B21-High Throughput SAXS
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Diamond Proposal Number(s):
[16207]
Open Access
Abstract: Talin is a mechanosensitive adapter protein that couples integrins to the cytoskeleton. Talin rod domain–containing protein 1 (TLNRD1) shares 22% homology with the talin R7R8 rod domains, and is highly conserved throughout vertebrate evolution, although little is known about its function. Here we show that TLNRD1 is an α-helical protein structurally homologous to talin R7R8. Like talin R7R8, TLNRD1 binds F-actin, but because it forms a novel antiparallel dimer, it also bundles F-actin. In addition, it binds the same LD motif–containing proteins, RIAM and KANK, as talin R7R8. In cells, TLNRD1 localizes to actin bundles as well as to filopodia. Increasing TLNRD1 expression enhances filopodia formation and cell migration on 2D substrates, while TLNRD1 down-regulation has the opposite effect. Together, our results suggest that TLNRD1 has retained the diverse interactions of talin R7R8, but has developed distinct functionality as an actin-bundling protein that promotes filopodia assembly.
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Sep 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Yakubu Princely
Abudu
,
Birendra
Kumar Shrestha
,
Wenxin
Zhang
,
Anthimi
Palara
,
Hanne Britt
Brenne
,
Kenneth Bowitz
Larsen
,
Deanna Lynn
Wolfson
,
Gianina
Dumitriu
,
Cristina Ionica
Øie
,
Balpreet Singh
Ahluwalia
,
Gahl
Levy
,
Christian
Behrends
,
Sharon A.
Tooze
,
Stephane
Mouilleron
,
Trond
Lamark
,
Terje
Johansen
Diamond Proposal Number(s):
[18566]
Abstract: Mitophagy is the degradation of surplus or damaged mitochondria by autophagy. In addition to programmed and stress-induced mitophagy, basal mitophagy processes exert organelle quality control. Here, we show that the sorting and assembly machinery (SAM) complex protein SAMM50 interacts directly with ATG8 family proteins and p62/SQSTM1 to act as a receptor for a basal mitophagy of components of the SAM and mitochondrial contact site and cristae organizing system (MICOS) complexes. SAMM50 regulates mitochondrial architecture by controlling formation and assembly of the MICOS complex decisive for normal cristae morphology and exerts quality control of MICOS components. To this end, SAMM50 recruits ATG8 family proteins through a canonical LIR motif and interacts with p62/SQSTM1 to mediate basal mitophagy of SAM and MICOS components. Upon metabolic switch to oxidative phosphorylation, SAMM50 and p62 cooperate to mediate efficient mitophagy.
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Aug 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[18069]
Open Access
Abstract: The kinetochore is the macromolecular protein complex that assembles onto centromeric DNA and binds spindle microtubules. Evolutionarily divergent kinetoplastids have an unconventional set of kinetochore proteins. It remains unknown how kinetochores assemble at centromeres in these organisms. Here, we characterize KKT2 and KKT3 in the kinetoplastid parasite Trypanosoma brucei. In addition to the N-terminal kinase domain and C-terminal divergent polo boxes, these proteins have a central domain of unknown function. We show that KKT2 and KKT3 are important for the localization of several kinetochore proteins and that their central domains are sufficient for centromere localization. Crystal structures of the KKT2 central domain from two divergent kinetoplastids reveal a unique zinc-binding domain (termed the CL domain for centromere localization), which promotes its kinetochore localization in T. brucei. Mutations in the equivalent domain in KKT3 abolish its kinetochore localization and function. Our work shows that the unique central domains play a critical role in mediating the centromere localization of KKT2 and KKT3.
