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Open Access
Abstract: The switch between planktonic and biofilm lifestyle correlates with intracellular concentration of the second messenger bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP). While bacteria possess cyclase and phosphodiesterase enzymes to catalyse formation or hydrolysis of c-di-GMP, both enzymatic domains often occur in a single protein. It is tacitly assumed that one of the two enzymatic activities is dominant, and that additional domains and protein interactions enable responses to environmental conditions and control activity. Here we report the structure of the phosphodiesterase domain of the membrane protein RbdA (regulator of biofilm dispersal) in a dimeric, activated state and show that phosphodiesterase activity is controlled by the linked cyclase. The phosphodiesterase region around helices α5/α6 forms the dimer interface, providing a rationale for activation, as this region was seen in contact with the cyclase domain in an auto-inhibited structure previously described. Kinetic analysis supports this model, as the activity of the phosphodiesterase alone is lower when linked to the cyclase. Analysis of a computed model of the RbdA periplasmatic domain reveals an all-helical architecture with a large binding pocket that could accommodate putative ligands. Unravelling the regulatory circuits in multi-domain phosphodiesterases like RbdA is important to develop strategies to manipulate or disperse bacterial biofilms.
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Aug 2024
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Ana
Martínez Gascueña
,
Haiyang
Wu
,
Rui
Wang
,
C. David
Owen
,
Pedro J.
Hernando
,
Serena
Monaco
,
Matthew
Penner
,
Ke
Xing
,
Gwenaelle
Le Gall
,
Richard
Gardner
,
Didier
Ndeh
,
Paulina A.
Urbanowicz
,
Daniel I. R.
Spencer
,
Martin
Walsh
,
Jesus
Angulo
,
Nathalie
Juge
Open Access
Abstract: Microbial α-L-fucosidases catalyse the hydrolysis of terminal α-L-fucosidic linkages and can perform transglycosylation reactions. Based on sequence identity, α-L-fucosidases are classified in glycoside hydrolases (GHs) families of the carbohydrate-active enzyme database. Here we explored the sequence-function space of GH29 fucosidases. Based on sequence similarity network (SSN) analyses, 15 GH29 α-L-fucosidases were selected for functional characterisation. HPAEC-PAD and LC-FD-MS/MS analyses revealed substrate and linkage specificities for α1,2, α1,3, α1,4 and α1,6 linked fucosylated oligosaccharides and glycoconjugates, consistent with their SSN clustering. The structural basis for the substrate specificity of GH29 fucosidase from Bifidobacterium asteroides towards α1,6 linkages and FA2G2 N-glycan was determined by X-ray crystallography and STD NMR. The capacity of GH29 fucosidases to carry out transfucosylation reactions with GlcNAc and 3FN as acceptors was evaluated by TLC combined with ESI–MS and NMR. These experimental data supported the use of SSN to further explore the GH29 sequence-function space through machine-learning models. Our lightweight protein language models could accurately allocate test sequences in their respective SSN clusters and assign 34,258 non-redundant GH29 sequences into SSN clusters. It is expected that the combination of these computational approaches will be used in the future for the identification of novel GHs with desired specificities.
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Jun 2024
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Gianluigi A.
Botton
,
Peter
Coll
,
Andrew J.
Dent
,
Sarnjeet
Dhesi
,
Sky
French
,
David R.
Hall
,
Joseph
Hriljac
,
Sarah
Macdonell
,
Sofia
Diaz-Moreno
,
Chris
Nicklin
,
Paul
Quinn
,
Robert
Rambo
,
Kawal
Sawhney
,
Richard P.
Walker
,
Martin A.
Walsh
,
Molly
Pekarik-Fry
Open Access
Abstract: A look at the Diamond-II upgrade programme
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Mar 2024
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I03-Macromolecular Crystallography
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Takashi
Miura
,
Tika R.
Malla
,
Lennart
Brewitz
,
Anthony
Tumber
,
Eidarus
Salah
,
Kang Ju
Lee
,
Naohiro
Terasaka
,
C. David
Owen
,
Claire
Strain-Damerell
,
Petra
Lukacik
,
Martin A.
