I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: CodB is a cytosine transporter from the Nucleobase-Cation-Symport-1 (NCS1) transporter family, a member of the widespread LeuT superfamily. Previous experiments with the nosocomial pathogen Pseudomonas aeruginosa have shown CodB as also important for the uptake of 5-fluorocytosine, which has been suggested as a novel drug to combat antimicrobial resistance by suppressing virulence. Here we solve the crystal structure of CodB from Proteus vulgaris, at 2.4 Å resolution in complex with cytosine. We show that CodB carries out the sodium-dependent uptake of cytosine and can bind 5-fluorocytosine. Comparison of the substrate-bound structures of CodB and the hydantoin transporter Mhp1, the only other NCS1 family member for which the structure is known, highlight the importance of the hydrogen bonds that the substrates make with the main chain at the breakpoint in the discontinuous helix, TM6. In contrast to other LeuT superfamily members, neither CodB nor Mhp1 makes specific interactions with residues on TM1. Comparison of the structures provides insight into the intricate mechanisms of how these proteins transport substrates across the plasma membrane.
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Jul 2022
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I04-Macromolecular Crystallography
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Open Access
Abstract: Gram-negative pathogens like Burkholderia pseudomallei use trimeric autotransporter adhesins such as BpaC as key molecules in their pathogenicity. Our 1.4 Å crystal structure of the membrane proximal part of the BpaC head domain shows that the domain is exclusively made of left-handed parallel β-roll repeats. This, the largest such structure solved, has two unique features. First, the core, rather than being composed of the canonical hydrophobic Ile and Val, is made up primarily of the hydrophilic Thr and Asn, with two different solvent channels. Second, comparing BpaC to all other left-handed parallel β-roll structures showed that the position of the head domain in the protein correlates with the number and type of charged residues. In BpaC, only negatively charged residues face the solvent – in stark contrast to the primarily positive surface charge of the left-handed parallel β-roll “type” protein, YadA. We propose extending the definitions of these head domains to include the BpaC-like head domain as a separate subtype, based on its unusual sequence, position and charge. We speculate that the function of left-handed parallel β-roll structures may differ depending on their position in the structure.
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Jun 2022
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I03-Macromolecular Crystallography
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Samuel L.
Freeman
,
Vera
Skafar
,
Hanna
Kwon
,
Alistair J.
Fielding
,
Peter C. E.
Moody
,
Alejandra
Martínez
,
Federico
Issoglio
,
Lucas
Inchausti
,
Pablo
Smircich
,
Ari
Zeida
,
Lucía
Piacenza
,
Rafael
Radi
,
Emma L.
Raven
Diamond Proposal Number(s):
[23269]
Open Access
Abstract: The protozoan parasite Trypanosoma cruzi is the causative agent of American trypanosomiasis, otherwise known as Chagas disease. To survive in the host, the T. cruzi parasite needs antioxidant defence systems. One of these is a hybrid heme peroxidase, the T. cruzi ascorbate peroxidase-cytochrome c peroxidase enzyme (TcAPx-CcP). TcAPx-CcP has high sequence identity to members of the class I peroxidase family, notably ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP), as well as a mitochondrial peroxidase from Leishmania major (LmP). The aim of this work was to solve the structure and examine the reactivity of the TcAPx-CcP enzyme. Low temperature electron paramagnetic resonance (EPR) spectra support the formation of an exchange-coupled [Fe(IV)=O Trp233•+] Compound I radical species, analogous to that used in CcP and LmP. We demonstrate that TcAPx-CcP is similar in overall structure to APX and CcP, but there are differences in the substrate binding regions. Furthermore, the electron transfer pathway from cytochrome c to the heme in CcP and LmP is preserved in the TcAPx-CcP structure. Integration of steady state kinetic experiments, molecular dynamic simulations, and bioinformatic analyses indicates that TcAPx-CcP preferentially oxidizes cytochrome c, but is still competent for oxididation of ascorbate. The results reveal that TcAPx-CcP is a credible cytochrome c peroxidase which can also bind and use ascorbate in host cells, where concentrations are in the millimolar range. Thus, kinetically and functionally TcAPx-CcP can be considered a hybrid peroxidase.
