I03-Macromolecular Crystallography
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Mauricio P.
Contreras
,
Hsuan
Pai
,
Muniyandi
Selvaraj
,
Amirali
Toghani
,
David M.
Lawson
,
Yasin
Tumtas
,
Cian
Duggan
,
Enoch Lok Him
Yuen
,
Clare E. M.
Stevenson
,
Adeline
Harant
,
Abbas
Maqbool
,
Chih-Hang
Wu
,
Tolga O.
Bozkurt
,
Sophien
Kamoun
,
Lida
Derevnina
Diamond Proposal Number(s):
[18565]
Open Access
Abstract: Parasites counteract host immunity by suppressing helper nucleotide binding and leucine-rich repeat (NLR) proteins that function as central nodes in immune receptor networks. Understanding the mechanisms of immunosuppression can lead to strategies for bioengineering disease resistance. Here, we show that a cyst nematode virulence effector binds and inhibits oligomerization of the helper NLR protein NRC2 by physically preventing intramolecular rearrangements required for activation. An amino acid polymorphism at the binding interface between NRC2 and the inhibitor is sufficient for this helper NLR to evade immune suppression, thereby restoring the activity of multiple disease resistance genes. This points to a potential strategy for resurrecting disease resistance in crop genomes.
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May 2023
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[26855]
Open Access
Abstract: Monkeypox virus (MPXV) is a double-stranded DNA virus from the family Poxviridae, which is endemic in West and Central Africa. Various human outbreaks occurred in the 1980s, resulting from a cessation of smallpox vaccination. Recently, MPXV cases have reemerged in non-endemic nations, and the 2022 outbreak has been declared a public health emergency. Treatment optionsare limited, and many countries lack the infrastructure to provide symptomatic treatments. The development of cost-effective antivirals could ease severe health outcomes. G-quadruplexes have been a target of interest in treating viral infections with different chemicals. In the present work, a genomic-scale mapping of different MPXV isolates highlighted two conserved putative quadruplex-forming sequences MPXV-exclusive in 590 isolates. Subsequently, we assessed the G-quadruplex formation using circular dichroism spectroscopy and solution small-angle X-ray scattering. Furthermore, biochemical assays indicated the ability of MPXV quadruplexes to be recognized by two specific G4-binding partners—Thioflavin T and DHX36. Additionally, our work also suggests that a quadruplex binding small-molecule with previously reported antiviral activity, TMPyP4, interacts with MPXV G-quadruplexes with nanomolar affinity in the presence and absence of DHX36. Finally, cell biology experiments suggests that TMPyP4 treatment substantially reduced gene expression of MPXV proteins. In summary, our work provides insights into the G-quadruplexes from the MPXV genome that can be further exploited to develop therapeutics.
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May 2023
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23620]
Open Access
Abstract: The cell cycle checkpoint kinase Mec1ATR and its integral partner Ddc2ATRIP are vital for the DNA damage and replication stress response. Mec1–Ddc2 “senses” single-stranded DNA (ssDNA) by being recruited to the ssDNA binding Replication Protein A (RPA) via Ddc2. In this study, we show that a DNA damage–induced phosphorylation circuit modulates checkpoint recruitment and function. We demonstrate that Ddc2–RPA interactions modulate the association between RPA and ssDNA and that Rfa1-phosphorylation aids in the further recruitment of Mec1–Ddc2. We also uncover an underappreciated role for Ddc2 phosphorylation that enhances its recruitment to RPA-ssDNA that is important for the DNA damage checkpoint in yeast. The crystal structure of a phosphorylated Ddc2 peptide in complex with its RPA interaction domain provides molecular details of how checkpoint recruitment is enhanced, which involves Zn2+. Using electron microscopy and structural modeling approaches, we propose that Mec1–Ddc2 complexes can form higher order assemblies with RPA when Ddc2 is phosphorylated. Together, our results provide insight into Mec1 recruitment and suggest that formation of supramolecular complexes of RPA and Mec1–Ddc2, modulated by phosphorylation, would allow for rapid clustering of damage foci to promote checkpoint signaling.