Walsh
,
Akane
Kawamura
,
Christopher J.
Schofield
,
Takayuki
Katoh
,
Hiroaki
Suga
Diamond Proposal Number(s):
[27088]
Open Access
Abstract: Due to their constrained conformations, cyclic β2,3-amino acids (cβAA) are key building blocks that can fold peptides into compact and rigid structures, improving peptidase resistance and binding affinity to target proteins, due to their constrained conformations. Although the translation efficiency of cβAAs is generally low, our engineered tRNA, referred to as tRNAPro1E2, enabled efficient incorporation of cβAAs into peptide libraries using the flexible in vitro translation (FIT) system. Here we report on the design and application of a macrocyclic peptide library incorporating three kinds of cβAAs: (1R,2S)-2-aminocyclopentane carboxylic acid (β1), (1S,2S)-2-aminocyclohexane carboxylic acid (β2), and (1R,2R)-2-aminocyclopentane carboxylic acid. This library was applied to an in vitro selection against the SARS-CoV-2 main protease (Mpro). The resultant peptides, BM3 and BM7, bearing one β2 and two β1, exhibited potent inhibitory activities with IC50 values of 40 nM and 20 nM, respectively. BM3 and BM7 also showed remarkable serum stability with half-lives of 48 h and >168 h, respectively. Notably, BM3A and BM7A, wherein the cβAAs were substituted with alanine, lost their inhibitory activities against Mpro and displayed substantially shorter serum half-lives. This observation underscores the significant contribution of cβAA to the activity and stability of peptides. Overall, our results highlight the potential of cβAA in generating potent and highly stable macrocyclic peptides with drug-like properties.
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Mar 2024
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Open Access
Abstract: The SARS-CoV-2 papain-like protease (PLpro) is an antiviral drug target that catalyzes the hydrolysis of the viral polyproteins pp1a/1ab, so releasing the non-structural proteins (nsps) 1–3 that are essential for the coronavirus lifecycle. The LXGG↓X motif in pp1a/1ab is crucial for recognition and cleavage by PLpro. We describe molecular dynamics, docking, and quantum mechanics/molecular mechanics (QM/MM) calculations to investigate how oligopeptide substrates derived from the viral polyprotein bind to PLpro. The results reveal how the substrate sequence affects the efficiency of PLpro-catalyzed hydrolysis. In particular, a proline at the P2′ position promotes catalysis, as validated by residue substitutions and mass spectrometry-based analyses. Analysis of PLpro catalyzed hydrolysis of LXGG motif-containing oligopeptides derived from human proteins suggests that factors beyond the LXGG motif and the presence of a proline residue at P2′ contribute to catalytic efficiency, possibly reflecting the promiscuity of PLpro. The results will help in identifying PLpro substrates and guiding inhibitor design.
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Oct 2023
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I03-Macromolecular Crystallography
I23-Long wavelength MX
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Kamel
El Omari
,
Ramona
Duman
,
Vitaliy
Mykhaylyk
,
Christian M.
Orr
,
Merlyn
Latimer-Smith
,
Graeme
Winter
,
Vinay
Grama
,
Feng
Qu
,
Kiran
Bountra
,
Hok Sau
Kwong
,
Maria
Romano
,
Rosana
Reis
,
Lutz
Vogeley
,
Luca
Vecchia
,
C. David
Owen
,
Sina
Wittmann
,
Max
Renner
,
Miki
Senda
,
Naohiro
Matsugaki
,
Yoshiaki
Kawano
,
Thomas A.
Bowden
,
Isabel
Moraes
,
Jonathan M.
Grimes
,
Erika J.
Mancini
,
Martin A.
Walsh
,
Cristiane R.
Guzzo
,
Raymond J.
Owens
,
E. Yvonne
Jones
,
David G.
Brown
,
Dave I.
