansom
Diamond Proposal Number(s):
[14856]
Open Access
Abstract: Polycystin-2 (PC2) is a transient receptor potential (TRP) channel present in ciliary membranes of the kidney. PC2 shares a transmembrane fold with other TRP channels, in addition to an extracellular domain found in TRPP and TRPML channels. Using molecular dynamics (MD) simulations and cryoelectron microscopy we identify and characterize PIP2 and cholesterol interactions with PC2. PC2 is revealed to have a PIP binding site close to the equivalent vanilloid/lipid binding site in the TRPV1 channel. A 3.0-Å structure reveals a binding site for cholesterol on PC2. Cholesterol interactions with the channel at this site are characterized by MD simulations. The two classes of lipid binding sites are compared with sites observed in other TRPs and in Kv channels. These findings suggest PC2, in common with other ion channels, may be modulated by both PIPs and cholesterol, and position PC2 within an emerging model of the roles of lipids in the regulation and organization of ciliary membranes.
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Dec 2019
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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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Open Access
Abstract: Kindlins co-activate integrins alongside talin. They possess, like talin, a FERM domain comprising F0-F3 subdomains, but with a pleckstrin homology (PH) domain inserted in the F2 subdomain that enables membrane association. We present the crystal structure of murine kindlin-3 PH domain determined at 2.23Å resolution and characterise its lipid binding using biophysical and computational approaches. Molecular dynamics (MD) simulations suggest flexibility in the PH domain loops connecting β-strands forming the putative phosphatidylinositol phosphate (PtdInsP) binding site. Simulations with PtdInsP-containing bilayers reveal that the PH domain associates with PtdInsP molecules mainly via the positively charged surface presented by the β1-β2 loop and that it binds with somewhat higher affinity to PtdIns(3,4,5)P3 compared to PtdIns(4,5)P2. Surface plasmon resonance (SPR) with lipid headgroups immobilised and the PH domain as analyte indicate affinities of 300 μM for PtdIns(3,4,5)P3 and 1mM for PtdIns(4,5)P2. In contrast, SPR studies with immobilised PH domain and lipid nanodiscs as analyte show affinities of 0.40 µM for PtdIns(3,4,5)P3 and no affinity for PtdIns(4,5)P2 when the inositol phosphate constitutes 5% of the total lipids (~5 molecules per nanodisc). Reducing the PtdIns(3,4,5)P3 composition to 1% abolishes nanodisc binding to the PH domain, as does site-directed mutagenesis of two lysines within the β1-β2 loop. Binding of PtdIns(3,4,5)P3 by a canonical PH domain, Grp1, is not similarly influenced by SPR experimental design. These data suggest a role for PtdIns(3,4,5)P3 clustering in the binding of some PH domains and not others, highlighting the importance lipid mobility and clustering for the biophysical assessment of protein-membrane interactions.
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Dec 2016
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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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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Verity A.
Jackson
,
Shahid
Mehmood
,
Matthieu
Chavent
,
Pietro
Roversi
,
Maria
Carrasquero
,
Daniel
Del Toro
,
Goenuel
Seyit-Bremer
,
Fanomezana M.
Ranaivoson
,
Davide
Comoletti
,
Mark S. P.
Sansom
,
Carol V.
Robinson
,
Rüdiger
Klein
,
Elena
Seiradake
Diamond Proposal Number(s):
[9306, 8423, 1747]
Open Access
Abstract: Latrophilin adhesion-GPCRs (Lphn1–3 or ADGRL1–3) and Unc5 cell guidance receptors (Unc5A–D) interact with FLRT proteins (FLRT1–3), thereby promoting cell adhesion and repulsion, respectively. How the three proteins interact and function simultaneously is poorly understood. We show that Unc5D interacts with FLRT2 in cis, controlling cell adhesion in response to externally presented Lphn3. The ectodomains of the three proteins bind cooperatively. Crystal structures of the ternary complex formed by the extracellular domains reveal that Lphn3 dimerizes when bound to FLRT2:Unc5, resulting in a stoichiometry of 1:1:2 (FLRT2:Unc5D:Lphn3). This 1:1:2 complex further dimerizes to form a larger ‘super-complex’ (2:2:4), using a previously undescribed binding motif in the Unc5D TSP1 domain. Molecular dynamics simulations, point-directed mutagenesis and mass spectrometry demonstrate the stability and molecular properties of these complexes. Our data exemplify how receptors increase their functional repertoire by forming different context-dependent higher-order complexes.
