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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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Anka
Lucic
,
Philip
Hinchliffe
,
Tika R.
Malla
,
Catherine L.
Tooke
,
Jurgen
Brem
,
Karina
Calvopina
,
Christopher T.
Lohans
,
Patrick
Rabe
,
Michael A.
Mcdonough
,
Timothy
Armistead
,
Allen M.
Orville
,
James
Spencer
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[17212, 23269, 18069]
Open Access
Abstract: Penems have demonstrated potential as antibacterials and β-lactamase inhibitors; however, their clinical use has been limited, especially in comparison with the structurally related carbapenems. Faropenem is an orally active antibiotic with a C2 tetrahydrofuran (THF) ring, which is resistant to hydrolysis by some β-lactamases. We report studies on the reactions of faropenem with carbapenem-hydrolysing β-lactamases, focusing on the class A serine β-lactamase KPC-2 and the metallo β-lactamases (MBLs) VIM-2 (a subclass B1 MBL) and L1 (a B3 MBL). Kinetic studies show that faropenem is a substrate for all three β-lactamases, though it is less efficiently hydrolysed by KPC-2. Crystallographic analyses on faropenem-derived complexes reveal the opening of the β-lactam ring with formation of an imine with KPC-2, VIM-2, and L1. In the cases of the KPC-2 and VIM-2 structures, the THF ring is opened to give an alkene, but with L1 the THF ring remains intact. Solution state studies, employing NMR, were performed on L1, KPC-2, VIM-2, VIM-1, NDM-1, OXA-23, OXA-10, and OXA-48. The solution results reveal, in all cases, formation of imine products in which the THF ring is opened; formation of a THF ring-closed imine product was only observed with VIM-1 and VIM-2. An enamine product with a closed THF ring was also observed in all cases, at varying levels. Combined with previous reports, the results exemplify the potential for different outcomes in the reactions of penems with MBLs and SBLs and imply further structure-activity relationship studies are worthwhile to optimise the interactions of penems with β-lactamases. They also exemplify how crystal structures of β-lactamase substrate/inhibitor complexes do not always reflect reaction outcomes in solution.
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Feb 2021
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B21-High Throughput SAXS
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[20229]
Open Access
Abstract: Thioredoxin reductases control the redox state of thioredoxins (Trxs)—ubiquitous proteins that regulate a spectrum of enzymes by dithiol-disulfide exchange reactions. In most organisms, Trx is reduced by NADPH via a thioredoxin reductase flavoenzyme (NTR), but in oxygenic photosynthetic organisms, this function can also be performed by an iron-sulfur ferredoxin (Fdx)-dependent thioredoxin reductase (FTR) that links light to metabolic regulation. We have recently found that some cyanobacteria, such as the thylakoid-less Gloeobacter and the ocean-dwelling green oxyphotobacterium Prochlorococcus, lack NTR and FTR but contain a thioredoxin reductase flavoenzyme (formerly tentatively called deeply-rooted thioredoxin reductase or DTR), whose electron donor remained undefined. Here we demonstrate that Fdx functions in this capacity and report the crystallographic structure of the transient complex between the plant-type Fdx1 and the thioredoxin reductase flavoenzyme from Gloeobacter violaceus. Thereby, our data demonstrate that this cyanobacterial enzyme belongs to the Fdx flavin-thioredoxin reductase (FFTR) family, originally described in the anaerobic bacterium Clostridium pasteurianum. Accordingly, the enzyme hitherto termed DTR is renamed FFTR. Our experiments further show that the redox sensitive peptide CP12 is modulated in vitro by the FFTR/Trx system, demonstrating that FFTR functionally substitutes for FTR in light-linked enzyme regulation in Gloeobacter. Altogether, we demonstrate the FFTR is spread within the cyanobacteria phylum and propose that, by substituting for FTR, it connects the reduction of target proteins to photosynthesis. Besides, the results indicate that FFTR acquisition constitutes a mechanism of evolutionary adaptation in marine phytoplankton such as Prochlorococcus that live in low-iron environments.
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Feb 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[23316, 17844, 13062]
Abstract: The human glucose transporters GLUT1 and GLUT3 have a central role in glucose uptake as canonical members of the Sugar Porter (SP) family. GLUT1 and GLUT3 share a fully conserved substrate-binding site with identical substrate coordination, but differ significantly in transport affinity in line with their physiological function. Here, we present a 2.4 Å crystal structure of GLUT1 in an inward open conformation and compare it with GLUT3 using both structural and functional data. Our work shows that interactions between a cytosolic “SP motif” and a conserved “A motif” stabilize the outward conformational state and increases substrate apparent affinity. Furthermore, we identify a previously undescribed Cl− ion site in GLUT1 and an endofacial lipid/glucose binding site which modulate GLUT kinetics. The results provide a possible explanation for the difference between GLUT1 and GLUT3 glucose affinity, imply a general model for the kinetic regulation in GLUTs and suggest a physiological function for the defining SP sequence motif in the SP family.
