I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[23269]
Abstract: The α-helix is pre-eminent in structural biology1 and widely exploited in protein folding2, design3 and engineering4. Although other helical peptide conformations do exist near to the α-helical region of conformational space—namely, 310-helices and π-helices5—these occur much less frequently in protein structures. Less favourable internal energies and reduced tendencies to pack into higher-order structures mean that 310-helices rarely exceed six residues in length in natural proteins, and that they tend not to form normal supersecondary, tertiary or quaternary interactions. Here we show that despite their absence in nature, synthetic peptide assemblies can be built from 310-helices. We report the rational design, solution-phase characterization and an X-ray crystal structure for water-soluble bundles of 310-helices with consolidated hydrophobic cores. The design uses six-residue repeats informed by analysing 310-helical conformations in known protein structures, and incorporates α-aminoisobutyric acid residues. Design iterations reveal a tipping point between α-helical and 310-helical folding, and identify features required for stabilizing assemblies of 310-helices. This work provides principles and rules to open opportunities for designing into this hitherto unexplored region of protein-structure space.
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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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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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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Owen A.
Davis
,
Kwai-Ming J.
Cheung
,
Alfie
Brennan
,
Matthew G.
Lloyd
,
Matthew J.
Rodrigues
,
Olivier A.
Pierrat
,
Gavin W.
Collie
,
Yann-Vai
Le Bihan
,
Rosemary
Huckvale
,
Alice C.
Harnden
,
Ana
Varela
,
Michael D.
Bright
,
Paul
Eve
,
Angela
Hayes
,
Alan T.
Henley
,
Michael D.
Carter
,
P. Craig
Mcandrew
,
Rachel
Talbot
,
Rosemary
Burke
,
Rob
Van Montfort
,
Florence I.
Raynaud
,
Olivia W.
Rossanese
,
Mirco
Meniconi
,
Benjamin R.
Bellenie
,
Swen
Hoelder
Open Access
Abstract: To identify new chemical series with enhanced binding affinity to the BTB domain of B-cell lymphoma 6 protein, we targeted a subpocket adjacent to Val18. With no opportunities for strong polar interactions, we focused on attaining close shape complementarity by ring fusion onto our quinolinone lead series. Following exploration of different sized rings, we identified a conformationally restricted core which optimally filled the available space, leading to potent BCL6 inhibitors. Through X-ray structure-guided design, combined with efficient synthetic chemistry to make the resulting novel core structures, a >300-fold improvement in activity was obtained by the addition of seven heavy atoms.
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Jun 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Maurice
Michel
,
Carlos
Benítez-Buelga
,
Patricia A.
Calvo
,
Bishoy M. F.
Hanna
,
Oliver
Mortusewicz
,
Geoffrey
Masuyer
,
Jonathan
Davies
,
Olov
Wallner
,
Sanjiv
Kumar
,
Julian J.
Albers
,
Sergio
Castañeda-Zegarra
,
Ann-Sofie
Jemth
,
Torkild
Visnes
,
Ana
Sastre-Perona
,
Akhilesh N.
Danda
,
Evert J.
Homan
,
Karthick
Marimuthu
,
Zhao
Zhenjun
,
Celestine N.
Chi
,
Antonio
Sarno
,
Elisée
Wiita
,
Catharina
Von Nicolai
,
Anna J.
Komor
,
Varshni
Rajagopal
,
Sarah
Müller
,
Emily C.
Hank
,
Marek
Varga
,
Emma R.
