I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Andrew
Poole
,
Vijaykumar
Karuppiah
,
Annabelle
Hartt
,
Jaafar N.
Haidar
,
Sylvie
Moureau
,
Tomasz
Dobrzycki
,
Conor
Hayes
,
Christopher
Rowley
,
Jorge
Dias
,
Stephen
Harper
,
Keir
Barnbrook
,
Miriam
Hock
,
Charlotte
Coles
,
Wei
Yang
,
Milos
Aleksic
,
Aimee Bence
Lin
,
Ross
Robinson
,
Joe D.
Dukes
,
Nathaniel
Liddy
,
Marc
Van Der Kamp
,
Gregory D.
Plowman
,
Annelise
Vuidepot
,
David K.
Cole
,
Andrew D.
Whale
,
Chandramouli
Chillakuri
Diamond Proposal Number(s):
[22870]
Open Access
Abstract: Neoantigens derived from somatic mutations are specific to cancer cells and are ideal targets for cancer immunotherapy. KRAS is the most frequently mutated oncogene and drives the pathogenesis of several cancers. Here we show the identification and development of an affinity-enhanced T cell receptor (TCR) that recognizes a peptide derived from the most common KRAS mutant, KRASG12D, presented in the context of HLA-A*11:01. The affinity of the engineered TCR is increased by over one million-fold yet fully able to distinguish KRASG12D over KRASWT. While crystal structures reveal few discernible differences in TCR interactions with KRASWT versus KRASG12D, thermodynamic analysis and molecular dynamics simulations reveal that TCR specificity is driven by differences in indirect electrostatic interactions. The affinity enhanced TCR, fused to a humanized anti-CD3 scFv, enables selective killing of cancer cells expressing KRASG12D. Our work thus reveals a molecular mechanism that drives TCR selectivity and describes a soluble bispecific molecule with therapeutic potential against cancers harboring a common shared neoantigen.
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Sep 2022
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Claire
Barber
,
Victoria Arena
De Souza
,
Rachel L.
Paterson
,
Magdalena
Martin-Urdiroz
,
Nitha Charles
Mulakkal
,
Velupillai
Srikannathasan
,
Mary
Connolly
,
Gwilym
Phillips
,
Tein
Foong-Leong
,
Robert
Pengelly
,
Vijaykumar
Karuppiah
,
Tressan
Grant
,
Marcin
Dembek
,
Anil
Verma
,
Dawn
Gibbs-Howe
,
Thomas H.
Blicher
,
Andrew
Knox
,
Ross A.
Robinson
,
David K.
Cole
,
Sarah
Leonard
Abstract: The non-polymorphic class Ib molecule, human leukocyte antigen (HLA)-E, primarily presents peptides from HLA class Ia leader peptides, providing an inhibitory signal to NK cells via CD94/NKG2 interactions.
Although peptides of pathogenic origin can also be presented by HLA-E to T cells, the molecular basis underpinning their role in antigen surveillance is largely unknown. Here, we solved a co-complex crystal structure of a T cell receptor (TCR) with an HLA-E presented peptide (pHLA-E) from bacterial (Mycobacterium tuberculosis) origin, and the first TCR-pHLA-E complex with a non-canonically presented peptide from viral (human immuno-deficiency virus; HIV) origin. The structures provided a molecular foundation to develop a novel method to introduce cysteine traps using non-natural amino acid chemistry that stabilized pHLA-E complexes whilst maintaining native interface contacts between the TCRs and different pHLA-E complexes. These pHLA-E monomers could be used to isolate pHLA-E specific T cells, with obvious utility for studying pHLA-E restricted T cells, and for the identification of putative therapeutic TCRs.
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Feb 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[12788]
Open Access
Abstract: In the native pathway to therapeutic cannabinoid biosynthesis in Cannabis sativa, the three‐step production of a key intermediate, olivetolic acid, is catalysed by the enzymes tetraketide synthase (TKS; linear tetraketide intermediate production in two stages) and olivetolic acid cyclase (OAC; final C2 → C7 aldol condensation). In the absence of OAC, a nonenzymatic C2 → C7 decarboxylative aldol condensation of the tetraketide intermediate occurs forming olivetol. TKS is a type III polyketide synthase, and the question arises why it is unable to form olivetolic acid directly, but instead forms this unwanted side product. We determined the TKS, CoA complex structure, and performed structurally guided mutagenesis studies to identify potential residues responsible for cyclization pathway discrimination in type III polyketide synthases. Prior studies suggested an ‘aldol switch’ is necessary to allow linear tetraketide intermediate release prior to cyclization, thereby enabling subsequent olivetolic acid production by OAC. However, our studies do not support the presence of a universal or predictable ‘aldol switch’ consensus sequence. Instead, we propose the mode of ordered active site water activation between type III polyketide synthases catalysing different cyclization mechanisms is subtle and homologue‐specific. Our work indicates that subtle structural variations between homologous enzymes can have a major mechanistic impact on the catalytic outcome. This highlights the importance of embedding high‐resolution structural analysis of multiple enzyme homologues with classical site‐directed mutagenesis studies when investigating highly similar enzymes with different mechanistic pathway outcomes.
