I03-Macromolecular Crystallography
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Samuel T.
Benedict
,
Kieran
Bowran
,
Eunice K. E.
Lee
,
Jean-Lou
Reyre
,
Huda
Ahmad
,
Aaron
Franklin
,
Nicole A.
Mietrach
,
Cheng-Ruei
Han
,
Jia Mun
Chan
,
Abigail J.
Layton
,
Emmanuele
Severi
,
Eleanor R.
Boardman
,
Kamilla
Anochshenko
,
Gregory
Goudge
,
Simon G.
Caulton
,
Todd L.
Lowary
,
Andrew L.
Lovering
,
Manuel
Banzhaf
,
Elisabeth C.
Lowe
,
Tracy
Palmer
,
Patrick J.
Moynihan
Diamond Proposal Number(s):
[26803]
Open Access
Abstract: Most bacteria have evolved mechanisms to compete with other bacteria, often through the specialised secretion of proteinaceous toxins. However, mycobacteria have not previously been reported to engage in this form of competition. The thick and unusual mycobacterial cell wall, comprised of peptidoglycan, arabinogalactan and mycolic acids, is generally thought to be highly protective to these bacteria. Here, we show that some mycobacteria can use endo-D-arabinanases of the GH183 family for inter-bacterial competition. These microorganisms secrete an endo-D-arabinanase effector via the type VII secretion system (T7SS) that cleaves the arabinogalactan layer of the mycobacterial cell envelope. We describe the molecular basis for this activity using structural biology and biochemistry, and identify a protein family that protects the bacterium from the activity of this toxin. The widespread presence of genes potentially encoding similar T7SS-secreted toxins in the Mycobacteriales suggests extensive inter-mycobacterial competition.
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Jun 2026
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I04-Macromolecular Crystallography
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Louise N.
Slope
,
Anokhi
Shah
,
Michael J.
Taylor
,
Valentina
Borghesani
,
Simon G.
Caulton
,
Nicholas J.
Brooks
,
Kate A.
Hadley
,
Georgina
Rose
,
Robert I.
Hunter
,
Hassane E. L.
Mkami
,
Graham M.
Smith
,
Aneika C.
Leney
,
Niklaas J.
Buurma
,
Andrew L.
Lovering
,
Janet E.
Lovett
,
Anna F. A.
Peacock
Diamond Proposal Number(s):
[34438]
Open Access
Abstract: Lanthanide-protein scaffolds hold significant promise for the design of functional biomaterials. Yet the selective incorporation of multiple lanthanide ions with distinct properties into discrete sites at tuneable distances within a single construct remains a key challenge. Here, we report the rational design and structural characterization of the first de novo coiled coil capable of binding two different lanthanide ions at independent, non-equivalent sites with defined intermetallic spacing. By installing orthogonal coordination environments, comprising Asn3Asp3 and Asp3-only motifs, at defined positions along the coiled coil axis, we achieve precise, site-specific metal binding across a series of constructs spanning 1 to 5 nm. Site occupancy and intermetallic distances were validated using luminescence, electron paramagnetic resonance (EPR) spectroscopy, mass spectrometry and X-ray crystallography. The latter reveals the first structure of a coiled coil bound to two Tb3+ ions, and the shortest non-bridged metal–metal distance reported to date in such a scaffold (11.9 Å). The chemically distinct coordination sites enable sequential and selective metal loading. Remarkably, this system is capable of binding two different lanthanides, Tb3+ and Yb3+, at distinct sites, despite their extremely similar coordination chemistries. These results establish a robust and modular platform for constructing nanometre-scale molecular rulers, and highlight new avenues for the rational design of multifunctional metalloproteins.
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May 2026
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I04-Macromolecular Crystallography
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Rebecca J.
Parr
,
Yoann G.
Santin
,
Giedrė
Ratkevičiūte
,
Simon G.
Caulton
,
Paul
Radford
,
Dominik
Gurvič
,
Matthew
Jenkins
,
Matthew T.
Doyle
,
Liam
Mead
,
Augustinas
Silale
,
Bert
Van Den Berg
,
Timothy J.
Knowles
,
R. Elizabeth
Sockett
,
Phillip J.
Stansfeld
,
Géraldine
Laloux
,
Andrew L.
Lovering
Diamond Proposal Number(s):
[19880]
Open Access
Abstract: Outer membrane proteins (OMPs) define the surface biology of Gram-negative bacteria, with roles in adhesion, transport, catalysis and signalling. Specifically, porin beta-barrels are common diffusion channels, predominantly monomeric/trimeric in nature. Here we show that the major OMP of the bacterial predator Bdellovibrio bacteriovorus, PopA, differs from this architecture, forming a pentameric porin-like superstructure. Our X-ray and cryo-EM structures reveal a bowl-shape composite outer β-wall, which houses a central chamber that encloses a section of the lipid bilayer. We demonstrate that PopA, reported to insert into prey inner membrane, causes defects when directed into Escherichia coli membranes. We discover widespread PopA homologues, including likely tetramers and hexamers, that retain the lipid chamber; a similar chamber is formed by an unrelated smaller closed-barrel family, implicating this as a general feature. Our work thus defines oligomeric OMP superfamilies, whose deviation from prior structures requires us to revisit existing membrane-interaction motifs and folding models.