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Aug 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18565, 25108]
Open Access
Abstract: ATP- and GTP-dependent molecular switches are extensively used to control functions of proteins in a wide range of biological processes. However, CTP switches are rarely reported. Here, we report that a nucleoid occlusion protein Noc is a CTPase enzyme whose membrane-binding activity is directly regulated by a CTP switch. In Bacillus subtilis, Noc nucleates on 16 bp NBS sites before associating with neighboring non-specific DNA to form large membrane-associated nucleoprotein complexes to physically occlude assembly of the cell division machinery. By in vitro reconstitution, we show that (1) CTP is required for Noc to form the NBS-dependent nucleoprotein complex, and (2) CTP binding, but not hydrolysis, switches Noc to a membrane-active state. Overall, we suggest that CTP couples membrane-binding activity of Noc to nucleoprotein complex formation to ensure productive recruitment of DNA to the bacterial cell membrane for nucleoid occlusion activity.
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Jul 2021
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Krios I-Titan Krios I at Diamond
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Mohinder
Pal
,
Hugo
Munoz-Hernandez
,
Dennis
Bjorklund
,
Lihong
Zhou
,
Gianluca
Degliesposti
,
J. Mark
Skehel
,
Emma L.
Hesketh
,
Rebecca F.
Thompson
,
Laurence H.
Pearl
,
Oscar
Llorca
,
Chrisostomos
Prodromou
Open Access
Abstract: The R2TP (RUVBL1-RUVBL2-RPAP3-PIH1D1) complex, in collaboration with heat shock protein 90 (HSP90), functions as a chaperone for the assembly and stability of protein complexes, including RNA polymerases, small nuclear ribonucleoprotein particles (snRNPs), and phosphatidylinositol 3-kinase (PI3K)-like kinases (PIKKs) such as TOR and SMG1. PIKK stabilization depends on an additional complex of TELO2, TTI1, and TTI2 (TTT), whose structure and function are poorly understood. The cryoelectron microscopy (cryo-EM) structure of the human R2TP-TTT complex, together with biochemical experiments, reveals the mechanism of TOR recruitment to the R2TP-TTT chaperone. The HEAT-repeat TTT complex binds the kinase domain of TOR, without blocking its activity, and delivers TOR to the R2TP chaperone. In addition, TTT regulates the R2TP chaperone by inhibiting RUVBL1-RUVBL2 ATPase activity and by modulating the conformation and interactions of the PIH1D1 and RPAP3 components of R2TP. Taken together, our results show how TTT couples the recruitment of TOR to R2TP with the regulation of this chaperone system.
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Jul 2021
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[16942]
Open Access
Abstract: Membrane remodeling and repair are essential for all cells. Proteins that perform these functions include Vipp1/IM30 in photosynthetic plastids, PspA in bacteria, and ESCRT-III in eukaryotes. Here, using a combination of evolutionary and structural analyses, we show that these protein families are homologous and share a common ancient evolutionary origin that likely predates the last universal common ancestor. This homology is evident in cryo-electron microscopy structures of Vipp1 rings from the cyanobacterium Nostoc punctiforme presented over a range of symmetries. Each ring is assembled from rungs that stack and progressively tilt to form dome-shaped curvature. Assembly is facilitated by hinges in the Vipp1 monomer, similar to those in ESCRT-III proteins, which allow the formation of flexible polymers. Rings have an inner lumen that is able to bind and deform membranes. Collectively, these data suggest conserved mechanistic principles that underlie Vipp1, PspA, and ESCRT-III-dependent membrane remodeling across all domains of life.
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Jul 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Daniel
Zaidman
,
Paul
Gehrtz
,
Mihajlo
Filep
,
Daren
Fearon
,
Ronen
Gabizon
,
Alice
Douangamath
,
Jaime
Prilusky
,
Shirly
Duberstein
,
Galit
Cohen
,
C. David
Owen
,
Efrat
Resnick
,
Claire
Strain-Damerell
,
Petra
Lukacik
,
Haim
Barr
,
Martin A.