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Oct 2019
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[8997]
Open Access
Abstract: Natural competence is the term used to describe the uptake of “naked” extracellular DNA by bacteria; it plays a significant role in horizontal genetic exchange. It is associated with type IV pili, and specialized competence pili mediate DNA uptake. Here, we show that the crystal structure of a competence-associated protein from Thermus thermophilus, ComZ, consists of a type II secretion pseudopilin-like domain, with a large β-solenoid domain inserted into the β-sheet of the pilin-like fold. ComZ binds with high affinity to another competence-associated pilin, PilA2, which lies adjacent to the comZ gene in the genome. The crystal structure of PilA2 revealed a similar type II secretion pseudopilin-like fold, with a small subdomain; docking simulations predicted that PilA2 binds between the pseudopilin-like and β-solenoid domains of ComZ. Electrophoretic shift analysis and DNase protection studies were used to show that ComZ alone and the ComZ/PilA2 complex are able to bind DNA. Protection against reductive dimethylation was used in combination with mass spectrometry and site-directed mutagenesis to identify two lysine residues in ComZ which are involved in DNA binding. They are located between the two domains in ComZ, on the opposite side from the predicted PilA2 binding site. These results suggest a model in which PilA2 assists ComZ in forming the competence pilus tip and DNA binds to the side of the fiber. The results demonstrate how a type IV pilin can be adapted to a specific function by domain insertion and provide the first structural insights into a tip-located competence pilin.
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Jun 2019
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[8997, 14509]
Open Access
Abstract: Type IV pili are responsible for a diverse range of functions, including twitching motility and cell adhesion. Assembly of the pilus fiber is driven by a cytoplasmic ATPase: it interacts with an inner membrane complex of biogenesis proteins which, in turn, bind to nascent pilin subunits and mediate fiber assembly. Here we report the structural characterization of the PilF TFP assembly ATPase from Thermus thermophilus. The crystal structure of a recombinant C-terminal fragment of PilF revealed bound, unhydrolysed ATP, although the full length complex was enzymatically active. 3D reconstructions were carried out by single particle cryoelectron microscopy for full length apoprotein PilF and in complex with AMPPNP. The structure forms an hourglass-like shape, with the ATPase domains in one half and the N1 domains in the second half which, we propose, interact with the other pilus biogenesis components. Molecular models for both forms were generated: binding of AMPPNP causes an upward shift of the N1 domains towards the ATPase domains of ~8 Å. We advocate a model in which ATP hydrolysis is linked to displacement of the N1 domains which is associated with lifting pilin subunits out of the inner membrane, and provide the activation energy needed to form the pilus fiber.
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Sep 2018
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[12788]
Abstract: Monoterpenoids offer potential as bio-derived monomer feedstocks for high performance renewable polymers. We describe a biocatalytic route to lactone monomers menthide and dihydrocarvide employing Baeyer-Villiger monooxygenases (BVMOs) from Pseudomonas sp. HI-70 (CPDMO) and Rhodococcus sp. Phi1 (CHMOPhi1) as an alternative to organic synthesis. The regio-selectivity of dihydrocarvide isomer formation was controlled by site-directed mutagenesis of three key active site residues in CHMOPhi1. A combination of crystal structure determination, molecular dynamics simulations and mechanistic modeling using density functional theory (DFT) on a range of models provides insight into the origins of discrimination of wild type (WT) and a variant CHMOPhi1 for producing different regio-isomers of the lactone product. Ring-opening polymerizations of the resultant lactones using mild metal-organic catalysts demonstrate their utility in polymer production. This semi-synthetic approach utilizing a biocatalytic step, non-petroleum feedstocks and mild polymerization catalysts, allows access to known and also to previously unreported and potentially novel lactone monomers and polymers.
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Mar 2018
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Vijaykumar
Karuppiah
,
Kara
Ranaghan
,
Nicole G. H.
Leferink
,
Linus O.
Johannissen
,
Muralidharan
Shanmugam
,
Aisling
Ni Cheallaigh
,
Nathan
Bennett
,
Lewis
Kearsey
,
Eriko
Takano
,
John
Gardiner
,
Marc
Van Der Kamp
,
Sam
Hay
,
Adrian J.