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Jul 2025
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I04-Macromolecular Crystallography
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Benjamin F.
Cooper
,
Giedrė
Ratkevičiūtė
,
Luke A.
Clifton
,
Hannah
Johnston
,
Rachel
Holyfield
,
David J.
Hardy
,
Simon G.
Caulton
,
William
Chatterton
,
Pooja
Sridhar
,
Peter
Wotherspoon
,
Gareth W.
Hughes
,
Stephen C. L.
Hall
,
Andrew L.
Lovering
,
Timothy J.
Knowles
Diamond Proposal Number(s):
[26803]
Open Access
Abstract: The E. coli Paraquat Inducible (Pqi) Pathway is a putative Gram-negative phospholipid transport system. The pathway comprises three components: an integral inner membrane protein (PqiA), a periplasmic spanning MCE family protein (PqiB) and an outer membrane lipoprotein (PqiC). Interactions between all complex components, including stoichiometry, remain uncharacterised; nevertheless, once assembled into their quaternary complex, the trio of Pqi proteins are anticipated to provide a continuous channel between the inner and outer membranes of diderms. Here, we present X-ray structures of both the native and a truncated, soluble construct of the PqiC lipoprotein, providing insight into its biological assembly, and utilise neutron reflectometry to characterise the nature of the PqiB-PqiC-membrane interaction. Finally, we employ phenotypic complementation assays to probe specific PqiC residues, which imply the interaction between PqiB and PqiC is less intimate than previously anticipated.
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Jan 2024
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Simon G.
Caulton
,
Carey
Lambert
,
Jess
Tyson
,
Paul
Radford
,
Asmaa
Al-Bayati
,
Samuel
Greenwood
,
Emma J.
Banks
,
Callum
Clark
,
Rob
Till
,
Elisabete
Pires
,
R. Elizabeth
Sockett
,
Andrew L.
Lovering
Diamond Proposal Number(s):
[26803, 19880]
Open Access
Abstract: Predatory bacteria, like the model endoperiplasmic bacterium Bdellovibrio bacteriovorus, show several adaptations relevant to their requirements for locating, entering and killing other bacteria. The mechanisms underlying prey recognition and handling remain obscure. Here we use complementary genetic, microscopic and structural methods to address this deficit. During invasion, the B. bacteriovorus protein CpoB concentrates into a vesicular compartment that is deposited into the prey periplasm. Proteomic and structural analyses of vesicle contents reveal several fibre-like proteins, which we name the mosaic adhesive trimer (MAT) superfamily, and show localization on the predator surface before prey encounter. These dynamic proteins indicate a variety of binding capabilities, and we confirm that one MAT member shows specificity for surface glycans from a particular prey. Our study shows that the B. bacteriovorus MAT protein repertoire enables a broad means for the recognition and handling of diverse prey epitopes encountered during bacterial predation and invasion.
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Jan 2024
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VMXm-Versatile Macromolecular Crystallography microfocus
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Leila T.
Alexander
,
Janani
Durairaj
,
Andriy
Kryshtafovych
,
Luciano A.
Abriata
,
Yusupha
Bayo
,
Gira
Bhabha
,
Cécile
Breyton
,
Simon G.
Caulton
,
James
Chen
,
Séraphine
Degroux
,
Damian C.
Ekiert
,
Benedikte S.
Erlandsen
,
Peter L.
Freddolino
,
Dominic
Gilzer
,
Chris
Greening
,
Jonathan M.
Grimes
,
Rhys
Grinter
,
Manickam
Gurusaran
,
Marcus D.
Hartmann
,
Charlie J.
Hitchman
,
Jeremy R.
Keown
,
Ashleigh
Kropp
,
Petri
Kursula
,
Andrew L.
Lovering
,
Bruno
Lemaitre
,
Andrea
Lia
,
Shiheng
Liu
,
Maria
Logotheti
,
Shuze
Lu
,
Sigurbjorn
Markusson
,
Mitchell D.
Miller
,
George
Minasov
,
Hartmut H.
Niemann
,
Felipe
Opazo
,
George N.
Phillips
,
Owen R.
Davies
,
Samuel
Rommelaere
,
Monica
Rosas‐lemus
,
Pietro
Roversi
,
Karla
Satchell
,
Nathan
Smith
,
Mark A.
Wilson
,
Kuan‐lin
Wu
,
Xian
Xia
,
Han
Xiao
,
Wenhua
Zhang
,
Z. Hong
Zhou
,
Krzysztof
Fidelis
,
Maya
Topf
,
John
Moult
,
Torsten
Schwede
Diamond Proposal Number(s):
[19946, 23570, 27314, 28534]
Open Access
Abstract: We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology.
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Jul 2023
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I24-Microfocus Macromolecular Crystallography
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Juliet
Morgan
,
Muhammad
Saleem
,
Ruiqi
Ng
,
Caroline
Armstrong
,
Szu S.
Wong
,
Simon G.
Caulton
,
Alice
Fickling
,
Huw E. L.