Scaletti
,
Monica
Pandey
,
Stella
Karsten
,
Hanne
Haslene-Hox
,
Simon
Loevenich
,
Petra
Marttila
,
Azita
Rasti
,
Kirill
Mamonov
,
Florian
Ortis
,
Fritz
Schömberg
,
Olga
Loseva
,
Josephine
Stewart
,
Nicholas
D’arcy-Evans
,
Tobias
Koolmeister
,
Martin
Henriksson
,
Dana
Michel
,
Ana
De Ory
,
Lucia
Acero
,
Oriol
Calvete
,
Martin
Scobie
,
Christian
Hertweck
,
Ivan
Vilotijevic
,
Christina
Kalderén
,
Ana
Osorio
,
Rosario
Perona
,
Alexandra
Stolz
,
Pal
Stenmark
,
Ulrika
Warpman Berglund
,
Miguel
De Vega
,
Thomas
Helleday
Diamond Proposal Number(s):
[15806, 21625]
Abstract: Oxidative DNA damage is recognized by 8-oxoguanine (8-oxoG) DNA glycosylase 1 (OGG1), which excises 8-oxoG, leaving a substrate for apurinic endonuclease 1 (APE1) and initiating repair. Here, we describe a small molecule (TH10785) that interacts with the phenylalanine-319 and glycine-42 amino acids of OGG1, increases the enzyme activity 10-fold, and generates a previously undescribed β,δ-lyase enzymatic function. TH10785 controls the catalytic activity mediated by a nitrogen base within its molecular structure. In cells, TH10785 increases OGG1 recruitment to and repair of oxidative DNA damage. This alters the repair process, which no longer requires APE1 but instead is dependent on polynucleotide kinase phosphatase (PNKP1) activity. The increased repair of oxidative DNA lesions with a small molecule may have therapeutic applications in various diseases and aging.
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Jun 2022
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Ana S.
Luis
,
Arnaud
Basle
,
Dominic P.
Byrne
,
Gareth S. A.
Wright
,
James A.
London
,
Chunsheng
Jin
,
Niclas G.
Karlsson
,
Gunnar C.
Hansson
,
Patrick A.
Eyers
,
Mirjam
Czjzek
,
Tristan
Barbeyron
,
Edwin A.
Yates
,
Eric C.
Martens
,
Alan
Cartmell
Diamond Proposal Number(s):
[21970, 18598]
Abstract: Sulfated glycans are ubiquitous nutrient sources for microbial communities that have coevolved with eukaryotic hosts. Bacteria metabolize sulfated glycans by deploying carbohydrate sulfatases that remove sulfate esters. Despite the biological importance of sulfatases, the mechanisms underlying their ability to recognize their glycan substrate remain poorly understood. Here, we use structural biology to determine how sulfatases from the human gut microbiota recognize sulfated glycans. We reveal seven new carbohydrate sulfatase structures spanning four S1 sulfatase subfamilies. Structures of S1_16 and S1_46 represent novel structures of these subfamilies. Structures of S1_11 and S1_15 demonstrate how non-conserved regions of the protein drive specificity toward related but distinct glycan targets. Collectively, these data reveal that carbohydrate sulfatases are highly selective for the glycan component of their substrate. These data provide new approaches for probing sulfated glycan metabolism while revealing the roles carbohydrate sulfatases play in host glycan catabolism.
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Jun 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Yuguang
Zhao
,
William
Mahy
,
Nicky J.
Willis
,
Hannah L.
Woodward
,
David
Steadman
,
Elliott D.
Bayle
,
Benjamin N.
Atkinson
,
James
Sipthorp
,
Luca
Vecchia
,
Reinis R.
Ruza
,
Karl
Harlos
,
Fiona
Jeganathan
,
Stefan
Constantinou
,
Artur
Costa
,
Svend
Kjær
,
Magda
Bictash
,
Patricia C.
Salinas
,
Paul
Whiting
,
Jean-Paul
Vincent
,
Paul V.
Fish
,
E. Yvonne
Jones
Diamond Proposal Number(s):
[16814]
Open Access
Abstract: The Wnt signaling suppressor Notum is a promising target for osteoporosis, Alzheimer’s disease, and colorectal cancers. To develop novel Notum inhibitors, we used an X-ray crystallographic fragment screen with the Diamond-SGC Poised Library (DSPL) and identified 59 fragment hits from the analysis of 768 data sets. Fifty-eight of the hits were found bound at the enzyme catalytic pocket with potencies ranging from 0.5 to >1000 μM. Analysis of the fragments’ diverse binding modes, enzymatic inhibitory activities, and chemical properties led to the selection of six hits for optimization, and five of these resulted in improved Notum inhibitory potencies. One hit, 1-phenyl-1,2,3-triazole 7, and its related cluster members, have shown promising lead-like properties. These became the focus of our fragment development activities, resulting in compound 7d with IC50 0.0067 μM. The large number of Notum fragment structures and their initial optimization provided an important basis for further Notum inhibitor development.
