I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Serena G.
Piticchio
,
Miriam
Martinez-Cartro
,
Salvatore
Scaffidi
,
Moira
Rachman
,
Sergio
Rodriguez-Arevalo
,
Ainoa
Sanchez-Arfelis
,
Carmen
Escolano
,
Sarah
Picaud
,
Tobias
Krojer
,
Panagis
Filippakopoulos
,
Frank
Von Delft
,
Carles
Galdeano
,
Xavier
Barril
Diamond Proposal Number(s):
[15433, 19301]
Abstract: Fragment-based drug discovery (FBDD) is a very effective hit identification method. However, the evolution of fragment hits into suitable leads remains challenging and largely artisanal. Fragment evolution is often scaffold-centric, meaning that its outcome depends crucially on the chemical structure of the starting fragment. Considering that fragment screening libraries cover only a small proportion of the corresponding chemical space, hits should be seen as probes highlighting privileged areas of the chemical space rather than actual starting points. We have developed an automated computational pipeline to mine the chemical space around any specific fragment hit, rapidly finding analogues that share a common interaction motif but are structurally novel and diverse. On a prospective application on the bromodomain-containing protein 4 (BRD4), starting from a known fragment, the platform yields active molecules with nonobvious scaffold changes. The procedure is fast and inexpensive and has the potential to uncover many hidden opportunities in FBDD.
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Dec 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Petra
Lukacik
,
C. David
Owen
,
Gemma
Harris
,
Jani Reddy
Bolla
,
Sarah
Picaud
,
Irfan
Alibay
,
Joanne E.
Nettleship
,
Louise E.
Bird
,
Raymond
Owens
,
Philip C.
Biggin
,
Panagis
Filippakopoulos
,
Carol V.
Robinson
,
Martin A.
Walsh
Diamond Proposal Number(s):
[4990, 5073, 4988]
Open Access
Abstract: Nontypeable Haemophilus influenzae (NTHi) is a significant pathogen in respiratory disease and otitis media. Important for NTHi survival, colonization and persistence in vivo is the Sap (sensitivity to antimicrobial peptides) ABC transporter system. Current models propose a direct role for Sap in heme and antimicrobial peptide (AMP) transport. Here, the crystal structure of SapA, the periplasmic component of Sap, in a closed, ligand bound conformation, is presented. Phylogenetic and cavity volume analysis predicts that the small, hydrophobic SapA central ligand binding cavity is most likely occupied by a hydrophobic di- or tri- peptide. The cavity is of insufficient volume to accommodate heme or folded AMPs. Crystal structures of SapA have identified surface interactions with heme and dsRNA. Heme binds SapA weakly (Kd 282 μM) through a surface exposed histidine, while the dsRNA is coordinated via residues which constitute part of a conserved motif (estimated Kd 4.4 μM). The RNA affinity falls within the range observed for characterized RNA/protein complexes. Overall, we describe in molecular-detail the interactions of SapA with heme and dsRNA and propose a role for SapA in the transport of di- or tri-peptides.
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Oct 2021
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[105433]
Open Access
Abstract: Extracellular signal-regulated kinase 3 (ERK3), known also as mitogen-activated protein kinase 6 (MAPK6), is an atypical member of MAPK kinase family, which has been poorly studied. Little is known regarding its function in biological processes, yet this atypical kinase has been suggested to play important roles in the migration and invasiveness of certain cancers. The lack of tools, such as a selective inhibitor, hampers the study of ERK3 biology. Here, we report the crystal structure of the kinase domain of this atypical MAPK kinase, providing molecular insights into its distinct ATP binding pocket compared to the classical MAPK ERK2, explaining differences in their inhibitor binding properties. Medium-scale small molecule screening identified a number of inhibitors, several of which unexpectedly exhibited remarkably high inhibitory potencies. The crystal structure of CLK1 in complex with CAF052, one of the most potent inhibitors identified for ERK3, revealed typical type-I binding mode of the inhibitor, which by structural comparison could likely be maintained in ERK3. Together with the presented structural insights, these diverse chemical scaffolds displaying both reversible and irreversible modes of action, will serve as a starting point for the development of selective inhibitors for ERK3, which will be beneficial for elucidating the important functions of this understudied kinase.