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Apr 2023
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12788]
Open Access
Abstract: The availability of an expanded genetic code opens exciting new opportunities in enzyme design and engineering. In this regard histidine analogues have proven particularly versatile, serving as ligands to augment metalloenzyme function and as catalytic nucleophiles in designed enzymes. The ability to genetically encode multiple functional residues could greatly expand the range of chemistry accessible within enzyme active sites. Here we develop mutually orthogonal translation components to selectively encode two structurally similar histidine analogues. Transplanting known mutations from a promiscuous Methanosarcina mazei pyrrolysyl-tRNA synthetase (MmPylRSIFGFF) into a single domain PylRS from Methanomethylophilus alvus (MaPylRSIFGFF) provided a variant with improved efficiency and specificity for 3-methyl-L-histidine (MeHis) incorporation. The MaPylRSIFGFF clone was further characterized using in vitro biochemical assays and X-ray crystallography. We subsequently engineered the orthogonal MmPylRS for activity and selectivity for 3-(3-pyridyl)-L-alanine (3-Pyr), which was used in combination with MaPylRSIFGFF to produce proteins containing both 3-Pyr and MeHis. Given the versatile roles played by histidine in enzyme mechanisms, we anticipate that the tools developed within this study will underpin the development of enzymes with new and enhanced functions.
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Apr 2023
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13597]
Open Access
Abstract: Bacteria use an array of sigma factors to regulate gene expression during different stages of their life cycles. Full-length, atomic-level structures of sigma factors have been challenging to obtain experimentally as a result of their many regions of intrinsic disorder. AlphaFold has now supplied plausible full-length models for most sigma factors. Here we discuss the current understanding of the structures and functions of sigma factors in the model organism, Bacillus subtilis, and present an X-ray crystal structure of a region of B. subtilis SigE, a sigma factor that plays a critical role in the developmental process of spore formation.
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Apr 2023
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I23-Long wavelength MX
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Open Access
Abstract: Despite being fundamental to multiple biological processes, phosphorus (P) availability in marine environments is often growth-limiting, with generally low surface concentrations. Picocyanobacteria strains encode a putative ABC-type phosphite/phosphate/phosphonate transporter, phnDCE, thought to provide access to an alternative phosphorus pool. This, however, is paradoxical given most picocyanobacterial strains lack known phosphite degradation or carbon-phosphate lyase pathway to utilise alternate phosphorus pools. To understand the function of the PhnDCE transport system and its ecological consequences, we characterised the PhnD1 binding proteins from four distinct marine Synechococcus isolates (CC9311, CC9605, MITS9220, and WH8102). We show the Synechococcus PhnD1 proteins selectively bind phosphorus compounds with a stronger affinity for phosphite than for phosphate or methyl phosphonate. However, based on our comprehensive ligand screening and growth experiments showing Synechococcus strains WH8102 and MITS9220 cannot utilise phosphite or methylphosphonate as a sole phosphorus source, we hypothesise that the picocyanobacterial PhnDCE transporter is a constitutively expressed, medium-affinity phosphate transporter, and the measured affinity of PhnD1 to phosphite or methyl phosphonate is fortuitous. Our MITS9220_PhnD1 structure explains the comparatively lower affinity of picocyanobacterial PhnD1 for phosphate, resulting from a more limited H-bond network. We propose two possible physiological roles for PhnD1. First, it could function in phospholipid recycling, working together with the predicted phospholipase, TesA, and alkaline phosphatase. Second, by having multiple transporters for P (PhnDCE and Pst), picocyanobacteria could balance the need for rapid transport during transient episodes of higher P availability in the environment, with the need for efficient P utilisation in typical phosphate-deplete conditions.
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Apr 2023
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Open Access
Abstract: The most advanced antiviral molecules addressing major SARS-CoV-2 targets (Main protease, Spike protein, and RNA polymerase), compared with proteins of other human pathogenic coronaviruses, may have a short-lasting clinical efficacy. Accumulating knowledge on the mechanisms underlying the target structural basis, its mutational progression, and the related biological significance to virus replication allows envisaging the development of better-targeted therapies in the context of COVID-19 epidemic and future coronavirus outbreaks. The identification of evolutionary patterns based solely on sequence information analysis for those targets can provide meaningful insights into the molecular basis of host–pathogen interactions and adaptation, leading to drug resistance phenomena. Herein, we will explore how the study of observed and predicted mutations may offer valuable suggestions for the application of the so-called “synthetic lethal” strategy to SARS-CoV-2 Main protease and Spike protein. The synergy between genetics evidence and drug discovery may prioritize the development of novel long-lasting antiviral agents.
