I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14043]
Abstract: The most abundant member of the collagen protein family, collagen I (also known as type I collagen; COL1), is composed of one unique (chain B) and two similar (chain A) polypeptides that self-assemble with one amino acid offset into a heterotrimeric triple helix. Given the offset, chain B can occupy either the leading (BAA), middle (ABA) or trailing (AAB) position of the triple helix, yielding three isomeric biomacromolecules with different protein recognition properties. Despite five decades of intensive research, there is no consensus on the position of chain B in COL1. Here, three triple-helical heterotrimers that each contain a putative von Willebrand factor (VWF) and discoidin domain receptor (DDR) recognition sequence from COL1 were designed with chain B permutated in all three positions. AAB demonstrated a strong preference for both VWF and DDR, and also induced higher levels of cellular DDR phosphorylation. Thus, we resolve this long-standing mystery and show that COL1 adopts an AAB register.
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Jan 2020
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I03-Macromolecular Crystallography
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Daniel
Del Toro
,
Maria A.
Carrasquero-ordaz
,
Amy
Chu
,
Tobias
Ruff
,
Meriam
Shahin
,
Verity A.
Jackson
,
Matthieu
Chavent
,
Miguel
Berbeira-santana
,
Goenuel
Seyit-bremer
,
Sara
Brignani
,
Rainer
Kaufmann
,
Edward
Lowe
,
Rüdiger
Klein
,
Elena
Seiradake
Diamond Proposal Number(s):
[12346, 1838]
Open Access
Abstract: Teneurins are ancient metazoan cell adhesion receptors that control brain development and neuronal wiring in higher animals. The extracellular C terminus binds the adhesion GPCR Latrophilin, forming a trans-cellular complex with synaptogenic functions. However, Teneurins, Latrophilins, and FLRT proteins are also expressed during murine cortical cell migration at earlier developmental stages. Here, we present crystal structures of Teneurin-Latrophilin complexes that reveal how the lectin and olfactomedin domains of Latrophilin bind across a spiraling beta-barrel domain of Teneurin, the YD shell. We couple structure-based protein engineering to biophysical analysis, cell migration assays, and in utero electroporation experiments to probe the importance of the interaction in cortical neuron migration. We show that binding of Latrophilins to Teneurins and FLRTs directs the migration of neurons using a contact repulsion-dependent mechanism. The effect is observed with cell bodies and small neurites rather than their processes. The results exemplify how a structure-encoded synaptogenic protein complex is also used for repulsive cell guidance.
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Jan 2020
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946]
Open Access
Abstract: Enteroviruses cause a range of human and animal diseases, some life-threatening, but there remain no licenced anti-enterovirus drugs. However, a benzene-sulfonamide derivative and related compounds have been shown recently to block infection of a range of enteroviruses by binding the capsid at a positively-charged surface depression conserved across many enteroviruses. It has also been established that glutathione is essential for the assembly of many enteroviruses, interacting with the capsid proteins to facilitate the formation of the pentameric assembly intermediate, although the mechanism is unknown. Here we show, by high resolution structure analyses of enterovirus F3, that reduced glutathione binds to the same interprotomer pocket as the benzene-sulfonamide derivative. Bound glutathione makes strong interactions with adjacent protomers, thereby explaining the underlying biological role of this druggable binding pocket and delineating the pharmacophore for potential antivirals.
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Jan 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18565]
Open Access
Abstract: Ferritins are multimers comprised of 4 α-helical bundle monomers that co-assemble to form protein shells surrounding an approximately spherical internal cavity. The assembled multimers acquire Fe2+ from their surroundings by utilising channels that penetrate the protein for the transportation of iron to diiron catalytic centres buried within the monomeric units. Here oxidation of the substrate to Fe3+ is coupled to the reduction of O2 and/or peroxide to yield the precursor to a ferric oxy hydroxide mineral that is stored within the internal cavity. The rhombic dodecahedral quaternary structure results in channels of 4-fold and 3-fold symmetry, located at the vertices, which are common to all 24mer-ferritins. Ferritins isolated from higher eukaryotes have been demonstrated to take up Fe2+ via the 3-fold channels. One of the defining features of ferritins isolated from prokaryotes is the presence of a further 24 channels, the B-channels, and these are thought to play an important role in Fe2+ uptake in this sub-family. SynFtn is an unusual ferritin isolated from the marine cyanobacterium Synechococcus CC9311. The reported structure of SynFtn derived from Fe2+ soaked crystals revealed the presence of a fully hydrated Fe2+ associated with three aspartate residues (Asp137 from each of the three symmetry related subunits) within each three-fold channel, suggesting that it might be the route for Fe2+ entry. Here, we present structural and spectro-kinetic data on two variants of SynFtn, D137A and E62A, designed to assess this possibility. Glu62 is equivalent to residues demonstrated to be important in the transfer of iron from the inner exit of the 3-fold channel to the catalytic centre in animal ferritins. As expected replacing Asp137 with a non-coordinating residue eliminated rapid iron oxidation by SynFtn. In contrast the rate of mineral core formation was severely impaired whilst the rate of iron transit into the catalytic centre was largely unaffected upon introducing a non-coordinating residue in place of Glu62 suggesting a role for this residue in release of the oxidised product. The identification of these two residues in SynFtn maps out major routes for Fe2+ entry to, and exit from, the catalytic ferroxidase centres.