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Feb 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[9948]
Open Access
Abstract: Cytochrome P450 CYP153AM.aq from Marinobacter aquaeolei serves as a model enzyme for the terminal (ω-) hydroxylation of medium- to long-chain fatty acids. We have engineered this enzyme using different mutagenesis approaches based on structure-sequence-alignments within the 3DM database and crystal structures of CYP153AM.aq and a homologue CYP153AP.sp. Applying these focused mutagenesis strategies and site-directed saturation mutagenesis, we created a variant that ω-hydroxylates octanoic acid. The M.aqRLT variant exhibited 151-fold improved catalytic efficiency and showed strongly improved substrate binding (25-fold reduced Km compared to the wild type). We then used molecular dynamics simulations to gain deeper insights into the dynamics of the protein. We found the tunnel modifications and the two loop regions showing greatly reduced flexibility in the engineered variant were the main features responsible for stabilizing the enzyme–substrate complex and enhancing the catalytic efficiency. Additionally, we showed that a previously known fatty acid anchor (Q129R) interacts significantly with the ligand to hold it in the reactive position, thereby boosting the activity of the variant M.aqRLT toward octanoic acid. The study demonstrates the significant effects of both substrate stabilization and the impact of enzyme flexibility on catalytic efficiency. These results could guide the future engineering of enzymes with deeply buried active sites to increase or even establish activities toward yet unknown types of substrates.
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Feb 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Mahima
Sharma
,
Palika
Abayakoon
,
Ruwan
Epa
,
Yi
Jin
,
James P.
Lingford
,
Tomohiro
Shimada
,
Masahiro
Nakano
,
Janice W.-y.
Mui
,
Akira
Ishihama
,
Ethan D.
Goddard-borger
,
Gideon J.
Davies
,
Spencer J.
Williams
Diamond Proposal Number(s):
[13587, 18598, 24948]
Open Access
Abstract: The sulfosugar sulfoquinovose (SQ) is produced by essentially all photosynthetic organisms on Earth and is metabolized by bacteria through the process of sulfoglycolysis. The sulfoglycolytic Embden–Meyerhof–Parnas pathway metabolizes SQ to produce dihydroxyacetone phosphate and sulfolactaldehyde and is analogous to the classical Embden–Meyerhof–Parnas glycolysis pathway for the metabolism of glucose-6-phosphate, though the former only provides one C3 fragment to central metabolism, with excretion of the other C3 fragment as dihydroxypropanesulfonate. Here, we report a comprehensive structural and biochemical analysis of the three core steps of sulfoglycolysis catalyzed by SQ isomerase, sulfofructose (SF) kinase, and sulfofructose-1-phosphate (SFP) aldolase. Our data show that despite the superficial similarity of this pathway to glycolysis, the sulfoglycolytic enzymes are specific for SQ metabolites and are not catalytically active on related metabolites from glycolytic pathways. This observation is rationalized by three-dimensional structures of each enzyme, which reveal the presence of conserved sulfonate binding pockets. We show that SQ isomerase acts preferentially on the β-anomer of SQ and reversibly produces both SF and sulforhamnose (SR), a previously unknown sugar that acts as a derepressor for the transcriptional repressor CsqR that regulates SQ-utilization. We also demonstrate that SF kinase is a key regulatory enzyme for the pathway that experiences complex modulation by the metabolites SQ, SLA, AMP, ADP, ATP, F6P, FBP, PEP, DHAP, and citrate, and we show that SFP aldolase reversibly synthesizes SFP. This body of work provides fresh insights into the mechanism, specificity, and regulation of sulfoglycolysis and has important implications for understanding how this biochemistry interfaces with central metabolism in prokaryotes to process this major repository of biogeochemical sulfur.
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Feb 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14692]
Open Access
Abstract: Bacterial hybrid malic enzymes (MaeB grouping, multidomain) catalyse the transformation of malate to pyruvate, and are a major contributor to cellular reducing power and carbon flux. Distinct from other malic enzyme subtypes, the hybrid enzymes are regulated by acetyl-CoA, a molecular indicator of the metabolic state of the cell. Here we solve the structure of a MaeB protein, which reveals hybrid enzymes use the appended phosphotransacetylase (PTA) domain to form a hexameric sensor that communicates acetyl-CoA occupancy to the malic enzyme active site, 60 Å away. We demonstrate that allostery is governed by a large-scale rearrangement that rotates the catalytic subunits 70° between the two states, identifying MaeB as a new model enzyme for the study of ligand-induced conformational change. Our work provides the mechanistic basis for metabolic control of hybrid malic enzymes, and identifies inhibition-insensitive variants that may find utility in synthetic biology.
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Feb 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diego A.
Leonardo
,
Italo A.
Cavini
,
Fernanda A.
Sala
,
Deborah C.
Mendonça
,
Higor
V. D. Rosa
,
Patricia S.
Kumagai
,
Edson
Crusca Jr
,
Napoleao F.
Valadares
,
Ivo A.
Marques
,
Jose
Brandao-neto
,
Claudia E.
Munte
,
Hans R.