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Jun 2022
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Ina
Pöhner
,
Antonio
Quotadamo
,
Joanna
Panecka-Hofman
,
Rosaria
Luciani
,
Matteo
Santucci
,
Pasquale
Linciano
,
Giacomo
Landi
,
Flavio
Di Pisa
,
Lucia
Dello Iacono
,
Cecilia
Pozzi
,
Stefano
Mangani
,
Sheraz
Gul
,
Gesa
Witt
,
Bernhard
Ellinger
,
Maria
Kuzikov
,
Nuno
Santarem
,
Anabela
Cordeiro-Da-Silva
,
Maria P.
Costi
,
Alberto
Venturelli
,
Rebecca C.
Wade
Open Access
Abstract: The optimization of compounds with multiple targets is a difficult multidimensional problem in the drug discovery cycle. Here, we present a systematic, multidisciplinary approach to the development of selective antiparasitic compounds. Computational fragment-based design of novel pteridine derivatives along with iterations of crystallographic structure determination allowed for the derivation of a structure–activity relationship for multitarget inhibition. The approach yielded compounds showing apparent picomolar inhibition of T. brucei pteridine reductase 1 (PTR1), nanomolar inhibition of L. major PTR1, and selective submicromolar inhibition of parasite dihydrofolate reductase (DHFR) versus human DHFR. Moreover, by combining design for polypharmacology with a property-based on-parasite optimization, we found three compounds that exhibited micromolar EC50 values against T. brucei brucei while retaining their target inhibition. Our results provide a basis for the further development of pteridine-based compounds, and we expect our multitarget approach to be generally applicable to the design and optimization of anti-infective agents.
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Jun 2022
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I24-Microfocus Macromolecular Crystallography
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Richard J.
Gildea
,
James
Beilsten-Edmands
,
Danny
Axford
,
Sam
Horrell
,
Pierre
Aller
,
James
Sandy
,
Juan
Sanchez-Weatherby
,
C. David
Owen
,
Petra
Lukacik
,
Claire
Strain-Damerell
,
Robin L.
Owen
,
Martin A.
Walsh
,
Graeme
Winter
Diamond Proposal Number(s):
[26986, 27088]
Open Access
Abstract: In macromolecular crystallography, radiation damage limits the amount of data that can be collected from a single crystal. It is often necessary to merge data sets from multiple crystals; for example, small-wedge data collections from micro-crystals, in situ room-temperature data collections and data collection from membrane proteins in lipidic mesophases. Whilst the indexing and integration of individual data sets may be relatively straightforward with existing software, merging multiple data sets from small wedges presents new challenges. The identification of a consensus symmetry can be problematic, particularly in the presence of a potential indexing ambiguity. Furthermore, the presence of non-isomorphous or poor-quality data sets may reduce the overall quality of the final merged data set. To facilitate and help to optimize the scaling and merging of multiple data sets, a new program, xia2.multiplex, has been developed which takes data sets individually integrated with DIALS and performs symmetry analysis, scaling and merging of multi-crystal data sets. xia2.multiplex also performs analysis of various pathologies that typically affect multi-crystal data sets, including non-isomorphism, radiation damage and preferential orientation. After the description of a number of use cases, the benefit of xia2.multiplex is demonstrated within a wider autoprocessing framework in facilitating a multi-crystal experiment collected as part of in situ room-temperature fragment-screening experiments on the SARS-CoV-2 main protease.
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Jun 2022
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I03-Macromolecular Crystallography
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Aekkachai
Tuekprakhon
,
Jiandong
Huo
,
Rungtiwa
Nutalai
,
Aiste
Dijokaite-Guraliuc
,
Daming
Zhou
,
Helen M.
Ginn
,
Muneeswaran
Selvaraj
,
Chang
Liu
,
Alexander J.
Mentzer
,
Piyada
Supasa
,
Helen M. E.
Duyvesteyn
,
Raksha
Das
,
Donal
Skelly
,
Thomas G.