Stuart
,
Konstantinos
Beis
,
Armin
Wagner
Open Access
Abstract: Despite recent advances in cryo-electron microscopy and artificial intelligence-based model predictions, a significant fraction of structure determinations by macromolecular crystallography still requires experimental phasing, usually by means of single-wavelength anomalous diffraction (SAD) techniques. Most synchrotron beamlines provide highly brilliant beams of X-rays of between 0.7 and 2 Å wavelength. Use of longer wavelengths to access the absorption edges of biologically important lighter atoms such as calcium, potassium, chlorine, sulfur and phosphorus for native-SAD phasing is attractive but technically highly challenging. The long-wavelength beamline I23 at Diamond Light Source overcomes these limitations and extends the accessible wavelength range to λ = 5.9 Å. Here we report 22 macromolecular structures solved in this extended wavelength range, using anomalous scattering from a range of elements which demonstrate the routine feasibility of lighter atom phasing. We suggest that, in light of its advantages, long-wavelength crystallography is a compelling option for experimental phasing.
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Oct 2023
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Vijil
Chenthamarakshan
,
Samuel C.
Hoffman
,
C. David
Owen
,
Petra
Lukacik
,
Claire
Strain-Damerell
,
Daren
Fearon
,
Tika R.
Malla
,
Anthony
Tumber
,
Christopher J.
Schofield
,
Helen M. E.
Duyvesteyn
,
Wanwisa
Dejnirattisai
,
Loic
Carrique
,
Thomas S.
Walter
,
Gavin R.
Screaton
,
Tetiana
Matviiuk
,
Aleksandra
Mojsilovic
,
Jason
Crain
,
Martin A.
Walsh
,
David I.
Stuart
,
Payel
Das
Diamond Proposal Number(s):
[27995]
Open Access
Abstract: Inhibitor discovery for emerging drug-target proteins is challenging, especially when target structure or active molecules are unknown. Here, we experimentally validate the broad utility of a deep generative framework trained at-scale on protein sequences, small molecules, and their mutual interactions—unbiased toward any specific target. We performed a protein sequence-conditioned sampling on the generative foundation model to design small-molecule inhibitors for two dissimilar targets: the spike protein receptor-binding domain (RBD) and the main protease from SARS-CoV-2. Despite using only the target sequence information during the model inference, micromolar-level inhibition was observed in vitro for two candidates out of four synthesized for each target. The most potent spike RBD inhibitor exhibited activity against several variants in live virus neutralization assays. These results establish that a single, broadly deployable generative foundation model for accelerated inhibitor discovery is effective and efficient, even in the absence of target structure or binder information.
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Jun 2023
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I03-Macromolecular Crystallography
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Takashi
Miura
,
Tika R.
Malla
,
C. David
Owen
,
Anthony
Tumber
,
Lennart
Brewitz
,
Michael A.
Mcdonough
,
Eidarus
Salah
,
Naohiro
Terasaka
,
Takayuki
Katoh
,
Petra
Lukacik
,
Claire
Strain-Damerell
,
Halina
Mikolajek
,
Martin A.
Walsh
,
Akane
Kawamura
,
Christopher J.
Schofield
,
Hiroaki
Suga
Diamond Proposal Number(s):
[27088]
Open Access
Abstract: γ-Amino acids can play important roles in the biological activities of natural products; however, the ribosomal incorporation of γ-amino acids into peptides is challenging. Here we report how a selection campaign employing a non-canonical peptide library containing cyclic γ2,4-amino acids resulted in the discovery of very potent inhibitors of the SARS-CoV-2 main protease (Mpro). Two kinds of cyclic γ2,4-amino acids, cis-3-aminocyclobutane carboxylic acid (γ1) and (1R,3S)-3-aminocyclopentane carboxylic acid (γ2), were ribosomally introduced into a library of thioether-macrocyclic peptides. One resultant potent Mpro inhibitor (half-maximal inhibitory concentration = 50 nM), GM4, comprising 13 residues with γ1 at the fourth position, manifests a 5.2 nM dissociation constant. An Mpro:GM4 complex crystal structure reveals the intact inhibitor spans the substrate binding cleft. The γ1 interacts with the S1′ catalytic subsite and contributes to a 12-fold increase in proteolytic stability compared to its alanine-substituted variant. Knowledge of interactions between GM4 and Mpro enabled production of a variant with a 5-fold increase in potency.
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May 2023
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Luiz Carlos
Saramago
,
Marcos V.
