I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Sites of protein-protein interaction (PPI) are potentially more selective binding sites for therapeutics than protein substrate-binding sites. PPIs include distinct regions frequently called “hotspots,” sites of key amino acid interactions. Prospective identification of these hotspots through X-ray crystallographic screening could assist in the identification of separation of function mutants for experimental validation, enhance confidence in AI-generated multiprotein complex predictions, and accelerate development of selective chemical probes. To explore these applications, we utilize the FragLite library to examine the binding surfaces of CDK2-cyclin A. The many protein- and peptide-CDK2-cyclin A complexes that have been structurally characterized make this complex an appropriate test case. We show that FragLites comprehensively map both known sites of protein-protein interaction on CDK2-cyclin A and identify a possible uncharacterized site, providing a structural method toward directing mechanistic studies and starting points for chemical probe design.
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Aug 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13587]
Open Access
Abstract: We describe the generation and characterization of camelid single-domain antibodies (nanobodies) raised against tumor suppressor protein p16INK4a (p16). p16 is a cell cycle regulator that inhibits cyclin-dependent kinases CDK4 and CDK6 and is inactivated in sporadic and familial cancers. The majority of p16 missense mutations cause loss of function by destabilizing the protein’s structure. We identify nanobodies that bind p16 with nanomolar affinities and restore the stability of many different cancer-associated p16 mutations located at sites throughout the protein. The crystal structure of a nanobody-p16 complex reveals that the nanobody binds to the opposite face of p16 to the CDK-binding interface permitting formation of a ternary complex. We confirm that nanobodies bind to p16 in a cellular setting and do not preclude p16 binding to CDK6 and its ability to induce cell-cycle arrest. These findings indicate that nanobodies merit testing as pharmacological chaperones for p16 reactivation in the cell.
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Aug 2025
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I04-Macromolecular Crystallography
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Pasquale A.
Morese
,
Ayaz
Ahmad
,
Mathewp.
Martin
,
Richard A.
Noble
,
Sara
Pintar
,
Lan Z.
Wang
,
Shangze
Xu
,
Andrew
Lister
,
Richard A.
Ward
,
Agnieszka K.
Bronowska
,
Martin E. M.
Noble
,
Hannah L.
Stewart
,
Michael J.
Waring
Diamond Proposal Number(s):
[30295]
Open Access
Abstract: The discovery of targeted covalent inhibitors is of increasing importance in drug discovery. Finding efficient covalent binders requires modulation of warhead reactivity and optimisation of warhead geometry and non-covalent interactions. Uncoupling the contributions that these factors make to potency is difficult and best practice for a testing cascade that is pragmatic and informative is yet to be fully established. We studied the structure-reactivity-activity relationships of a series of analogues of the EGFR inhibitor poziotinib with point changes in two substructural regions as well as variations in warhead reactivity and geometry. This showed that a simple probe displacement assay that is appropriately tuned in respect of timing and reagent concentrations can reveal structural effects on all three factors: non-covalent affinity, warhead reactivity and geometry. These effects include the detection of potency differences between an enantiomeric pair that differ greatly in their activity and their capacity to form a covalent bond. This difference is rationalised by X-ray crystallography and computational studies and the effect translates quantitatively into cellular mechanistic and phenotypic effects.
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Apr 2025
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Cellular retinoic acid binding protein 2 (CRABP2) transports retinoic acid from the cytoplasm to the nucleus where it then transfers its cargo to retinoic acid receptor-containing complexes leading to activation of gene transcription. We demonstrate using purified proteins that CRABP2 is also a cyclin D3-specific binding protein and that the CRABP2 cyclin D3 binding site and the proposed CRABP2 nuclear localization sequence overlap. Both sequences are within the helix-loop-helix motif that forms a lid to the retinoic acid binding pocket. Mutations within this sequence that block both cyclin D3 and retinoic acid binding promote formation of a CRABP2 structure in which the retinoic acid binding pocket is occupied by an alternative lid conformation. Structural and functional analysis of CRABP2 and cyclin D3 mutants combined with AlphaFold models of the ternary CDK4/6-cyclin D3-CRABP2 complex supports the identification of an α-helical protein binding site on the cyclin D3 C-terminal cyclin box fold.
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Oct 2024
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[28576]
Open Access
Abstract: The cell division cycle 25 phosphatases CDC25A, B and C regulate cell cycle transitions by dephosphorylating residues in the conserved glycine-rich loop of CDKs to activate their activity. Here, we present the cryo-EM structure of CDK2-cyclin A in complex with CDC25A at 2.7 Å resolution, providing a detailed structural analysis of the overall complex architecture and key protein-protein interactions that underpin this 86 kDa complex. We further identify a CDC25A C-terminal helix that is critical for complex formation. Sequence conservation analysis suggests CDK1/2-cyclin A, CDK1-cyclin B and CDK2/3-cyclin E are suitable binding partners for CDC25A, whilst CDK4/6-cyclin D complexes appear unlikely substrates. A comparative structural analysis of CDK-containing complexes also confirms the functional importance of the conserved CDK1/2 GDSEID motif. This structure improves our understanding of the roles of CDC25 phosphatases in CDK regulation and may inform the development of CDC25-targeting anticancer strategies.
