I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[26793]
Open Access
Abstract: Bacteria coexist in polymicrobial communities where they engage in complex interactions, including interbacterial antagonism. The environmental bacterial pathogen Chromobacterium violaceum possesses an active type VI secretion system (T6SS), which relies mainly on VgrG3 for its activity and role in interbacterial competition. However, the arsenal of toxic effectors delivered by this T6SS remains unknown. Here, we identify the repertoire of C. violaceum T6SS effectors and characterize a novel antibacterial Rhs-family effector, RhsF (Rhs with a FIX domain), and its cognate immunity protein, RhsFi. Using mass spectrometry analyses of secreted proteins and proteins co-immunoprecipitated with VgrG3, we identified six novel effector candidates, namely four phospholipases, a protein of unknown function, and the previously-uncharacterized Rhs protein, RhsF (CV_1431). RhsF contains an N-terminal FIX domain and can intoxicate susceptible bacteria in a T6SS-dependent manner. The action of the C-terminal toxin domain of RhsF (RhsF-CT) is prevented by RhsFi (CV_1430), confirming that RhsF-RhsFi comprises an effector-immunity pair. The structure of the RhsF-CT/RhsFi complex determined by X-ray crystallography (1.85 Å resolution) revealed that RhsF-CT shares structural similarity with ADP-ribosyltransferase toxins and that RhsFi inhibits toxicity via direct occlusion of the RhsF-CT catalytic site. Functional assays indicated that RhsF-CT ADP-ribosylates RNA in vitro and that RhsF toxicity requires a catalytic triad composed of R1403, Y1456, and E1497 residues. Overall, our findings reveal effectors secreted by the T6SS of C. violaceum, establish RhsF as a potent antibacterial toxin, and confirm T6SS-dependent delivery of a FIX-containing Rhs protein, expanding the known repertoire of bacterial arms involved in microbial competition.
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Jun 2026
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19844, 33658]
Open Access
Abstract: TonB-dependent uptake systems of gram-negative bacterial pathogens constitute prominent virulence factors, allowing nutrients—primarily siderophore-bound iron—to cross the highly impermeable outer membrane. Remarkably, the ferredoxin uptake system (Fus) of certain soft rot plant pathogens imports an entire host protein into the periplasm and extracts its bound iron for growth. The inner membrane protein FusB, a TonB homolog, plays two roles in facilitating import. First, like other TonBs, it remodels the globular plug domain obstructing the lumen of the outer membrane receptor FusA to allow ferredoxin passage. Unusually for a TonB protein, FusB then interacts directly with the FusA-bound ferredoxin substrate to facilitate its transport into the periplasm. Here, we describe structures of the FusB homodimer as well as the FusB-ferredoxin complex and, using biophysical, biochemical, and mutagenesis approaches, we determine the key features of the binding interfaces formed by FusB with FusA and ferredoxin. The C-terminal domain of FusB (FusB-CTD) exists in a monomer-dimer equilibrium in vitro, with the homodimer stabilized by an intermolecular R241-D322 salt bridge. The “FusB-box” of FusA interacts with monomeric FusB-CTD, and FusA D53 outcompetes FusB D322 to bind R241. Upon ferredoxin binding, FusB-CTD undergoes a structural rearrangement, expanding its β-sheet from three to four strands. In agreement with the proposed sequence of events, ferredoxin binding displaces FusA from FusB with R241 forming an intramolecular salt bridge with D322 to stabilize the newly formed β-hairpin of FusB. We propose a mechanistic model for ferredoxin import where FusB R241 acts as a molecular switch.