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Jan 2020
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Abstract: Heterocycles, a class of molecules that includes oxazoles, constitute one of the most common building blocks in current pharmaceuticals and are common in medicinally important natural products. The antitumor natural product nataxazole is a model for a large class of benzoxazole containing molecules that are made by a pathway that is not characterized. We report structural, biochemical and chemical evidence that benzoxazole biosynthesis proceeds through an ester generated by an ATP dependent adenylating enzyme. The ester re‐arranges via a tetrahedral hemiorthoamide to yield an amide which is a shunt product and not, as previously thought, an intermediate in the pathway. A second zinc dependent enzyme catalyses formation of hemiorthoamide from the ester but, by shuttling protons, the enzyme eliminates water, a reverse hydrolysis reaction, to yield the benzoxazole and avoids the amide. These insights have allowed us to harness the pathway to synthesize a series of novel halogenated benzoxazoles.
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Jan 2020
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I03-Macromolecular Crystallography
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Christopher R.
Wellaway
,
Dominique
Amans
,
Paul
Bamborough
,
Heather
Barnett
,
Rino A.
Bit
,
Jack A.
Brown
,
Neil R.
Carlson
,
Chun-wa
Chung
,
Anthony W. J.
Cooper
,
Peter D.
Craggs
,
Robert P.
Davis
,
Tony W.
Dean
,
John P.
Evans
,
Laurie
Gordon
,
Isobel L.
Harada
,
David J.
Hirst
,
Philip G.
Humphreys
,
Katherine L.
Jones
,
Antonia J.
Lewis
,
Matthew J.
Lindon
,
Dave
Lugo
,
Mahnoor
Mahmood
,
Scott
Mccleary
,
Patricia
Medeiros
,
Darren J.
Mitchell
,
Michael
O’sullivan
,
Armelle
Le Gall
,
Vipulkumar K.
Patel
,
Chris
Patten
,
Darren L.
Poole
,
Rishi R.
Shah
,
Jane E.
Smith
,
Kayleigh A. J.
Stafford
,
Pamela J.
Thomas
,
Mythily
Vimal
,
Ian D.
Wall
,
Robert J.
Watson
,
Natalie
Wellaway
,
Gang
Yao
,
Rab K.
Prinjha
Abstract: The bromodomain and extraterminal (BET) family of bromodomain-containing proteins are important regulators of the epigenome through their ability to recognize N-acetyl lysine (KAc) post-translational modifications on histone tails. These interactions have been implicated in various disease states and, consequently, disruption of BET–KAc binding has emerged as an attractive therapeutic strategy with a number of small molecule inhibitors now under investigation in the clinic. However, until the utility of these advanced candidates is fully assessed by these trials, there remains scope for the discovery of inhibitors from new chemotypes with alternative physicochemical, pharmacokinetic, and pharmacodynamic profiles. Herein, we describe the discovery of a candidate-quality dimethylpyridone benzimidazole compound which originated from the hybridization of a dimethylphenol benzimidazole series, identified using encoded library technology, with an N-methyl pyridone series identified through fragment screening. Optimization via structure- and property-based design led to I-BET469, which possesses favorable oral pharmacokinetic properties, displays activity in vivo, and is projected to have a low human efficacious dose.
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Jan 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Storm
Hassell-hart
,
Andrew
Runcie
,
Tobias
Krojer
,
Jordan
Doyle
,
Ella
Lineham
,
Cory A.
Ocasio
,
Brenno A. D.
Neto
,
Oleg
Fedorov
,
Graham
Marsh
,
Hannah
Maple
,
Robert
Felix
,
Rebecca
Banks
,
Alessio
Ciulli
,
Sarah
Picaud
,
Panagis
Filippakopoulos
,
Frank
Von Delft
,
Paul
Brennan
,
Helen J. S.
Stewart
,
Timothy J.
Chevassut
,
Martin
Walker
,
Carol
Austin
,
Simon
Morley
,
John
Spencer
Diamond Proposal Number(s):
[19301]
Abstract: (+)-JD1, a rationally designed ferrocene analogue of the BET bromodomain (BRD) probe molecule (+)-JQ1, has been synthesized and evaluated in biophysical, cell-based assays as well as in pharmacokinetic studies. It displays nanomolar activity against BRD isoforms, and its cocrystal structure was determined in complex with the first bromodomain of BRD4 and compared with that of (+)-JQ1, a known BRD4 small-molecule probe. At 1 μM concentration, (+)-JD1 was able to inhibit c-Myc, a key driver in cancer and an indirect target of BRD4.