Ritter
,
Ali
Amini
,
Sagida
Bibi
,
Sandra
Adele
,
Sile Ann
Johnson
,
Bede
Constantinides
,
Hermione
Webster
,
Nigel
Temperton
,
Paul
Klenerman
,
Eleanor
Barnes
,
Susanna J.
Dunachie
,
Derrick
Crook
,
Andrew J.
Pollard
,
Teresa
Lambe
,
Philip
Goulder
,
Neil G.
Paterson
,
Mark A.
Williams
,
David R.
Hall
,
Elizabeth E.
Fry
,
Juthathip
Mongkolsapaya
,
Jingshan
Ren
,
David I.
Stuart
,
Gavin R.
Screaton
,
Christopher
Conlon
,
Alexandra
Deeks
,
John
Frater
,
Lisa
Frending
,
Siobhan
Gardiner
,
Anni
Jämsén
,
Katie
Jeffery
,
Tom
Malone
,
Eloise
Phillips
,
Lucy
Rothwell
,
Lizzie
Stafford
Diamond Proposal Number(s):
[27009]
Open Access
Abstract: The Omicron lineage of SARS-CoV-2, first described in November 2021, spread rapidly to become globally dominant and has split into a number of sub-lineages. BA.1 dominated the initial wave but has been replaced by BA.2 in many countries. Recent sequencing from South Africa’s Gauteng region uncovered two new sub-lineages, BA.4 and BA.5 which are taking over locally, driving a new wave. BA.4 and BA.5 contain identical spike sequences and, although closely related to BA.2, contain further mutations in the receptor binding domain of spike. Here, we study the neutralization of BA.4/5 using a range of vaccine and naturally immune serum and panels of monoclonal antibodies. BA.4/5 shows reduced neutralization by serum from triple AstraZeneca or Pfizer vaccinated individuals compared to BA.1 and BA.2. Furthermore, using serum from BA.1 vaccine breakthrough infections there are likewise, significant reductions in the neutralization of BA.4/5, raising the possibility of repeat Omicron infections.
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Jun 2022
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[13775]
Open Access
Abstract: Solution and solid-state NMR spectroscopy are highly complementary techniques for studying structure and dynamics in very high molecular weight systems. Here we have analysed the dynamics of HIV-1 capsid (CA) assemblies in presence of the cofactors IP6 and ATPγS and the host-factor CPSF6 using a combination of solution state and cross polarisation magic angle spinning (CP-MAS) solid-state NMR. In particular, dynamical effects on ns to µs and µs to ms timescales are observed revealing diverse motions in assembled CA.
Using CP-MAS NMR, we exploited the sensitivity of the amide/Cα-Cβ backbone chemical shifts in DARR and NCA spectra to observe the plasticity of the HIV-1 CA tubular assemblies and also map the binding of cofactors and the dynamics of cofactor-CA complexes. In solution, we measured how the addition of host- and co-factors to CA -hexamers perturbed the chemical shifts and relaxation properties of CA-Ile and -Met methyl groups using transverse-relaxation-optimized NMR spectroscopy to exploit the sensitivity of methyl groups as probes in high-molecular weight proteins. These data show how dynamics of the CA protein assembly over a range of spatial and temporal scales play a critical role in CA function. Moreover, we show that binding of IP6, ATPγS and CPSF6 results in local chemical shift as well as dynamic changes for a significant, contiguous portion of CA, highlighting how allosteric pathways communicate ligand interactions between adjacent CA protomers.
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Jun 2022
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Abstract: Nitroreductases activate nitroaromatic antibiotics and cancer prodrugs to cytotoxic hydroxylamines and reduce quinones to quinols. Using steady-state and stopped-flow kinetics we show that the E. coli nitroreductase NfsA is 20-50 fold more active with NADPH than with NADH and that product release may be rate-limiting. The crystal structure of NfsA with NADP+ shows that a mobile loop forms a phosphate-binding pocket. The nicotinamide ring and nicotinamide ribose are mobile, as confirmed in molecular dynamics (MD) simulations. We present a model of NADPH bound to NfsA. Only one NADP+ is seen bound to the NfsA dimers, and MD simulations show that binding of a second NADP(H) cofactor is unfavourable, suggesting that NfsA and other members of this protein superfamily may have a half-of-sites mechanism.
