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Instruments / Technical Area	Publication Details	Published
I04-1-Macromolecular Crystallography (fixed wavelength)	Defining substrate selection by rhinoviral 2A proteinase through its crystal structure with the inhibitor zVAM.fmk Karin M. Deutschmann-Olek , Wyatt W. Yue , Gustavo A. Bezerra , Tim Skern Virology 562, 128 - 141 DOI: 10.1016/j.virol.2021.07.008 Diamond Proposal Number(s): [20221] Open Access Show Abstract ▼ Abstract: Picornavirus family members cause disease in humans. Human rhinoviruses (RV), the main causative agents of the common cold, increase the severity of asthma and COPD; hence, effective agents against RVs are required. The 2A proteinase (2Apro), found in all enteroviruses, represents an attractive target; inactivating mutations in poliovirus 2Apro result in an extension of the VP1 protein preventing infectious virion assembly. Variations in sequence and substrate specificity on eIF4G isoforms between RV 2Apro of genetic groups A and B hinder 2Apro as drug targets. Here, we demonstrate that although RV-A2 and RV-B4 2Apro cleave the substrate GAB1 at different sites, the 2Apro from both groups cleave equally efficiently an artificial site containing P1 methionine. We determined the RV-A2 2Apro structure complexed with zVAM.fmk, containing P1 methionine. Analysis of this first 2Apro-inhibitor complex reveals a conserved hydrophobic P4 pocket among enteroviral 2Apro as a potential target for broad-spectrum anti-enteroviral inhibitors.	Oct 2021
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Fragment screening reveals starting points for rational design of galactokinase 1 inhibitors to treat classic galactosemia Sabrina R. Mackinnon , Tobias Krojer , William R. Foster , Laura Diaz-Saez , Manshu Tang , Kilian V. M. Huber , Frank Von Delft , Kent Lai , Paul Brennan , Gustavo Arruda Bezerra , Wyatt W. Yue Acs Chemical Biology 152 DOI: 10.1021/acschembio.0c00498 Diamond Proposal Number(s): [18145] Show Abstract ▼ Abstract: Classic galactosemia is caused by loss-of-function mutations in galactose-1-phosphate uridylyltransferase (GALT) that lead to toxic accumulation of its substrate, galactose-1-phosphate. One proposed therapy is to inhibit the biosynthesis of galactose-1-phosphate, catalyzed by galactokinase 1 (GALK1). Existing inhibitors of human GALK1 (hGALK1) are primarily ATP-competitive with limited clinical utility to date. Here, we determined crystal structures of hGALK1 bound with reported ATP- competitive inhibitors of the spiro-benzoxazole series, to reveal their binding mode in the active site. Spurred by the need for additional chemotypes of hGALK1 inhibitors, desirably targeting a nonorthosteric site, we also performed crystallography-based screening by soaking hundreds of hGALK1 crystals, already containing active site ligands, with fragments from a custom library. Two fragments were found to bind close to the ATP binding site, and a further eight were found in a hotspot distal from the active site, highlighting the strength of this method in identifying previously uncharacterized allosteric sites. To generate inhibitors of improved potency and selectivity targeting the newly identified binding hotspot, new compounds were designed by merging overlapping fragments. This yielded two micromolar inhibitors of hGALK1 that were not competitive with respect to either substrate (ATP or galactose) and demonstrated good selectivity over hGALK1 homologues, galactokinase 2 and mevalonate kinase. Our findings are therefore the first to demonstrate inhibition of hGALK1 from an allosteric site, with potential for further development of potent and selective inhibitors to provide novel therapeutics for classic galactosemia.	Mar 2021
B21-High Throughput SAXS I03-Macromolecular Crystallography	Crystal structure and interaction studies of human DHTKD1 provide insight into a mitochondrial megacomplex in lysine catabolism Gustavo A. Bezerra , William R. Foster , Henry J. Bailey , Kevin G. Hicks , Sven W. Sauer , Bianca Dimitrov , Thomas J. Mccorvie , Jürgen G. Okun , Jared Rutter , Stefan Kölker , Wyatt W. Yue Iucrj 7 DOI: 10.1107/S205225252000696X Open Access Show Abstract ▼ Abstract: DHTKD1 is a lesser-studied E1 enzyme among the family of 2-oxoacid dehydrogenases. In complex with E2 (dihydrolipoamide succinyltransferase, DLST) and E3 (dihydrolipoamide dehydrogenase, DLD) components, DHTKD1 is involved in lysine and tryptophan catabolism by catalysing the oxidative decarboxylation