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Nov 2020
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[15433]
Open Access
Abstract: Wnt signalling is dependent on dishevelled proteins (DVL1-3), which assemble an intracellular Wnt signalosome at the plasma membrane. The levels of DVL1-3 are regulated by multiple Cullin-RING E3 ligases that mediate their ubiquitination and degradation. The BTB-Kelch protein KLHL12 was the first E3 ubiquitin ligase to be identified for DVL1-3, but the molecular mechanisms determining its substrate interactions have remained unknown. Here, we mapped the interaction of DVL1-3 to a ‘PGXPP' motif that is conserved in other known partners and substrates of KLHL12, including PLEKHA4, PEF1, SEC31 and DRD4. To determine the binding mechanism, we solved a 2.4 Å crystal structure of the Kelch domain of KLHL12 in complex with a DVL1 peptide that bound with low micromolar affinity. The DVL1 substrate adopted a U-shaped turn conformation that enabled hydrophobic interactions with all six blades of the Kelch domain β-propeller. In cells, the mutation or deletion of this motif reduced the binding and ubiquitination of DVL1 and increased its stability confirming this sequence as a degron motif for KLHL12 recruitment. These results define the molecular mechanisms determining DVL regulation by KLHL12 and establish the KLHL12 Kelch domain as a new protein interaction module for a novel proline-rich motif.
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Jun 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
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[15433]
Open Access
Abstract: BTB-Kelch proteins form the largest subfamily of Cullin-RING E3 ligases, yet their substrate complexes are mapped and structurally characterized only for KEAP1 and KLHL3. KLHL20 is a related CUL3-dependent ubiquitin ligase linked to autophagy, cancer, and Alzheimer's disease that promotes the ubiquitination and degradation of substrates including DAPK1, PML, and ULK1. We identified an “LPDLV”-containing motif in the DAPK1 death domain that determines its recruitment and degradation by KLHL20. A 1.1-Å crystal structure of a KLHL20 Kelch domain-DAPK1 peptide complex reveals DAPK1 binding as a loose helical turn that inserts deeply into the central pocket of the Kelch domain to contact all six blades of the β propeller. Here, KLHL20 forms salt-bridge and hydrophobic interactions including tryptophan and cysteine residues ideally positioned for covalent inhibitor development. The structure highlights the diverse binding modes of β-propeller domains versus linear grooves and suggests a new target for structure-based drug design.
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Jul 2019
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Krios I-Titan Krios I at Diamond
Krios II-Titan Krios II at Diamond
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Chiara
Marabelli
,
Biagina
Marrocco
,
Simona
Pilotto
,
Sagar
Chittori
,
Sarah
Picaud
,
Sara
Marchese
,
Giuseppe
Ciossani
,
Federico
Forneris
,
Panagis
Filippakopoulos
,
Guy
Schoehn
,
Daniela
Rhodes
,
Sriram
Subramaniam
,
Andrea
Mattevi
Diamond Proposal Number(s):
[16082]
Open Access
Abstract: LSD1 and LSD2 are homologous histone demethylases with opposite biological outcomes related to chromatin silencing and transcription elongation, respectively. Unlike LSD1, LSD2 nucleosome-demethylase activity relies on a specific linker peptide from the multidomain protein NPAC. We used single-particle cryoelectron microscopy (cryo-EM), in combination with kinetic and mutational analysis, to analyze the mechanisms underlying the function of the human LSD2/NPAC-linker/nucleosome complex. Weak interactions between LSD2 and DNA enable multiple binding modes for the association of the demethylase to the nucleosome. The demethylase thereby captures mono- and dimethyl Lys4 of the H3 tail to afford histone demethylation. Our studies also establish that the dehydrogenase domain of NPAC serves as a catalytically inert oligomerization module. While LSD1/CoREST forms a nucleosome docking platform at silenced gene promoters, LSD2/NPAC is a multifunctional enzyme complex with flexible linkers, tailored for rapid chromatin modification, in conjunction with the advance of the RNA polymerase on actively transcribed genes.
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Apr 2019
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B21-High Throughput SAXS
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Jean-Philippe
Lambert
,
Sarah
Picaud
,
Takao
Fujisawa
,
Huayun
Hou
,
Pavel
Savitsky
,
Liis
Uusküla-Reimand
,
Gagan D.