Mulholland
,
David
Leys
,
Nigel S.
Scrutton
Diamond Proposal Number(s):
[12788]
Open Access
Abstract: Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an important class of terpenes and are industrially important as flavours and fragrances. We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthase (bCinS) and show that these are active biocatalysts for monoterpene production using biocatalysis and metabolic engineering platforms. In metabolically engineered monoterpene-producing E. coli strains use of bLinS leads to 300-fold higher linalool production compared with the corresponding plant monoterpene synthase. With bCinS, 1,8-cineole is produced with 96% purity compared to 67% from plant species. Structures of bLinS and bCinS, and their complexes with fluorinated substrate analogues, show that these bacterial monoterpene synthases are similar to previously characterised sesquiterpene synthases. Molecular dynamics simulations suggest that these monoterpene synthases do not undergo large-scale conformational changes during the reaction cycle making them attractive targets for structured-based protein engineering to expand the catalytic scope of these enzymes towards alternative monoterpene scaffolds. Comparison of the bLinS and bCinS structures indicates how their active sites steer reactive carbocation intermediates to the desired acyclic linalool (bLinS) or bicyclic 1,8-cineole (bCinS) products. The work reported here provides the analysis of structures for this important class of monoterpene synthase. This should now guide exploitation of the bacterial enzymes as gateway biocatalysts for the production of other monoterpenes and monoterpenoids.
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Aug 2017
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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John
Waller
,
Helen
Toogood
,
Vijaykumar
Karuppiah
,
Nicholas John William
Rattray
,
David
Mansell
,
David
Leys
,
John M.
Gardiner
,
Anna
Fryszkowska
,
Syed
Ahmed
,
Rakeshwar
Bandichhor
,
Prashanth Reddy
Gaddameedhi
,
Nigel
Scrutton
Diamond Proposal Number(s):
[12788]
Open Access
Abstract: Reduction of double bonds of α,β-unsaturated carboxylic acids and esters by ene-reductases remains challenging and it typically requires activation by a second electron-withdrawing moiety, such as a halide or second carboxylate group. We showed that profen precursors, 2-arylpropenoic acids and their esters, were efficiently reduced by Old Yellow Enzymes (OYEs). The XenA and GYE enzymes showed activity towards acids, while a wider range of enzymes were active towards the equivalent methyl esters. Comparative co-crystal structural analysis of profen-bound OYEs highlighted key interactions important in determining substrate binding in a catalytically active conformation. The general utility of ene reductases for the synthesis of (R)-profens was established and this work will now drive future mutagenesis studies to screen for the production of pharmaceutically-active (S)-profens.
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Apr 2017
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Type IV pilins are proteins which form polymers that extend from the surface of the bacterial cell; they are involved in mediating a wide variety of functions, including adhesion, motility and natural competence. Here we describe the determination of the crystal structures of three type IVa pilins proteins from the thermophile Thermus thermophilus. They form part of a cluster of pilus-like proteins within the genome; our results show that one, Tt1222, is very closely related to the main structural type IV pilin, PilA4. The other two, Tt1218 and Tt1219, also adopt canonical pilin-like folds but, interestingly, are most closely related to the structures of the type II secretion system pseudopilins, EpsI/GspI and XcpW/GspJ. GspI and GspJ have been shown to form a complex with another pseudopilin, GspK, and this heterotrimeric complex is known to play a key role in initiating assembly of a pseudopilus which is thought to drive the secretion process. The structural similarity of Tt1218 and Tt1219 to GspI and GspJ suggests that they might work in a similar way, to deliver functions associated with type IV pili in T. thermophilus, such as natural competence.
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Dec 2016
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8997]
Open Access
Abstract: Three enzymes of the Mentha essential oil biosynthetic pathway are highly homologous, namely the ketoreductases (−)-menthone:(−)-menthol reductase and (−)-menthone:(+)-neomenthol reductase, and the “ene” reductase isopiperitenone reductase. We identified a rare catalytic residue substitution in the last two, and performed comparative crystal structure analyses and residue-swapping mutagenesis to investigate whether this determines the reaction outcome. The result was a complete loss of native activity and a switch between ene reduction and ketoreduction. This suggests the importance of a catalytic glutamate vs. tyrosine residue in determining the outcome of the reduction of α,β-unsaturated alkenes, due to the substrate occupying different binding conformations, and possibly also to the relative acidities of the two residues. This simple switch in mechanism by a single amino acid substitution could potentially generate a large number of de novo ene reductases.
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Aug 2016
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