Kalbitzer
,
Nicolas
Soler
,
Isabel
Uson
,
Ingemar
André
,
Ana
P. U. Araujo
,
Humberto
D'muniz Pereira
,
Richard C.
Garratt
Abstract: Septins are an example of subtle molecular recognition whereby different paralogues must correctly assemble into functional filaments important for essential cellular events such as cytokinesis. Most possess C-terminal domains capable of forming coiled coils which are believed to be involved in filament formation and bundling. Here, we report an integrated structural approach which aims to unravel their architectural diversity and in so doing provide direct structural information for the coiled-coil regions of five human septins. Unexpectedly, we encounter dimeric structures presenting both parallel and antiparallel arrangements which are in consonance with molecular modelling suggesting that both are energetically accessible. These sequences therefore code for two metastable states of different orientations which employ different but overlapping interfaces. The antiparallel structures present a mixed coiled-coil interface, one side of which is dominated by a continuous chain of core hydrophilic residues. This unusual type of coiled coil could be used to expand the toolkit currently available to the protein engineer for the design of previously unforeseen coiled-coil based assemblies. Within a physiological context, our data provide the first atomic details related to the assumption that the parallel orientation is likely formed between septin monomers from the same filament whilst antiparallelism may participate in the widely described interfilament cross-bridges necessary for higher order structures and thereby septin function.
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Feb 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Yuguang
Zhao
,
Laura-nadine
Schuhmacher
,
Morgan
Roberts
,
Satoshi
Kakugawa
,
Ganka
Bineva-todd
,
Steve
Howell
,
Nicola
O'reilly
,
Christine
Perret
,
Ambrosius P.
Snijders
,
Jean-paul
Vincent
,
E. Yvonne
Jones
Diamond Proposal Number(s):
[14744]
Open Access
Abstract: Objectives: The only proteins known to be modified by O-linked lipidation are Wnts and ghrelin, and enzymatic removal of this post-translational modification inhibits ligand activity. Indeed, the Wnt-deacylase activity of Notum is the basis of its ability to act as a feedback inhibitor of Wnt signalling. Whether Notum also deacylates ghrelin has not been determined. Methods: We used mass-spectrometry to assay ghrelin deacylation by Notum and co-crystallisation to reveal enzyme-substrate interactions at the atomic level. CRISPR/Cas technology was used to tag endogenous Notum and assess its localisation in mice while liver-specific Notum knock-out mice allowed us to investigate the physiological role of Notum in modulating the level of ghrelin deacylation. Results: Mass-spectrometry detected the removal of octanoyl from ghrelin by purified active Notum, but not by an inactive mutant. The 2.2 Å resolution crystal structure of the Notum-ghrelin complex shows the octanoyl lipid is accommodated in the hydrophobic pocket of Notum. The knock-in allele expressing HA-tagged Notum reveals that Notum is produced in the liver and present in the bloodstream, albeit at a low level. Liver-specific inactivation of Notum in animals fed with a high fat diet leads to a small but significant increase in acylated ghrelin in the circulation, while no such increase is seen in wildtype animals on the same diet. Conclusions: Overall our data demonstrate Notum can act as a ghrelin deacylase, and that this may be physiologically relevant under high fat diet conditions. Our work therefore adds Notum to the list of enzymes, including butylcholineasterase and other carboxylesterases, that modulate the acylation state of ghrelin. The contribution of multiple enzymes could help tune the activity of this important hormone to a wide range of physiological conditions.
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Feb 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Declan A.
Gray
,
Joshua B. R.
White
,
Abraham O.
Oluwole
,
Parthasarathi
Rath
,
Amy J.
Glenwright
,
Adam
Mazur
,
Michael
Zahn
,
Arnaud
Basle
,
Carl
Morland
,
Sasha L.
Evans
,
Alan
Cartmell
,
Carol V.
Robinson
,
Sebastian
Hiller
,
Neil A.
Ranson
,
David N.
Bolam
,
Bert
Van Den Berg
Diamond Proposal Number(s):
[13587, 18598]
Open Access
Abstract: In Bacteroidetes, one of the dominant phyla of the mammalian gut, active uptake of large nutrients across the outer membrane is mediated by SusCD protein complexes via a “pedal bin” transport mechanism. However, many features of SusCD function in glycan uptake remain unclear, including ligand binding, the role of the SusD lid and the size limit for substrate transport. Here we characterise the β2,6 fructo-oligosaccharide (FOS) importing SusCD from Bacteroides thetaiotaomicron (Bt1762-Bt1763) to shed light on SusCD function. Co-crystal structures reveal residues involved in glycan recognition and suggest that the large binding cavity can accommodate several substrate molecules, each up to ~2.5 kDa in size, a finding supported by native mass spectrometry and isothermal titration calorimetry. Mutational studies in vivo provide functional insights into the key structural features of the SusCD apparatus and cryo-EM of the intact dimeric SusCD complex reveals several distinct states of the transporter, directly visualising the dynamics of the pedal bin transport mechanism.
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Jan 2021
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