Williams
,
Adam D.
Munday
,
José A.
López
,
Mark S.
Searle
,
Jonas
Emsley
Abstract: Cell-surface receptor interactions between leukocyte integrin macrophage-1 antigen (Mac-1, also known as CR3, αMβ2, CD11b/CD18) and platelet glycoprotein Ibα (GPIbα) are critical to vascular inflammation. To define the key residues at the binding interface, we used nuclear magnetic resonance (NMR) to assign the spectra of the mouse Mac-1 I-domain and mapped the residues contacting the mouse GPIbα N-terminal domain (GPIbαN) to the locality of the integrin metal ion-dependant adhesion site (MIDAS) surface. We next determined the crystal structures of the mouse GPIbαN and Mac-1 I-domain to 2 Å and 2.5 Å resolution, respectively. The mouse Mac-1 I-domain crystal structure reveals an active conformation that is stabilized by a crystal contact from the α7-helix with a glutamate side chain completing the octahedral coordination sphere of the MIDAS Mg2+ ion. The amino acid sequence of the α7-helix and disposition of the glutamic acid matches the C-terminal capping region α-helix of GPIbα effectively acting as a ligand mimetic. Using these crystal structures in combination with NMR measurements and docking analysis, we developed a model whereby an acidic residue from the GPIbα leucine-rich repeat (LRR) capping α-helix coordinates directly to the Mac-1 MIDAS Mg2+ ion. The Mac-1:GPIbαN complex involves additional interactions consolidated by an elongated pocket flanking the GPIbαN LRR capping α-helix. The GPIbαN α-helix has an HxxxE motif, which is equivalent by homology to RxxxD from the human GPIbαN. Subsequent mutagenesis of residues at this interface, coupled with surface plasmon resonance studies, confirmed the importance of GPIbαN residues H218, E222, and the Mac-1 MIDAS residue T209 to formation of the complex.
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May 2019
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I02-Macromolecular Crystallography
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Open Access
Abstract: Ubiquitin specific proteases (USPs) reverse ubiquitination and regulate virtually all cellular processes. Defined non-catalytic domains in USP4 and USP15 are known to interact with E3 ligases and substrate recruitment factors. No such interactions have been reported for these domains in the paralog USP11, a key regulator of DNA double-strand break repair by homologous recombination (HR). We hypothesized that USP11 domains adjacent to its protease domain harbour unique peptide-binding sites. Here, using a next-generation phage display (NGPD) strategy, combining phage display library screening with next generation sequencing, we discovered unique USP11 interacting peptide motifs. Isothermal titration calorimetry disclosed that the highest affinity peptides (KD of ~10 μM) exhibit exclusive selectivity for USP11 over USP4 and USP15 in vitro. Furthermore, a crystal structure of a USP11-peptide complex revealed a previously unknown binding site in USP11’s non-catalytic ubiquitin-like (UBL) region. This site interacted with a helical motif and is absent in USP4 and USP15. Reporter assays using USP11-WT versus a binding pocket-deficient double mutant disclosed that this binding site modulates USP11’s function in HR-mediated DNA repair. The highest affinity USP11 peptide binder fused to a cellular delivery sequence induced significant nuclear localization and cell cycle arrest in S phase, affecting the viability of different mammalian cell lines. The USP11 peptide ligands and the paralog-specific functional site in USP11 identified here provide a framework for the development of new biochemical tools and therapeutic agents. We propose that an NGPD-based strategy for identifying interacting peptides may be applied also to other cellular targets.
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Oct 2018
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14692]
Open Access
Abstract: Ubiquitin specific protease 15 (USP15) regulates important cellular processes, including transforming growth factor β (TGF-β) signaling, mitophagy, mRNA processing, and innate immune responses; however, structural information on USP15’s catalytic domain is currently unavailable. Here, we determined crystal structures of the USP15 catalytic core domain, revealing a canonical USP fold, including a finger, palm, and thumb region. Unlike for the structure of paralog USP4, the catalytic triad is in an inactive configuration with the catalytic cysteine ~10Å apart from the catalytic histidine. This conformation is atypical, and a similar misaligned catalytic triad has so far been observed only for USP7, although USP15 and USP7 are differently regulated. Moreover, we found that the active site loops are flexible, resulting in a largely open ubiquitin tail binding channel. Comparison of the USP15 and USP4 structures points to a possible activation mechanism. Sequence differences between these two USPs mainly map to the S1’ region likely to confer specificity, whereas the S1 ubiquitin-binding pocket is highly conserved. Isothermal titration calorimetry monoubiquitin and linear diubiquitin binding experiments showed significant differences in their thermodynamic profiles, with USP15 displaying a lower affinity for monoubiquitin than USP4. Moreover, we report that USP15 is weakly inhibited by the antineoplastic agent mitoxantrone in vitro. A USP15-mitoxantrone complex structure disclosed that the anthracenedione interacts with the S1’ binding site. Our results reveal first insights into USP15’s catalytic domain structure, conformational changes, differences between paralogs, and small molecule interactions and establish a framework for cellular probe and inhibitor development.
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Sep 2018
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