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Jun 2022
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[20145]
Abstract: During the initiation of DNA replication, oligonucleotide primers are synthesized de novo by primases and are subsequently extended by replicative polymerases to complete genome duplication. The primase-polymerase (Prim-Pol) superfamily is a diverse grouping of primases, which includes replicative primases and CRISPR-associated primase-polymerases (CAPPs) involved in adaptive immunity1,2,3. Although much is known about the activities of these enzymes, the precise mechanism used by primases to initiate primer synthesis has not been elucidated. Here we identify the molecular bases for the initiation of primer synthesis by CAPP and show that this mechanism is also conserved in replicative primases. The crystal structure of a primer initiation complex reveals how the incoming nucleotides are positioned within the active site, adjacent to metal cofactors and paired to the templating single-stranded DNA strand, before synthesis of the first phosphodiester bond. Furthermore, the structure of a Prim-Pol complex with double-stranded DNA shows how the enzyme subsequently extends primers in a processive polymerase mode. The structural and mechanistic studies presented here establish how Prim-Pol proteins instigate primer synthesis, revealing the requisite molecular determinants for primer synthesis within the catalytic domain. This work also establishes that the catalytic domain of Prim-Pol enzymes, including replicative primases, is sufficient to catalyse primer formation.
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May 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[16816, 18582, 19190, 25586, 30588]
Open Access
Abstract: Polyomaviruses are a family of ubiquitous double-stranded DNA viruses many of which are human pathogens. These include BK polyomavirus which causes severe urinary tract infection in immunocompromised patients and Merkel cell polyomavirus associated with aggressive cancers. The small genome of polyomaviruses lacks conventional drug targets, and no specific drugs are available at present. Here we focus on the main structural protein VP1 of BK polyomavirus which is responsible for icosahedral capsid formation. To provide a foundation towards rational drug design, we crystallized truncated VP1 pentamers and subjected them to a high-throughput screening for binding drug-like fragments through a direct X-ray analysis. To enable a highly performant screening, rigorous optimization of the crystallographic pipeline and processing with the latest generation PanDDA2 software were necessary. As a result, a total of 144 binding hits were established. Importantly, the hits are well clustered in six surface pockets. Three pockets are located on the outside of the pentamer and map on the regions where the ‘invading’ C-terminal arm of another pentamer is attached upon capsid assembly. Another set of three pockets is situated within the wide pore along the five-fold axis of the VP1 pentamer. These pockets are situated at the interaction interface with the minor capsid protein VP2 which is indispensable for normal functioning of the virus. Here we systematically analyse the three outside pockets which are highly conserved across various polyomaviruses, while point mutations in these pockets are detrimental for viral replication. We show that one of the pockets can accommodate antipsychotic drug trifluoperazine. For each pocket, we derive pharmacophore features which enable the design of small molecules preventing the interaction between VP1 pentamers and therefore inhibiting capsid assembly. Our data lay a foundation towards a rational development of first-in-class drugs targeting polyomavirus capsid.
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May 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: UPF3 is a key nonsense-mediated mRNA decay (NMD) factor required for mRNA surveillance and eukaryotic gene expression regulation. UPF3 exists as two paralogs (A and B) which are differentially expressed depending on cell type and developmental stage and believed to regulate NMD activity based on cellular requirements. UPF3B mutations cause intellectual disability. The underlying molecular mechanisms remain elusive, as many of the mutations lie in the poorly characterized middle-domain of UPF3B. Here, we show that UPF3A and UPF3B share structural and functional homology to paraspeckle proteins comprising an RNA-recognition motif-like domain (RRM-L), a NONA/paraspeckle-like domain (NOPS-L), and extended α-helical domain. These domains are essential for RNA/ribosome-binding, RNA-induced oligomerization and UPF2 interaction. Structures of UPF2′s third middle-domain of eukaryotic initiation factor 4G (MIF4GIII) in complex with either UPF3B or UPF3A reveal unexpectedly intimate binding interfaces. UPF3B’s disease-causing mutation Y160D in the NOPS-L domain displaces Y160 from a hydrophobic cleft in UPF2 reducing the binding affinity ∼40-fold compared to wildtype. UPF3A, which is upregulated in patients with the UPF3B-Y160D mutation, binds UPF2 with ∼10-fold higher affinity than UPF3B reliant mainly on NOPS-L residues. Our characterization of RNA- and UPF2-binding by UPF3′s middle-domain elucidates its essential role in NMD.
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May 2022
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