I24-Microfocus Macromolecular Crystallography
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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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I02-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Y. Y.
Dong
,
Ashley
Pike
,
Alexandra
Mackenzie
,
C.
Mcclenaghan
,
P.
Aryal
,
L.
Dong
,
Andrew
Quigley
,
Mariana
Grieben
,
S.
Goubin
,
S.
Mukhopadhyay
,
G. F.
Ruda
,
M. V.
Clausen
,
L.
Cao
,
P. E.
Brennan
,
N. A.
Burgess-brown
,
M. S. P.
Sansom
,
S. J.
Tucker
,
E. P.
Carpenter
Diamond Proposal Number(s):
[8421, 10619]
Abstract: TREK-2 (KCNK10/K2P10), a two-pore domain potassium (K2P) channel, is gated by multiple stimuli such as stretch, fatty acids, and pH and by several drugs. However, the mechanisms that control channel gating are unclear. Here we present crystal structures of the human TREK-2 channel (up to 3.4 angstrom resolution) in two conformations and in complex with norfluoxetine, the active metabolite of fluoxetine (Prozac) and a state-dependent blocker of TREK channels. Norfluoxetine binds within intramembrane fenestrations found in only one of these two conformations. Channel activation by arachidonic acid and mechanical stretch involves conversion between these states through movement of the pore-lining helices. These results provide an explanation for TREK channel mechanosensitivity, regulation by diverse stimuli, and possible off-target effects of the serotonin reuptake inhibitor Prozac.
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Mar 2015
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I24-Microfocus Macromolecular Crystallography
|
Open Access
Abstract: Sodiumproton antiporters rapidly exchange protons and sodium ions across the membrane to regulate intracellular pH, cell volume, and sodium concentration. How ion binding and release is coupled to the conformational changes associated with transport is not clear. Here, we report a crystal form of the prototypical sodiumproton antiporter NhaA from Escherichia coli in which the protein is seen as a dimer. In this new structure, we observe a salt bridge between an essential aspartic acid (Asp163) and a conserved lysine (Lys300). An equivalent salt bridge is present in the homologous transporter NapA, but not in the only other known crystal structure of NhaA, which provides the foundation of most existing structural models of electrogenic sodiumproton antiport. Molecular dynamics simulations show that the stability of the salt bridge is weakened by sodium ions binding to Asp164 and the neighboring Asp163. This suggests that the transport mechanism involves Asp163 switching between forming a salt bridge with Lys300 and interacting with the sodium ion. pKa calculations suggest that Asp163 is highly unlikely to be protonated when involved in the salt bridge. As it has been previously suggested that Asp163 is one of the two residues through which proton transport occurs, these results have clear implications to the current mechanistic models of sodiumproton antiport in NhaA.
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Nov 2014
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I24-Microfocus Macromolecular Crystallography
|
Jingquan
Tan
,
Sarah L.
Rouse
,
Dianfan
Li
,
Valerie
Pye
,
Lutz
Vogeley
,
Alette
Brinth
,
Toufic
El Arnaout
,
John C.
Whitney
,
P. Lynne
Howell
,
Mark S. P.
Sansom
,
Martin
Caffrey
Diamond Proposal Number(s):
[8224]
Open Access
Abstract: The exopolysaccharide alginate is an important component of biofilms produced by Pseudomonas aeruginosa, a major pathogen that contributes to the demise of cystic fibrosis patients. Alginate exits the cell via the outer membrane porin AlgE. X-ray structures of several AlgE crystal forms are reported here. Whilst all share a common β-barrel constitution, they differ in the degree to which loops L2 and T8 are ordered. L2 and T8 have been identified as an extracellular gate (E-gate) and a periplasmic gate (P-gate), respectively, that reside on either side of an alginate-selectivity pore located midway through AlgE. Passage of alginate across the membrane is proposed to be regulated by the sequential opening and closing of the two gates. In one crystal form, the selectivity pore contains a bound citrate. Because citrate mimics the uronate monomers of alginate, its location is taken to highlight a route through AlgE taken by alginate as it crosses the pore. Docking and molecular-dynamics simulations support and extend the proposed transport mechanism. Specifically, the P-gate and E-gate are flexible and move between open and closed states. Citrate can leave the selectivity pore bidirectionally. Alginate docks stably in a linear conformation through the open pore. To translate across the pore, a force is required that presumably is provided by the alginate-synthesis machinery. Accessing the open pore is facilitated by complex formation between AlgE and the periplasmic protein AlgK. Alginate can thread through a continuous pore in the complex, suggesting that AlgK pre-orients newly synthesized exopolysaccharide for delivery to AlgE.
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Aug 2014
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
Katie J
Simmons
,
Scott
Jackson
,
Florian
Brueckner
,
Simon
Patching
,
O.
Beckstein
,
Ekaterina
Ivanova
,
Tian
Geng
,
Simone
Weyand
,
David
Drew
,
J.
Lanigan
,
D. J.
Sharples
,
M. S.
Sansom
,
So
Iwata
,
C. W.
Fishwick
,
A. P.
Johnson
,
Alex
Cameron
,
Peter
Henderson
Open Access
Abstract: The hydantoin transporter Mhp1 is a sodium-coupled secondary active transport protein of the nucleobase-cation-symport family and a member of the widespread 5-helix inverted repeat superfamily of transporters. The structure of Mhp1 was previously solved in three different conformations providing insight into the molecular basis of the alternating access mechanism. Here, we elucidate detailed events of substrate binding, through a combination of crystallography, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the design and synthesis of novel ligands. We show precisely where 5-substituted hydantoin substrates bind in an extended configuration at the interface of the bundle and hash domains. They are recognised through hydrogen bonds to the hydantoin moiety and the complementarity of the 5-substituent for a hydrophobic pocket in the protein. Furthermore, we describe a novel structure of an intermediate state of the protein with the external thin gate locked open by an inhibitor, 5-(2-naphthylmethyl)-L-hydantoin, which becomes a substrate when leucine 363 is changed to an alanine. We deduce the molecular events that underlie acquisition and transport of a ligand by Mhp1.
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Aug 2014
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