Gupta
,
Hala
Abdouni
,
Zhen-Yuan
Lin
,
Monika
Tucholska
,
James D. R.
Knight
,
Beatriz
Gonzalez-Badillo
,
Nicole
St-Denis
,
Joseph A.
Newman
,
Manuel
Stucki
,
Laurence
Pelletier
,
Nuno
Bandeira
,
Michael D.
Wilson
,
Panagis
Filippakopoulos
,
Anne-Claude
Gingras
Diamond Proposal Number(s):
[6391, 10619, 15433]
Open Access
Abstract: Targeting bromodomains (BRDs) of the bromo-and-extra-terminal (BET) family offers opportunities for therapeutic intervention in cancer and other diseases. Here, we profile the interactomes of BRD2, BRD3, BRD4, and BRDT following treatment with the pan-BET BRD inhibitor JQ1, revealing broad rewiring of the interaction landscape, with three distinct classes of behavior for the 603 unique interactors identified. A group of proteins associate in a JQ1-sensitive manner with BET BRDs through canonical and new binding modes, while two classes of extra-terminal (ET)-domain binding motifs mediate acetylation-independent interactions. Last, we identify an unexpected increase in several interactions following JQ1 treatment that define negative functions for BRD3 in the regulation of rRNA synthesis and potentially RNAPII-dependent gene expression that result in decreased cell proliferation. Together, our data highlight the contributions of BET protein modules to their interactomes allowing for a better understanding of pharmacological rewiring in response to JQ1.
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Dec 2018
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I04-Macromolecular Crystallography
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Matous
Hrdinka
,
Lisa
Schlicher
,
Bing
Dai
,
Daniel M.
Pinkas
,
Joshua C.
Bufton
,
Sarah
Picaud
,
Jennifer A.
Ward
,
Catherine
Rogers
,
Chalada
Suebsuwong
,
Sameer
Nikhar
,
Gregory D.
Cuny
,
Kilian V. M.
Huber
,
Panagis
Filippakopoulos
,
Alex N.
Bullock
,
Alexei
Degterev
,
Mads
Gyrd‐hansen
Diamond Proposal Number(s):
[15433]
Open Access
Abstract: RIPK2 mediates inflammatory signaling by the bacteria‐sensing receptors NOD1 and NOD2. Kinase inhibitors targeting RIPK2 are a proposed strategy to ameliorate NOD‐mediated pathologies. Here, we reveal that RIPK2 kinase activity is dispensable for NOD2 inflammatory signaling and show that RIPK2 inhibitors function instead by antagonizing XIAP‐binding and XIAP‐mediated ubiquitination of RIPK2. We map the XIAP binding site on RIPK2 to the loop between β2 and β3 of the N‐lobe of the kinase, which is in close proximity to the ATP‐binding pocket. Through characterization of a new series of ATP pocket‐binding RIPK2 inhibitors, we identify the molecular features that determine their inhibition of both the RIPK2‐XIAP interaction, and of cellular and in vivoNOD2 signaling. Our study exemplifies how targeting of the ATP‐binding pocket in RIPK2 can be exploited to interfere with the RIPK2‐XIAP interaction for modulation of NOD signaling.
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Jul 2018
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[15433]
Abstract: HLA-I molecules play a central role in antigen presentation. They typically bind 9- to 12-mer peptides, and their canonical binding mode involves anchor residues at the second and last positions of their ligands. To investigate potential noncanonical binding modes, we collected in-depth and accurate HLA peptidomics datasets covering 54 HLA-I alleles and developed algorithms to analyze these data. Our results reveal frequent (442 unique peptides) and statistically significant C-terminal extensions for at least eight alleles, including the common HLA-A03:01, HLA-A31:01, and HLA-A68:01. High resolution crystal structure of HLA-A68:01 with such a ligand uncovers structural changes taking place to accommodate C-terminal extensions and helps unraveling sequence and structural properties predictive of the presence of these extensions. Scanning viral proteomes with the C-terminal extension motifs identifies many putative epitopes and we demonstrate direct recognition by human CD8+ T cells of a 10-mer epitope from cytomegalovirus predicted to follow the C-terminal extension binding mode.
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May 2018
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