I02-Macromolecular Crystallography
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David R.
Chisholm
,
Charles W. E.
Tomlinson
,
Garr-layy
Zhou
,
Claire
Holden
,
Valerie
Affleck
,
Rebecca
Lamb
,
Katherine
Newling
,
Peter
Ashton
,
Roy
Valentine
,
Christopher
Redfern
,
János
Erostyák
,
Geza
Makkai
,
Carrie A.
Ambler
,
Andrew
Whiting
,
Ehmke
Pohl
Abstract: Retinoids, such as all-trans-retinoic acid (ATRA), are endogenous signalling molecules derived from Vitamin A that influ-ence a variety of cellular processes through mediation of transcription events in the cell nucleus. Due to these wide-ranging and powerful biological activities, retinoids have emerged as therapeutic candidates of enormous potential. However, their use has been limited, to date, due to a lack of understanding of the complex and intricate signaling pathways that they con-trol. We have designed and synthesized a family of synthetic retinoids that exhibit strong, intrinsic, solvatochromatic fluo-rescence as multifunctional tools to interrogate these important biological activities. We utilized the unique photophysical characteristics of these fluorescent retinoids to develop a novel in vitro fluorometric binding assay to characterize and quanti-fy their binding to their cellular targets, including Cellular Retinoid Binding Protein II (CRABPII). The dihydroquinoline retinoid, DC360, exhibited particularly strong binding (Kd = 34.0 ± 2.5 nM) and we further used X-ray crystallography to solve the structure of the DC360-CRABPII complex to 1.8 Å, which showed that DC360 occupies the known hydrophobic retinoid-binding pocket. Finally, we used confocal fluorescence microscopy to image the cellular behaviour of the com-pounds in cultured human epithelial cells, highlighting a fascinating nuclear localisation, and used RNA sequencing to con-firm that the compounds regulate similar cellular processes to ATRA. We anticipate that the unique properties of these fluo-rescent retinoids can now be used to shed new light on the vital and highly complex retinoid signalling pathway.
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Feb 2019
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13062]
Abstract: The 54 kDa protein properdin, also known as factor P (FP), plays a major role in the complement system through the stabilization of the alternative pathway convertases. FP circulates in the blood as cyclic dimers, trimers and tetramers, and this heterogeneity challenges detailed structural insight into the mechanism of convertase stabilization by FP. Here, the generation of an intact FP monomer and a variant monomer with the third thrombospondin repeat liberated is described. Both FP monomers were excised from recombinant full-length FP containing internal cleavage sites for TEV protease. These FP monomers could be crystallized, and complete data sets extending to 2.8 Å resolution for the intact FP monomer and to 3.5 Å resolution for the truncated variant were collected. The principle of specific monomer excision and domain removal by the insertion of a protease cleavage site may be broadly applicable to structural studies of oligomeric, flexible and modular proteins.
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Feb 2019
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I02-Macromolecular Crystallography
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M. Teresa
Bertran
,
Stephane
Mouilleron
,
Yanxiang
Zhou
,
Rakhi
Bajaj
,
Federico
Uliana
,
Ganesan Senthil
Kumar
,
Audrey
Van Drogen
,
Rebecca
Lee
,
Jennifer J.
Banerjee
,
Simon
Hauri
,
Nicola
O’reilly
,
Matthias
Gstaiger
,
Rebecca
Page
,
Wolfgang
Peti
,
Nicolas
Tapon
Diamond Proposal Number(s):
[9826]
Open Access
Abstract: Serine/threonine phosphatases such as PP1 lack substrate specificity and associate with a large array of targeting subunits to achieve the requisite selectivity. The tumour suppressor ASPP (apoptosis-stimulating protein of p53) proteins associate with PP1 catalytic subunits and are implicated in multiple functions from transcriptional regulation to cell junction remodelling. Here we show that Drosophila ASPP is part of a multiprotein PP1 complex and that PP1 association is necessary for several in vivo functions of Drosophila ASPP. We solve the crystal structure of the human ASPP2/PP1 complex and show that ASPP2 recruits PP1 using both its canonical RVxF motif, which binds the PP1 catalytic domain, and its SH3 domain, which engages the PP1 C-terminal tail. The ASPP2 SH3 domain can discriminate between PP1 isoforms using an acidic specificity pocket in the n-Src domain, providing an exquisite mechanism where multiple motifs are used combinatorially to tune binding affinity to PP1.
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Feb 2019
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I02-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
Krios I-Titan Krios I at Diamond
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Kamel
El Omari
,
Sai
Li
,
Abhay
Kotecha
,
Thomas S.
Walter
,
Eduardo A.
Bignon
,
Karl
Harlos
,
Pentti
Somerharju
,
Felix
De Haas
,
Daniel K.