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Jun 2026
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[31850]
Open Access
Abstract: Chordin is a cysteine-rich protein which acts as a regulator of bone morphogenetic protein (BMP) signalling in the extracellular matrix. Acting in concert with twisted gastrulation (TWSG1), chordin works as an antagonist of BMP signalling by binding tightly to the growth factor and is a vital component of the network of interactions that establish developmental signalling gradients. Chordin is known to interact with BMP ligands via its four von-Willebrand factor type C domains, but the function of the large central four CHRD domains were previously unknown. Here we show that these domains interact strongly with sulphated glycosaminoglycans (GAGs) and provide evidence for the location of the binding site using X-ray crystallographic analysis combined with mutagenesis and biophysical techniques. Additionally, we report the first recombinant expression and purification of the complete functional chordin, TWSG1, BMP2, BMP7 complex which was used to demonstrate that the four CHRD domains are largely redundant with respect to the role of chordin as an inhibitor of BMP ligands. We therefore propose that the four CHRD domains of chordin have relevance in the diffusion and localisation of chordin-TWSG1-BMP complexes at the tissue and organismal level, mediated by their interaction with GAGs or proteoglycans.
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Jun 2026
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[39641]
Open Access
Abstract: The β-barrel assembly machinery (BAM) inserts outer membrane proteins (OMPs) into the outer membrane of Gram-negative bacteria. In well-studied organisms such as Escherichia coli, BAM consists of BamA and four accessory lipoproteins (BamB-E) that coordinate substrate recognition, membrane remodeling, and β-barrel insertion. In the Lyme disease spirochete Borrelia burgdorferi, however, the BAM system is simplified, comprising only BamA (BB0795), BamD (BB0324), and BamB (BB0028), while lacking BamC and BamE. In addition, B. burgdorferi encodes a TamB homolog (BB0794), suggesting a hybrid BAM-TAM organization.
Here, we define the structural architecture of the spirochetal BAM-TAM system. AlphaFold3 modeling predicts that BamD binds to BamA periplasmic domains POTRA4-5, while BamB interacts with POTRA1, POTRA3, POTRA4, and POTRA5, forming a closed periplasmic ring distinct from the enterobacterial arrangement. Microscale thermophoresis confirms micromolar-affinity binding of BamD to BamA POTRA3-5, no detectable interaction with POTRA1-3, and loss of POTRA3-5 interaction upon mutation of key interface residues. Crystal structures of BamA POTRA1-2 and POTRA2-3 further define the architecture and flexibility of the periplasmic region.
Strikingly, structural modeling of the full complex suggests that the C-terminal β-strands of TamB complete the BamA β-barrel through β-strand augmentation, forming a continuous hybrid barrel with a hydrophobic exterior and hydrophilic interior. This arrangement may provide a direct structural link between periplasmic protein handling and membrane insertion.
Together, these findings support a reorganized BAM architecture in B. burgdorferi and suggest that the hybrid BAM-TAM organization is conserved across Lyme disease-associated Borrelia species, providing a framework for understanding outer membrane assembly in spirochetes.
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Jun 2026
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[21035]
Open Access
Abstract: The protein kinase C-related kinase (PKN) family of serine/threonine kinases consists of PKN1, PKN2 and PKN3, all of which are Rho family GTPase effectors. PKNs have three N-terminal Homology Region 1 (HR1) domains (HR1a, HR1b and HR1c), which form antiparallel coiled coils, which in two cases interact with Rho family GTPases, activating the kinase. The PKNs are implicated in several important cellular processes, including cytoskeletal regulation, cell adhesion, gene expression and cell cycle progression, and are also implicated in cancer. Here we have investigated the roles of the HR1 domains in PKN oligomerisation. We show that PKN1 HR1a is a dimer and that the HR1c domain drives further oligomerization. We have mapped the interactions between the HR1 domains and used an integrative approach to model HR1-containing PKN1 dimers. Biophysical analysis shows that RhoA forms a 1:2 complex with HR1a, resulting in a rearrangement of the HR1a dimer, an outcome supported by SAXS models. In contrast, Rac1 binds to monomeric HR1a, suggesting that this GTPase activates PKN1 via a different mechanism. These data provide structural insight into interactions between HR1 domains and the Rho family proteins and their potential consequences for PKN1 activation.