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Apr 2016
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I24-Microfocus Macromolecular Crystallography
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Dianfan
Li
,
Phillip J.
Stansfeld
,
Mark S. P.
Sansom
,
Aaron
Keogh
,
Lutz
Vogeley
,
Nicole
Howe
,
Joseph
Lyons
,
David
Aragao
,
Petra
Fromme
,
Raimund
Fromme
,
Shibom
Basu
,
Ingo
Grotjohann
,
Christopher
Kupitz
,
Kimberley
Rendek
,
Uwe
Weierstall
,
Nadia A.
Zatsepin
,
Vadim
Cherezov
,
Wei
Liu
,
Sateesh
Bandaru
,
Niall J.
English
,
Cornelius
Gati
,
Anton
Barty
,
Oleksandr
Yefanov
,
Henry N.
Chapman
,
Kay
Diederichs
,
Marc
Messerschmidt
,
Sébastien
Boutet
,
Garth J.
Williams
,
M.
Marvin Seibert
,
Martin
Caffrey
Open Access
Abstract: Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution.
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Dec 2015
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I04-Macromolecular Crystallography
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Christos
Pliotas
,
A Caroline E.
Dahl
,
Tim
Rasmussen
,
Kozhinjampara R
Mahendran
,
Terry K
Smith
,
Phedra
Marius
,
Joseph
Gault
,
Thandiwe
Banda
,
Akiko
Rasmussen
,
Samantha
Miller
,
Carol V.
Robinson
,
Hagan
Bayley
,
Mark S. P.
Sansom
,
Ian R.
Booth
,
James H
Naismith
Abstract: The ability of proteins to sense membrane tension is pervasive in biology. A higher-resolution structure of the Escherichia coli small-conductance mechanosensitive channel MscS identifies alkyl chains inside pockets formed by the transmembrane helices (TMs). Purified MscS contains E. coli lipids, and fluorescence quenching demonstrates that phospholipid acyl chains exchange between bilayer and TM pockets. Molecular dynamics and biophysical analyses show that the volume of the pockets and thus the number of lipid acyl chains within them decreases upon channel opening. Phospholipids with one acyl chain per head group (lysolipids) displace normal phospholipids (with two acyl chains) from MscS pockets and trigger channel opening. We propose that the extent of acyl-chain interdigitation in these pockets determines the conformation of MscS. When interdigitation is perturbed by increased membrane tension or by lysolipids, the closed state becomes unstable, and the channel gates.
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Nov 2015
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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John
Beale
,
Joanne
Parker
,
Firdaus
Samsudin
,
Anne l.
Barrett
,
Anish
Senan
,
Louise
Bird
,
David
Scott
,
Raymond
Owens
,
Mark S. P
Sansom
,
Stephen
Tucker
,
David
Meredith
,
Philip W.
Fowler
,
Simon
Newstead
Diamond Proposal Number(s):
[10627]
Open Access
Abstract: Mammals obtain nitrogen via the uptake of di- and tri- peptides in the gastrointestinal tract through the action of PepT1 and PepT2, which are members of the POT family of proton-coupled oligopeptide trans- porters. PepT1 and PepT2 also play an important role in drug transport in the human body. Recent crystal structures of bacterial homologs revealed a conserved peptide-binding site and mechanism of transport. However, a key structural difference exists between bacterial and mammalian homologs with only the latter containing a large extracellular domain, the function of which is currently unknown. Here, we present the crystal structure of the extracellular domain from both PepT1 and PepT2 that reveal two immunoglobulin-like folds connected in tandem, providing structural insight into mammalian pep- tide transport. Functional and biophysical studies demonstrate that these domains interact with the intestinal protease trypsin, suggesting a role in clus- tering proteolytic activity to the site of peptide trans- port in eukaryotic cells.
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Oct 2015
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