Clare
,
Mika
Molin
,
Felipe
Hurtado
,
Mengqiu
Li
,
Jonathan
Grimes
,
Dennis H.
Bamford
,
Nicole D.
Tischler
,
Juha T.
Huiskonen
,
Dave I.
Stuart
,
Elina
Roine
Diamond Proposal Number(s):
[10627]
Open Access
Abstract: Lipid membrane fusion is an essential function in many biological processes. Detailed mechanisms of membrane fusion and the protein structures involved have been mainly studied in eukaryotic systems, whereas very little is known about membrane fusion in prokaryotes. Haloarchaeal pleomorphic viruses (HRPVs) have a membrane envelope decorated with spikes that are presumed to be responsible for host attachment and membrane fusion. Here we determine atomic structures of the ectodomains of the 57-kDa spike protein VP5 from two related HRPVs revealing a previously unreported V-shaped fold. By Volta phase plate cryo-electron tomography we show that VP5 is monomeric on the viral surface, and we establish the orientation of the molecules with respect to the viral membrane. We also show that the viral membrane fuses with the host cytoplasmic membrane in a process mediated by VP5. This sheds light on protein structures involved in prokaryotic membrane fusion.
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Feb 2019
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I02-Macromolecular Crystallography
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Megan J.
Agajanian
,
Matthew P.
Walker
,
Alison D.
Axtman
,
Roberta R.
Ruela-de-sousa
,
D. Stephen
Serafin
,
Alex D.
Rabinowitz
,
David M.
Graham
,
Meagan B.
Ryan
,
Tigist
Tamir
,
Yuko
Nakamichi
,
Melissa V.
Gammons
,
James M.
Bennett
,
Rafael M.
Counago
,
David H.
Drewry
,
Jonathan M.
Elkins
,
Carina
Gileadi
,
Opher
Gileadi
,
Paulo H.
Godoi
,
Nirav
Kapadia
,
Susanne
Müller
,
André S.
Santiago
,
Fiona J.
Sorrell
,
Carrow I.
Wells
,
Oleg
Fedorov
,
Timothy M.
Willson
,
William J.
Zuercher
,
Michael B.
Major
Open Access
Abstract: β-Catenin-dependent WNT signal transduction governs development, tissue homeostasis, and a vast array of human diseases. Signal propagation through a WNT-Frizzled/LRP receptor complex requires proteins necessary for clathrin-mediated endocytosis (CME). Paradoxically, CME also negatively regulates WNT signaling through internalization and degradation of the receptor complex. Here, using a gain-of-function screen of the human kinome, we report that the AP2 associated kinase 1 (AAK1), a known CME enhancer, inhibits WNT signaling. Reciprocally, AAK1 genetic silencing or its pharmacological inhibition using a potent and selective inhibitor activates WNT signaling. Mechanistically, we show that AAK1 promotes clearance of LRP6 from the plasma membrane to suppress the WNT pathway. Time-course experiments support a transcription-uncoupled, WNT-driven negative feedback loop; prolonged WNT treatment drives AAK1-dependent phosphorylation of AP2M1, clathrin-coated pit maturation, and endocytosis of LRP6. We propose that, following WNT receptor activation, increased AAK1 function and CME limits WNT signaling longevity.
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Jan 2019
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[9475, 13467]
Open Access
Abstract: The introduction of azole heterocycles into a peptide backbone is the principal step in the biosynthesis of numerous compounds with therapeutic potential. One of them is microcin B17, a bacterial topoisomerase inhibitor whose activity depends on the conversion of selected serine and cysteine residues of the precursor peptide to oxazoles and thiazoles by the McbBCD synthetase complex. Crystal structures of McbBCD reveal an octameric B4C2D2 complex with two bound substrate peptides. Each McbB dimer clamps the N-terminal recognition sequence, while the C-terminal heterocycle of the modified peptide is trapped in the active site of McbC. The McbD and McbC active sites are distant from each other, which necessitates alternate shuttling of the peptide substrate between them, while remaining tethered to the McbB dimer. An atomic-level view of the azole synthetase is a starting point for deeper understanding and control of biosynthesis of a large group of ribosomally synthesized natural products.
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Jan 2019
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Stuart
Francis
,
Daniel
Croft
,
Alexander W.
Schuettelkopf
,
Charles
Parry
,
Angelo
Pugliese
,
Ken
Cameron
,
Sophie
Claydon
,
Martin
Drysdale
,
Claire
Gardner
,
Andrea
Gohlke
,
Gillian
Goodwin
,
Christopher H.