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Jun 2022
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NONE-No attached Diamond beamline
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Charles J.
Buchanan
,
Ben
Gaunt
,
Peter J.
Harrison
,
Yun
Yang
,
Jiwei
Liu
,
Aziz
Khan
,
Andrew M.
Giltrap
,
Audrey
Le Bas
,
Philip N.
Ward
,
Kapil
Gupta
,
Maud
Dumoux
,
Tiong Kit
Tan
,
Lisa
Schimaski
,
Sergio
Daga
,
Nicola
Picchiotti
,
Margherita
Baldassarri
,
Elisa
Benetti
,
Chiara
Fallerini
,
Francesca
Fava
,
Annarita
Giliberti
,
Panagiotis I.
Koukos
,
Matthew J.
Davy
,
Abirami
Lakshminarayanan
,
Xiaochao
Xue
,
Georgios
Papadakis
,
Lachlan P.
Deimel
,
Virgínia
Casablancas-Antràs
,
Timothy D. W.
Claridge
,
Alexandre M. J. J.
Bonvin
,
Quentin J.
Sattentau
,
Simone
Furini
,
Marco
Gori
,
Jiandong
Huo
,
Raymond J.
Owens
,
Christiane
Schaffitzel
,
Imre
Berger
,
Alessandra
Renieri
,
James H.
Naismith
,
Andrew J.
Baldwin
,
Benjamin G.
Davis
Open Access
Abstract: Many pathogens exploit host cell-surface glycans. However, precise analyses of glycan ligands binding with heavily-modified pathogen proteins can be confounded by overlapping sugar signals and/or compound with known experimental constraints. ‘Universal saturation transfer analysis’ (uSTA) builds on existing nuclear magnetic resonance spectroscopy to provide an automated workflow for quantitating protein-ligand interactions. uSTA reveals that early-pandemic, B-origin lineage SARS-CoV-2 spike trimer binds sialoside sugars in an ‘end-on’ manner. uSTA-guided modelling and a high-resolution cryo-electron microscopy structure implicate the spike N-terminal domain (NTD) and confirm end-on binding. This finding rationalizes the effect of NTD mutations that abolish sugar-binding in SARS CoV 2 variants of concern. Together with genetic variance analyses in early pandemic patient cohorts, this binding implicates a sialylated polylactosamine motif found on tetraantennary N-linked glycoproteins in deeper human lung as potentially relevant to virulence and/or zoonosis.
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Jun 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19458, 23459]
Open Access
Abstract: β-Lactams are the most important class of antibacterials, but their use is increasingly compromised by resistance, most importantly via serine β-lactamase (SBL)-catalyzed hydrolysis. The scope of β-lactam antibacterial activity can be substantially extended by coadministration with a penicillin-derived SBL inhibitor (SBLi), i.e., the penam sulfones tazobactam and sulbactam, which are mechanism-based inhibitors working by acylation of the nucleophilic serine. The new SBLi enmetazobactam, an N-methylated tazobactam derivative, has recently completed clinical trials. Biophysical studies on the mechanism of SBL inhibition by enmetazobactam reveal that it inhibits representatives of all SBL classes without undergoing substantial scaffold fragmentation, a finding that contrasts with previous reports on SBL inhibition by tazobactam and sulbactam. We therefore reinvestigated the mechanisms of tazobactam and sulbactam using mass spectrometry under denaturing and nondenaturing conditions, X-ray crystallography, and NMR spectroscopy. The results imply that the reported extensive fragmentation of penam sulfone–derived acyl–enzyme complexes does not substantially contribute to SBL inhibition. In addition to observation of previously identified inhibitor-induced SBL modifications, the results reveal that prolonged reaction of penam sulfones with SBLs can induce dehydration of the nucleophilic serine to give a dehydroalanine residue that undergoes reaction to give a previously unobserved lysinoalanine cross-link. The results clarify the mechanisms of action of widely clinically used SBLi, reveal limitations on the interpretation of mass spectrometry studies concerning mechanisms of SBLi, and will inform the development of new SBLi working by reaction to form hydrolytically stable acyl–enzyme complexes.
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May 2022
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