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Aug 2024
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Jon
Agirre
,
Mihaela
Atanasova
,
Haroldas
Bagdonas
,
Charles B.
Ballard
,
Arnaud
Basle
,
James
Beilsten-Edmands
,
Rafael J.
Borges
,
David G.
Brown
,
J. Javier
Burgos-Marmol
,
John M.
Berrisford
,
Paul S.
Bond
,
Iracema
Caballero
,
Lucrezia
Catapano
,
Grzegorz
Chojnowski
,
Atlanta G.
Cook
,
Kevin D.
Cowtan
,
Tristan I.
Croll
,
Judit É.
Debreczeni
,
Nicholas E.
Devenish
,
Eleanor J.
Dodson
,
Tarik R.
Drevon
,
Paul
Emsley
,
Gwyndaf
Evans
,
Phil R.
Evans
,
Maria
Fando
,
James
Foadi
,
Luis
Fuentes-Montero
,
Elspeth F.
Garman
,
Markus
Gerstel
,
Richard J.
Gildea
,
Kaushik
Hatti
,
Maarten L.
Hekkelman
,
Philipp
Heuser
,
Soon Wen
Hoh
,
Michael A.
Hough
,
Huw T.
Jenkins
,
Elisabet
Jiménez
,
Robbie P.
Joosten
,
Ronan M.
Keegan
,
Nicholas
Keep
,
Eugene B.
Krissinel
,
Petr
Kolenko
,
Oleg
Kovalevskiy
,
Victor S.
Lamzin
,
David M.
Lawson
,
Andrey
Lebedev
,
Andrew G. W.
Leslie
,
Bernhard
Lohkamp
,
Fei
Long
,
Martin
Maly
,
Airlie
Mccoy
,
Stuart J.
Mcnicholas
,
Ana
Medina
,
Claudia
Millán
,
James W.
Murray
,
Garib N.
Murshudov
,
Robert A.
Nicholls
,
Martin E. M.
Noble
,
Robert
Oeffner
,
Navraj S.
Pannu
,
James M.
Parkhurst
,
Nicholas
Pearce
,
Joana
Pereira
,
Anastassis
Perrakis
,
Harold R.
Powell
,
Randy J.
Read
,
Daniel J.
Rigden
,
William
Rochira
,
Massimo
Sammito
,
Filomeno
Sanchez Rodriguez
,
George M.
Sheldrick
,
Kathryn L.
Shelley
,
Felix
Simkovic
,
Adam J.
Simpkin
,
Pavol
Skubak
,
Egor
Sobolev
,
Roberto A.
Steiner
,
Kyle
Stevenson
,
Ivo
Tews
,
Jens M. H.
Thomas
,
Andrea
Thorn
,
Josep Triviño
Valls
,
Ville
Uski
,
Isabel
Uson
,
Alexei
Vagin
,
Sameer
Velankar
,
Melanie
Vollmar
,
Helen
Walden
,
David
Waterman
,
Keith S.
Wilson
,
Martyn
Winn
,
Graeme
Winter
,
Marcin
Wojdyr
,
Keitaro
Yamashita
Open Access
Abstract: The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world.
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Jun 2023
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Gemma
Davison
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Mathew P.
Martin
,
Shannon
Turberville
,
Selma
Dormen
,
Richard
Heath
,
Amy B.
Heptinstall
,
Marie
Lawson
,
Duncan C.
Miller
,
Yi Min
Ng
,
James N.
Sanderson
,
Ian
Hope
,
Daniel
Wood
,
Céline
Cano
,
Jane A.
Endicott
,
Ian R.
Hardcastle
,
Martin E. M.
Noble
,
Michael J.
Waring
Open Access
Abstract: The development of ligands for biological targets is critically dependent on the identification of sites on proteins that bind molecules with high affinity. A set of compounds, called FragLites, can identify such sites, along with the interactions required to gain affinity, by X-ray crystallography. We demonstrate the utility of FragLites in mapping the binding sites of bromodomain proteins BRD4 and ATAD2 and demonstrate that FragLite mapping is comparable to a full fragment screen in identifying ligand binding sites and key interactions. We extend the FragLite set with analogous compounds derived from amino acids (termed PepLites) that mimic the interactions of peptides. The output of the FragLite maps is shown to enable the development of ligands with leadlike potency. This work establishes the use of FragLite and PepLite screening at an early stage in ligand discovery allowing the rapid assessment of tractability of protein targets and informing downstream hit-finding.
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Nov 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Mélanie
Uguen
,
Gemma
Davison
,
Lukas J.