Gray
,
Jennifer
Konczal
,
Laura
Mcdonald
,
Mokdad
Mezna
,
Andrew
Pannifer
,
Nikki
Paul
,
Laura
Machesky
,
Heather
Mckinnon
,
Justin
Bower
Diamond Proposal Number(s):
[6683, 8659, 11651]
Open Access
Abstract: Fascin is an actin binding and bundling protein that is not expressed in normal epithelial tissues but overexpressed in a variety of invasive epithelial tumors. It has a critical role in cancer cell metastasis by promoting cell migration and invasion. Here we report the crystal structures of fascin in complex with a series of novel and potent inhibitors. Subsequent structure-based elaboration of this and related compounds enabled the development of a series with nanomolar affinities for fascin, good physicochemical properties and the ability to inhibit fascin-mediated bundling of filamentous actin. These compounds provide promising starting points for fascin-targeted anti-metastatic therapies.
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Jan 2019
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Mark
Elliott
,
Christine
Favre-guilmard
,
Sai Man
Liu
,
Jacquie
Maignel
,
Geoffrey
Masuyer
,
Matthew
Beard
,
Christopher
Boone
,
Denis
Carré
,
Mikhail
Kalinichev
,
Stephane
Lezmi
,
Imran
Mir
,
Camille
Nicoleau
,
Shilpa
Palan
,
Cindy
Perier
,
Elsa
Raban
,
Sicai
Zhang
,
Min
Dong
,
Pal
Stenmark
,
Johannes
Krupp
Diamond Proposal Number(s):
[11265, 15806]
Open Access
Abstract: Although botulinum neurotoxin serotype A (BoNT/A) products are common treatments for various disorders, there is only one commercial BoNT/B product, whose low potency, likely stemming from low affinity toward its human receptor synaptotagmin 2 (hSyt2), has limited its therapeutic usefulness. We express and characterize two full-length recombinant BoNT/B1 proteins containing designed mutations E1191M/S1199Y (rBoNT/B1MY) and E1191Q/S1199W (rBoNT/B1QW) that enhance binding to hSyt2. In preclinical models including human-induced pluripotent stem cell neurons and a humanized transgenic mouse, this increased hSyt2 affinity results in high potency, comparable to that of BoNT/A. Last, we solve the cocrystal structure of rBoNT/B1MY in complex with peptides of hSyt2 and its homolog hSyt1. We demonstrate that neuronal surface receptor binding limits the clinical efficacy of unmodified BoNT/B and that modified BoNT/B proteins have promising clinical potential.
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Jan 2019
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12346]
Abstract: The RAS gene family is frequently mutated in human cancers, and the quest for compounds that bind to mutant RAS remains a major goal, as it also does for inhibitors of protein–protein interactions. We have refined crystallization conditions for KRAS169Q61H-yielding crystals suitable for soaking with compounds and exploited this to assess new RAS-binding compounds selected by screening a protein–protein interaction-focused compound library using surface plasmon resonance. Two compounds, referred to as PPIN-1 and PPIN-2, with related structures from 30 initial RAS binders showed binding to a pocket where compounds had been previously developed, including RAS effector protein–protein interaction inhibitors selected using an intracellular antibody fragment (called Abd compounds). Unlike the Abd series of RAS binders, PPIN-1 and PPIN-2 compounds were not competed by the inhibitory anti-RAS intracellular antibody fragment and did not show any RAS-effector inhibition properties. By fusing the common, anchoring part from the two new compounds with the inhibitory substituents of the Abd series, we have created a set of compounds that inhibit RAS-effector interactions with increased potency. These fused compounds add to the growing catalog of RAS protein–protein inhibitors and show that building a chemical series by crossing over two chemical series is a strategy to create RAS-binding small molecules.
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Jan 2019
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Gemma V.
White
,
Emma V.
Edgar
,
Duncan S.
Holmes
,
Xiao Qing
Lewell
,
John
Liddle
,
Oxana
Polyakova
,
Kathrine J.
Smith
,
James H.
Thorpe
,
Ann L.
Walker
,
Yichen
Wang
,
Robert J.
Young
,
Alain
Hovnanian
Diamond Proposal Number(s):
[5799]
Abstract: Netherton syndrome (NS) is a rare and debilitating severe autosomal recessive genetic skin disease with high mortality rates particularly in neonates. NS is caused by loss-of-function SPINK5 mutations leading to unregulated kallikrein 5 (KLK5) and kallikrein 7 (KLK7) activity. Furthermore, KLK5 inhibition has been proposed as a potential therapeutic treatment for NS. Identification of potent and selective KLK5 inhibitors would enable further exploration of the disease biology and could ultimately lead to a treatment for NS. This publication describes how fragmentation of known trypsin-like serine protease (TLSP) inhibitors resulted in the identification of a series of phenolic amidine-based KLK5 inhibitors 1. X-ray crystallography was used to find alternatives to the phenol interaction leading to identification of carbonyl analogues such as lactam 13 and benzimidazole 15. These reversible inhibitors, with selectivity over KLK1 (10–100 fold), provided novel starting points for the guided growth towards suitable tool molecules for the exploration of KLK5 biology.
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Jan 2019
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