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Apr 2026
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[23269]
Open Access
Abstract: The malarial parasite, Plasmodium falciparum (Pf), utilizes aminopeptidases in the breakdown of hemoglobin-derived oligopeptides to release amino acids for protein synthesis during growth and asexual reproduction of erythrocytic stages of the parasite. However, a N-terminal peptide bond that involves proline is difficult to hydrolyze. Aminopeptidase P (APP) is capable of cleaving peptide bonds with proline in the second position. Inhibition of PfAPP is therefore an attractive strategy for developing therapeutics for the treatment of malaria by limiting the supply of amino acids at the erythrocytic stage. We employed the structure-activity relationship of an existing APP inhibitor, apstatin, to design a more potent PfAPP inhibitor by introducing a hydroxamic acid metal-binding group in place of the amino-alcohol of apstatin, and an aromatic P4′ moiety. A hydroxamic tetrapeptide with phenylalanine at P4′ (6d) greatly increased the inhibitory potency (apstatin Ki, 16 μM; 6d, Ki 685 nM). Replacing the P3′ proline of 6d with a 2-substituted piperidine (6e) further improved the potency (Ki, 24 nM). Crystal structure analysis of PfAPP in complex with 6d and 6e showed binding at the active site with coordination of the hydroxamic acid metal binding group to the di-metal center, and several protein-inhibitor interactions involving domains II and III. A comparison of PfAPP-6e with human APP1 indicated that the P4′ phenylalanine drives inhibitor potency and selectivity towards PfAPP, by forming an interaction with Tyr617 of the adjacent monomer within the dimer. The details presented here should be useful for the future design of potent and selective PfAPP inhibitors.
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Mar 2026
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14692, 19880]
Open Access
Abstract: The innate immune protein human surfactant protein D (SP-D) recognises pathogens in the lungs via binding to carbohydrate surface structures. SP-D targets gram-negative bacterial lipopolysaccharide via calcium-dependent binding, preferentially to the inner core heptose (HepI). To further investigate this recognition, we have determined the high-resolution crystal structures of a trimeric recombinant fragment of human SP-D complexed with synthetic di- and trisaccharides, HepI-Kdo, HepIII-HepII-HepI, and HepII-HepI phosphorylated at either HepI or HepII, inner core lipopolysaccharide motifs common to many gram-negative bacteria. In contrast to acid-hydrolysed lipopolysaccharide used in several previous studies, these synthetic saccharides allow presentation of both the innermost Kdo in its natural pyranose form and heptose phosphorylation. The structures confirm the flexibility of SP-D to adopt alternative binding modes when the preferred epitope is not available, reveal a preference for recognition of the reducing terminal heptose (HepI) via the glyceryl group, indicate that a single Kdo attached to HepI does not have a significant role in ligand recognition, and provide evidence that heptose phosphorylation is a major determinant of recognition. The disaccharide with HepII O4’ phosphorylation binds via the preferred HepI glyceryl-hydroxyls, while HepI O4’ phosphorylation reveals HepII binding via the pyranose ring O3’ and O4’ hydroxyls, which would not be possible with the usual HepII O3’ link to the outer core. The ability of HepI O4’ phosphorylation to prevent preferred HepI recognition suggests a role for heptose phosphorylation in shielding the bacterial LPS inner core from immune recognition.