Sprenger
,
James H.
Hunter
,
Mathew P.
Martin
,
Shannon
Turberville
,
Jessica
Watt
,
Bernard T.
Golding
,
Martin E. N.
Noble
,
Hannah L.
Stewart
,
Michael J.
Waring
Open Access
Abstract: High-throughput screening provides one of the most common ways of finding hit compounds. Lead-like libraries, in particular, provide hits with compatible functional groups and vectors for structural elaboration and physical properties suitable for optimization. Library synthesis approaches can lead to a lack of chemical diversity because they employ parallel derivatization of common building blocks using single reaction types. We address this problem through a “build–couple–transform” paradigm for the generation of lead-like libraries with scaffold diversity. Nineteen transformations of a 4-oxo-2-butenamide scaffold template were optimized, including 1,4-cyclizations, 3,4-cyclizations, reductions, and 1,4-additions. A pool-transformation approach efficiently explored the scope of these transformations for nine different building blocks and synthesized a >170-member library with enhanced chemical space coverage and favorable drug-like properties. Screening revealed hits against CDK2. This work establishes the build–couple–transform concept for the synthesis of lead-like libraries and provides a differentiated approach to libraries with significantly enhanced scaffold diversity.
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Aug 2022
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Duncan C.
Miller
,
Tristan
Reuillon
,
Lauren
Molyneux
,
Timothy
Blackburn
,
Simon J.
Cook
,
Noel
Edwards
,
Jane A.
Endicott
,
Bernard T.
Golding
,
Roger J.
Griffin
,
Ian
Hardcastle
,
Suzannah J.
Harnor
,
Amy
Heptinstall
,
Pamela
Lochhead
,
Mathew P.
Martin
,
Nick C.
Martin
,
Stephanie
Myers
,
David R.
Newell
,
Richard A.
Noble
,
Nicole
Phillips
,
Laurent
Rigoreau
,
Huw
Thomas
,
Julie A.
Tucker
,
Lan-Zhen
Wang
,
Michael J.
Waring
,
Ai-Ching
Wong
,
Stephen R.
Wedge
,
Martin E. M.
Noble
,
Celine
Cano
Diamond Proposal Number(s):
[9948, 13587]
Open Access
Abstract: The nonclassical extracellular signal-related kinase 5 (ERK5) mitogen-activated protein kinase pathway has been implicated in increased cellular proliferation, migration, survival, and angiogenesis; hence, ERK5 inhibition may be an attractive approach for cancer treatment. However, the development of selective ERK5 inhibitors has been challenging. Previously, we described the development of a pyrrole carboxamide high-throughput screening hit into a selective, submicromolar inhibitor of ERK5 kinase activity. Improvement in the ERK5 potency was necessary for the identification of a tool ERK5 inhibitor for target validation studies. Herein, we describe the optimization of this series to identify nanomolar pyrrole carboxamide inhibitors of ERK5 incorporating a basic center, which suffered from poor oral bioavailability. Parallel optimization of potency and in vitro pharmacokinetic parameters led to the identification of a nonbasic pyrazole analogue with an optimal balance of ERK5 inhibition and oral exposure.
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Apr 2022
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B21-High Throughput SAXS
I04-1-Macromolecular Crystallography (fixed wavelength)
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Marco
Salamina
,
Bailey C.
Montefiore
,
Mengxi
Liu
,
Daniel J.
Wood
,
Richard
Heath
,
James R.
Ault
,
Lan-Zhen
Wang
,
Svitlana
Korolchuk
,
Arnaud
Basle
,
Martyna
Pastok
,
Judith
Reeks
,
Natalie J.
Tatum
,
Frank
Sobott
,
Stefan T.
Arold
,
Michele
Pagano
,
Martin E. M.
Noble
,
Jane A.
Endicott
Diamond Proposal Number(s):
[13587, 16970]
Open Access
Abstract: The SCFSKP2 ubiquitin ligase relieves G1 checkpoint control of CDK-cyclin complexes by promoting p27KIP1 degradation. We describe reconstitution of stable complexes containing SKP1-SKP2 and CDK1-cyclin B or CDK2-cyclin A/E, mediated by the CDK regulatory subunit CKS1. We further show that a direct interaction between a SKP2 N-terminal motif and cyclin A can stabilize SKP1-SKP2-CDK2-cyclin A complexes in the absence of CKS1. We identify the SKP2 binding site on cyclin A and demonstrate the site is not present in cyclin B or cyclin E. This site is distinct from but overlapping with features that mediate binding of p27KIP1 and other G1 cyclin regulators to cyclin A. We propose that the capacity of SKP2 to engage with CDK2-cyclin A by more than one structural mechanism provides a way to fine tune the degradation of p27KIP1 and distinguishes cyclin A from other G1 cyclins to ensure orderly cell cycle progression.
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Mar 2021
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