Santana
,
Bárbara Figueira
Gomes
,
Rafael Ferreira
Dantas
,
Mario R.
Senger
,
Pedro Henrique
Oliveira Borges
,
Vivian Neuza
Dos Santos Ferreira
,
Alice
Dos Santos Rosa
,
Amanda Resende
Tucci
,
Milene
Dias Miranda
,
Petra
Lukacik
,
Claire
Strain-Damerell
,
C. David
Owen
,
Martin A.
Walsh
,
Sabrina
Baptista Ferreira
,
Floriano Paes
Silva-Junior
Abstract: SARS-CoV-2 is the causative agent of COVID-19 and is responsible for the current global pandemic. The viral genome contains 5 major open reading frames of which the largest ORF1ab codes for two polyproteins, pp1ab and pp1a, which are subsequently cleaved into 16 nonstructural proteins (nsp) by two viral cysteine proteases encoded within the polyproteins. The main protease (Mpro, nsp5) cleaves the majority of the nsp’s, making it essential for viral replication and has been successfully targeted for the development of antivirals. The first oral Mpro inhibitor, nirmatrelvir, was approved for treatment of COVID-19 in late December 2021 in combination with ritonavir as Paxlovid. Increasing the arsenal of antivirals and development of protease inhibitors and other antivirals with a varied mode of action remains a priority to reduce the likelihood for resistance emerging. Here, we report results from an artificial intelligence-driven approach followed by in vitro validation, allowing the identification of five fragment-like Mpro inhibitors with IC50 values ranging from 1.5 to 241 μM. The three most potent molecules (compounds 818, 737, and 183) were tested against SARS-CoV-2 by in vitro replication in Vero E6 and Calu-3 cells. Compound 818 was active in both cell models with an EC50 value comparable to its measured IC50 value. On the other hand, compounds 737 and 183 were only active in Calu-3, a preclinical model of respiratory cells, showing selective indexes twice as high as those for compound 818. We also show that our in silico methodology was successful in identifying both reversible and covalent inhibitors. For instance, compound 818 is a reversible chloromethylamide analogue of 8-methyl-γ-carboline, while compound 737 is an N-pyridyl-isatin that covalently inhibits Mpro. Given the small molecular weights of these fragments, their high binding efficiency in vitro and efficacy in blocking viral replication, these compounds represent good starting points for the development of potent lead molecules targeting the Mpro of SARS-CoV-2.
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Apr 2023
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VMXm-Versatile Macromolecular Crystallography microfocus
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Lennart
Brewitz
,
Leo
Dumjahn
,
Yilin
Zhao
,
C. David
Owen
,
Stephen M.
Laidlaw
,
Tika R.
Malla
,
Dung
Nguyen
,
Petra
Lukacik
,
Eidarus
Salah
,
Adam D.
Crawshaw
,
Anna J.
Warren
,
Jose
Trincao
,
Claire
Strain-Damerell
,
Miles W.
Carroll
,
Martin A.
Walsh
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[27088]
Open Access
Abstract: Nirmatrelvir (PF-07321332) is a nitrile-bearing small-molecule inhibitor that, in combination with ritonavir, is used to treat infections by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Nirmatrelvir interrupts the viral life cycle by inhibiting the SARS-CoV-2 main protease (Mpro), which is essential for processing viral polyproteins into functional nonstructural proteins. We report studies which reveal that derivatives of nirmatrelvir and other Mpro inhibitors with a nonactivated terminal alkyne group positioned similarly to the electrophilic nitrile of nirmatrelvir can efficiently inhibit isolated Mpro and SARS-CoV-2 replication in cells. Mass spectrometric and crystallographic evidence shows that the alkyne derivatives inhibit Mpro by apparent irreversible covalent reactions with the active site cysteine (Cys145), while the analogous nitriles react reversibly. The results highlight the potential for irreversible covalent inhibition of Mpro and other nucleophilic cysteine proteases by alkynes, which, in contrast to nitriles, can be functionalized at their terminal position to optimize inhibition and selectivity, as well as pharmacodynamic and pharmacokinetic properties.
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Feb 2023
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