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Feb 2026
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23459, 31353]
Open Access
Abstract: Traboulsi Syndrome is an autosomal recessive hereditary disease associated with developmental defects, in particular of the ocular system. Single nucleotide polymorphisms affecting the ASPH gene, which encodes for the 2-oxoglutarate (2OG)-dependent oxygenase aspartate/asparagine-β-hydroxylase (AspH), are associated with Traboulsi Syndrome. AspH catalyzes hydroxylations of conserved aspartate/asparagine residues in epidermal growth factor-like domain (EGFD) proteins. We report studies on the clinically-observed Traboulsi Syndrome-associated R688Q, R735Q, and R735W AspH variants. The results reveal that pathogenic active site substitutions substantially reduce, though do not ablate, EGFD hydroxylase activity compared to wildtype AspH. They imply that efficient AspH catalyzed EGFD hydroxylation is important during human development. Crystallographic studies reveal conservation of the overall AspH fold, but that the preferred conformations of 2OG in complex with the R735Q and R735W AspH variants differ from that with wildtype AspH. Screening of potential 2OG cosubstrate substitutes reveals certain 2-oxoacids, including naturally present metabolites, manifest enhanced catalytic efficiency of Traboulsi Syndrome-associated AspH variants compared to 2OG. The results thus provide proof-of-principle for a therapeutic strategy involving rescue of impaired activities of pathogenic active site AspH variants by use of 2-oxoacids, or 2-oxoacid precursors, other than 2OG.
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Dec 2025
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I04-Macromolecular Crystallography
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Will
Scott
,
Esther
Ivorra-Molla
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Dipayan
Akhuli
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Teresa
Massam-Wu
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Pawel K.
Lysyganicz
,
Rylie
Walsh
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Matthew
Parent
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Jonathan
Cook
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Lijiang
Song
,
Abhishek
Kumar
,
Falk
Schneider
,
Masanori
Mishima
,
Allister
Crow
,
Mohan K.
Balasubramanian
Open Access
Abstract: Photobleaching of fluorescent proteins often limits the acquisition of high-quality images in microscopy. StayGold, a novel dimeric green fluorescent protein recently monomerised through sequence engineering, addresses this challenge with its high photostability. There is now focus on producing different colour StayGold derivatives to facilitate concurrent tagging of multiple targets. The unnatural amino acid 3-aminotyrosine has previously been shown to red-shift superfolder GFP upon incorporation into its chromophore via genetic code expansion. Here we apply the same strategy to red-shift StayGold through substitution of Tyrosine-58 with 3-aminotyrosine. The resultant red fluorescent protein, StayRose, shows an excitation wavelength maximum of 530 nm and an emission wavelength maximum of 588 nm. Importantly, the monomeric mStayRose retains the favourable photostability in vivo in E. coli and zebrafish embryos. A high-resolution crystal structure of StayRose confirms the modified structure of the amino chromophore within an unperturbed 3D fold. Although reliant on genetic code expansion, StayRose provides an important step towards developing red-shifted StayGold derivatives.
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Oct 2025
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[31440, 37593]
Open Access
Abstract: α-Methylacyl-CoA racemase (AMACR; P504S) is a pivotal enzyme involved in the β-oxidation of branched-chain fatty acids and bile acid intermediates, catalyzing the conversion between (2R)- and (2S)-2-methylacyl-CoA thioester epimers. The AMACR reaction enables downstream catabolism of these thioesters via stereospecific enzymes within the β-oxidation pathway. The AMACR homolog in Mycobacterium tuberculosis (MCR) has emerged as a tractable model for dissecting the mechanistic underpinnings of the racemization reaction and presents a promising therapeutic target given the pathogen’s dependence on lipid metabolism for persistence and virulence. Previously we reported the detailed molecular structure of wild-type MCR and in complex with a diverse set of acyl-CoA substrates. They revealed conserved active site residues that mediate substrate anchoring and epimerization and highlighted distinct molecular interactions that confer selectivity toward 2-methyl-branched substrates. Complementing these results, in this report we present high-resolution structures for 2-arylthiopropanoyl-CoA inhibitors in complex with MCR and a comprehensive set of enzyme inhibition assays to delineate structure-activity relationships and probe competitive binding modes. Our findings underscore the importance of inhibitor side-chain branching and CoA anchoring in modulating enzymatic turnover and inhibition. Together, these data enhance our understanding of the racemization mechanism of MCR and establish a structural foundation for the rational design of selective inhibitors. Targeting MCR could represent a novel future therapeutic strategy for M. tuberculosis based on impairing fatty acid utilization.
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Oct 2025
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