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Mar 2023
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Krios I-Titan Krios I at Diamond
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Phong Quoc
Nguyen
,
Sonia
Huecas
,
Amna
Asif-Laidin
,
Adrián
Plaza-Pegueroles
,
Beatrice
Capuzzi
,
Noé
Palmic
,
Christine
Conesa
,
Joël
Acker
,
Juan
Reguera
,
Pascale
Lesage
,
Carlos
Fernandez-Tornero
Diamond Proposal Number(s):
[22006]
Open Access
Abstract: The yeast Ty1 retrotransposon integrates upstream of genes transcribed by RNA polymerase III (Pol III). Specificity of integration is mediated by an interaction between the Ty1 integrase (IN1) and Pol III, currently uncharacterized at the atomic level. We report cryo-EM structures of Pol III in complex with IN1, revealing a 16-residue segment at the IN1 C-terminus that contacts Pol III subunits AC40 and AC19, an interaction that we validate by in vivo mutational analysis. Binding to IN1 associates with allosteric changes in Pol III that may affect its transcriptional activity. The C-terminal domain of subunit C11, involved in RNA cleavage, inserts into the Pol III funnel pore, providing evidence for a two-metal mechanism during RNA cleavage. Additionally, ordering next to C11 of an N-terminal portion from subunit C53 may explain the connection between these subunits during termination and reinitiation. Deletion of the C53 N-terminal region leads to reduced chromatin association of Pol III and IN1, and a major fall in Ty1 integration events. Our data support a model in which IN1 binding induces a Pol III configuration that may favor its retention on chromatin, thereby improving the likelihood of Ty1 integration.
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Mar 2023
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[16637]
Open Access
Abstract: CRISPR-Cas is a prokaryotic adaptive immune system, classified into six different types, each characterised by a signature protein. Type III systems, classified based on the presence of a Cas10 subunit, are rather diverse multi-subunit assemblies with a range of enzymatic activities and downstream ancillary effectors. The broad array of current biotechnological CRISPR applications is mainly based on proteins classified as Type II, however recent developments established the feasibility and efficacy of multi-protein Type III CRISPR-Cas effector complexes as RNA-targeting tools in eukaryotes. The crenarchaeon Saccharolobus solfataricus has two type III system subtypes (III–B and III-D). Here, we report the cryo-EM structure of the Csm Type III-D complex from S. solfataricus (SsoCsm), which uses CRISPR RNA to bind target RNA molecules, activating the Cas10 subunit for antiviral defence. The structure reveals the complex organisation, subunit/subunit connectivity and protein/guide RNA interactions of the SsoCsm complex, one of the largest CRISPR effectors known.
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Feb 2023
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Krios IV-Titan Krios IV at Diamond
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Yang
Yang
,
Holly J.
Garringer
,
Yang
Shi
,
Sofia
Lovestam
,
Sew
Peak-Chew
,
Xianjun
Zhang
,
Abhay
Kotecha
,
Mehtap
Bacioglu
,
Atsuo
Koto
,
Masaki
Takao
,
Maria Grazia
Spillantini
,
Bernardino
Ghetti
,
Ruben
Vidal
,
Alexey G.
Murzin
,
Sjors H. W.
Scheres
,
Michel
Goedert
Diamond Proposal Number(s):
[23268]
Open Access
Abstract: A 21-nucleotide duplication in one allele of SNCA was identified in a previously described disease with abundant α-synuclein inclusions that we now call juvenile-onset synucleinopathy (JOS). This mutation translates into the insertion of MAAAEKT after residue 22 of α-synuclein, resulting in a protein of 147 amino acids. Both wild-type and mutant proteins were present in sarkosyl-insoluble material that was extracted from frontal cortex of the individual with JOS and examined by electron cryo-microscopy. The structures of JOS filaments, comprising either a single protofilament, or a pair of protofilaments, revealed a new α-synuclein fold that differs from the folds of Lewy body diseases and multiple system atrophy (MSA). The JOS fold consists of a compact core, the sequence of which (residues 36–100 of wild-type α-synuclein) is unaffected by the mutation, and two disconnected density islands (A and B) of mixed sequences. There is a non-proteinaceous cofactor bound between the core and island A. The JOS fold resembles the common substructure of MSA Type I and Type II dimeric filaments, with its core segment approximating the C-terminal body of MSA protofilaments B and its islands mimicking the N-terminal arm of MSA protofilaments A. The partial similarity of JOS and MSA folds extends to the locations of their cofactor-binding sites. In vitro assembly of recombinant wild-type α-synuclein, its insertion mutant and their mixture yielded structures that were distinct from those of JOS filaments. Our findings provide insight into a possible mechanism of JOS fibrillation in which mutant α-synuclein of 147 amino acids forms a nucleus with the JOS fold, around which wild-type and mutant proteins assemble during elongation.
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Feb 2023
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