of 2-oxoadipate (2OA) in mitochondria. Here, the 1.9 Å resolution crystal structure of human DHTKD1 is solved in complex with the thiamine diphosphate co-factor. The structure reveals how the DHTKD1 active site is modelled upon the well characterized homologue 2-oxoglutarate (2OG) dehydrogenase but engineered specifically to accommodate its preference for the longer substrate of 2OA over 2OG. A 4.7 Å resolution reconstruction of the human DLST catalytic core is also generated by single-particle electron microscopy, revealing a 24-mer cubic scaffold for assembling DHTKD1 and DLD protomers into a megacomplex. It is further demonstrated that missense DHTKD1 variants causing the inborn error of 2-aminoadipic and 2-oxoadipic aciduria impact on the complex formation, either directly by disrupting the interaction with DLST, or indirectly through destabilizing the DHTKD1 protein. This study provides the starting framework for developing DHTKD1 modulators to probe the intricate mitochondrial energy metabolism.	Jul 2020
B21-High Throughput SAXS I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Human aminolevulinate synthase structure reveals a eukaryotic-specific autoinhibitory loop regulating substrate binding and product release Henry J. Bailey , Gustavo A. Bezerra , Jason R. Marcero , Siladitya Padhi , William R. Foster , Elzbieta Rembeza , Arijit Roy , David F. Bishop , Robert J. Desnick , Gopalakrishnan Bulusu , Harry A. Dailey , Wyatt W. Yue Nature Communications 11 DOI: 10.1038/s41467-020-16586-x Open Access Show Abstract ▼ Abstract: 5′-aminolevulinate synthase (ALAS) catalyzes the first step in heme biosynthesis, generating 5′-aminolevulinate from glycine and succinyl-CoA. Inherited frameshift indel mutations of human erythroid-specific isozyme ALAS2, within a C-terminal (Ct) extension of its catalytic core that is only present in higher eukaryotes, lead to gain-of-function X-linked protoporphyria (XLP). Here, we report the human ALAS2 crystal structure, revealing that its Ct-extension folds onto the catalytic core, sits atop the active site, and precludes binding of substrate succinyl-CoA. The Ct-extension is therefore an autoinhibitory element that must re-orient during catalysis, as supported by molecular dynamics simulations. Our data explain how Ct deletions in XLP alleviate autoinhibition and increase enzyme activity. Crystallography-based fragment screening reveals a binding hotspot around the Ct-extension, where fragments interfere with the Ct conformational dynamics and inhibit ALAS2 activity. These fragments represent a starting point to develop ALAS2 inhibitors as substrate reduction therapy for porphyria disorders that accumulate toxic heme intermediates.	Jun 2020
B21-High Throughput SAXS I04-1-Macromolecular Crystallography (fixed wavelength)	Structural basis for the regulation of human 5,10-methylenetetrahydrofolate reductase by phosphorylation and S-adenosylmethionine inhibition D. Sean Froese , Jolanta Kopec , Elzbieta Rembeza , Gustavo Arruda Bezerra , Anselm Erich Oberholzer , Terttu Suormala , Seraina Lutz , Rod Chalk , Oktawia Borkowska , Matthias R. Baumgartner , Wyatt W. Yue Nature Communications 9 DOI: 10.1038/s41467-018-04735-2 Diamond Proposal Number(s): [10619, 51433] Open Access Show Abstract ▼ Abstract: The folate and methionine cycles are crucial for biosynthesis of lipids, nucleotides and proteins, and production of the methyl donor S-adenosylmethionine (SAM). 5,10-methylenetetrahydrofolate reductase (MTHFR) represents a key regulatory connection between these cycles, generating 5-methyltetrahydrofolate for initiation of the methionine cycle, and undergoing allosteric inhibition by its end product SAM. Our 2.5 Å resolution crystal structure of human MTHFR reveals a unique architecture, appending the well-conserved catalytic TIM-barrel to a eukaryote-only SAM-binding domain. The latter domain of novel fold provides the predominant interface for MTHFR homo-dimerization, positioning the N-terminal serine-rich phosphorylation region near the C-terminal SAM-binding domain. This explains how MTHFR phosphorylation, identified on 11 N-terminal residues (16 in total), increases sensitivity to SAM binding and inhibition. Finally, we demonstrate that the 25-amino-acid inter-domain linker enables conformational plasticity and propose it to be a key mediator of SAM regulation. Together, these results provide insight into the molecular regulation of MTHFR.	Jun 2018

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