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Dec 2019
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18069]
Open Access
Abstract: Background: Malaria kills over 400,000 people each year and nearly half the world’s population live in at-risk areas. Progress against malaria has recently stalled, highlighting the need for developing novel therapeutics. The parasite haemoglobin degradation pathway, active in the blood stage of the disease where malaria symptoms and lethality manifest, is a well-established drug target. A key enzyme in this pathway is the papain-type protease falcipain-2. Methods: The crystallographic structure of falcipain-2 at 3.45 Å resolution was resolved in complex with an (E)-chalcone small-molecule inhibitor. The falcipain-2–(E)-chalcone complex was analysed with reference to previous falcipain complexes and their similarity to human cathepsin proteases. Results: The (E)-chalcone inhibitor binds falcipain-2 to the rear of the substrate-binding cleft. This is the first structure of a falcipain protease where the rear of the substrate cleft is bound by a small molecule. In this manner, the (E)-chalcone inhibitor mimics interactions observed in protein-based falcipain inhibitors, which can achieve high interaction specificity. Conclusions: This work informs the search for novel anti-malaria therapeutics that target falcipain-2 by showing the binding site and interactions of the medically privileged (E)-chalcone molecule. Furthermore, this study highlights the possibility of chemically combining the (E)-chalcone molecule with an existing active-site inhibitor of falcipain, which may yield a potent and selective compound for blocking haemoglobin degradation by the malaria parasite.
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Dec 2019
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I03-Macromolecular Crystallography
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James P.
Holt‐martyn
,
Rasheduzzaman
Chowdhury
,
Anthony
Tumber
,
Tzu‐lan
Yeh
,
Martine I.
Abboud
,
Kerstin
Lippl
,
Christopher T.
Lohans
,
Gareth W.
Langley
,
William
Figg
,
Michael A.
Mcdonough
,
Christopher W.
Pugh
,
Peter J.
Ratcliffe
,
Christopher J.
Schofield
Abstract: The 2‐oxoglutarate‐dependent hypoxia inducible factor prolyl hydroxylases (PHDs) are targets for treatment of a variety of diseases including anaemia. One PHD inhibitor is approved for use for the treatment of renal anaemia and others are in late stage clinical trials. The number of reported templates for PHD inhibition is limited. We report structure–activity relationship and crystallographic studies on a promising class of 4‐hydroxypyrimidine‐containing PHD inhibitors.
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Dec 2019
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Paula
Gimenez Mascarell
,
Iker
Oyenarte
,
Irene
Gonzalez Recio
,
Carmen
Fernandez-rodriguez
,
María Ángeles
Corral-rodríguez
,
Igone
Campos-zarraga
,
Jorge
Simón
,
Elie
Kostantin
,
Serge
Hardy
,
Antonio
Diaz Quintana
,
Mara
Zubillaga Lizeaga
,
Nekane
Merino
,
Tammo
Diercks
,
Francisco J.
Blanco
,
Irene
Diaz Moreno
,
María Luz
Martínez-chantar
,
Michel L.
Tremblay
,
Dominik
Müller
,
Dritan
Siliqi
,
Luis Alfonso
Martinez-cruz
Diamond Proposal Number(s):
[15832]
Open Access
Abstract: The four member family of “Cyclin and Cystathionine β-synthase (CBS) domain divalent metal cation transport mediators”, CNNMs, are the least-studied mammalian magnesium transport mediators. CNNM4 is abundant in the brain and the intestinal tract, and its abnormal activity causes Jalili Syndrome. Recent findings show that suppression of CNNM4 in mice promotes malignant progression of intestinal polyps and is linked to infertility. The association of CNNM4 with phosphatases of the regenerating liver, PRLs, abrogates its Mg2+-efflux capacity, thus resulting in an increased intracellular Mg2+ concentration that favors tumor growth. Here we present the crystal structures of the two independent intracellular domains of human CNNM4, i.e., the Bateman module and the cyclic nucleotide binding-like domain (cNMP). We also derive a model structure for the full intracellular region in the absence and presence of MgATP and the oncogenic interacting partner, PRL-1. We find that only the Bateman module interacts with ATP and Mg2+, at non-overlapping sites facilitating their positive cooperativity. Furthermore, both domains dimerize autonomously, where the cNMP domain dimer forms a rigid cleft to restrict the Mg2+ induced sliding of the inserting CBS1 motives of the Bateman module, from a twisted to a flat disk shaped dimer.
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Dec 2019
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