Walsh
,
Frank
Von Delft
,
Nir
London
Diamond Proposal Number(s):
[18145, 27963]
Abstract: Designing covalent inhibitors is increasingly important, although it remains challenging. Here, we present covalentizer, a computational pipeline for identifying irreversible inhibitors based on structures of targets with non-covalent binders. Through covalent docking of tailored focused libraries, we identify candidates that can bind covalently to a nearby cysteine while preserving the interactions of the original molecule. We found ∼11,000 cysteines proximal to a ligand across 8,386 complexes in the PDB. Of these, the protocol identified 1,553 structures with covalent predictions. In a prospective evaluation, five out of nine predicted covalent kinase inhibitors showed half-maximal inhibitory concentration (IC50) values between 155 nM and 4.5 μM. Application against an existing SARS-CoV Mpro reversible inhibitor led to an acrylamide inhibitor series with low micromolar IC50 values against SARS-CoV-2 Mpro. The docking was validated by 12 co-crystal structures. Together these examples hint at the vast number of covalent inhibitors accessible through our protocol.
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Jun 2021
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I03-Macromolecular Crystallography
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Chang
Liu
,
Helen M.
Ginn
,
Wanwisa
Dejnirattisai
,
Piyada
Supasa
,
Beibei
Wang
,
Aekkachai
Tuekprakhon
,
Rungtiwa
Nutalai
,
Daming
Zhou
,
Alexander J.
Mentzer
,
Yuguang
Zhao
,
Helen M. E.
Duyvesteyn
,
César
López-Camacho
,
Jose
Slon-Campos
,
Thomas
Walter
,
Donal
Skelly
,
Sile Ann
Johnson
,
Thomas G.
Ritter
,
Chris
Mason
,
Sue Ann
Costa Clemens
,
Felipe Gomes
Naveca
,
Valdinete
Nascimento
,
Fernanda
Nascimento
,
Cristiano
Fernandes Da Costa
,
Paola Cristina
Resende
,
Alex
Pauvolid-Correa
,
Marilda M.
Siqueira
,
Christina
Dold
,
Nigel
Temperton
,
Tao
Dong
,
Andrew J.
Pollard
,
Julian C.
Knight
,
Derrick
Crook
,
Teresa
Lambe
,
Elizabeth
Clutterbuck
,
Sagida
Bibi
,
Amy
Flaxman
,
Mustapha
Bittaye
,
Sandra
Belij-Rammerstorfer
,
Sarah C.
Gilbert
,
Tariq
Malik
,
Miles W.
Carroll
,
Paul
Klenerman
,
Eleanor
Barnes
,
Susanna J.
Dunachie
,
Vicky
Baillie
,
Natali
Serafin
,
Zanele
Ditse
,
Kelly
Da Silva
,
Neil G.
Paterson
,
Mark A.
Williams
,
David R.
Hall
,
Shabir
Madhi
,
Marta C.
Nunes
,
Philip
Goulder
,
Elizabeth E.
Fry
,
Juthathip
Mongkolsapaya
,
Jingshan
Ren
,
David I.
Stuart
,
Gavin R.
Screaton
Diamond Proposal Number(s):
[27009]
Abstract: SARS-CoV-2 has undergone progressive change with variants conferring advantage rapidly becoming dominant lineages e.g. B.1.617. With apparent increased transmissibility variant B.1.617.2 has contributed to the current wave of infection ravaging the Indian subcontinent and has been designated a variant of concern in the UK. Here we study the ability of monoclonal antibodies, convalescent and vaccine sera to neutralize B.1.617.1 and B.1.617.2 and complement this with structural analyses of Fab/RBD complexes and map the antigenic space of current variants. Neutralization of both viruses is reduced when compared with ancestral Wuhan related strains but there is no evidence of widespread antibody escape as seen with B.1.351. However, B.1.351 and P.1 sera showed markedly more reduction in neutralization of B.1.617.2 suggesting that individuals previously infected by these variants may be more susceptible to reinfection by B.1.617.2. This observation provides important new insight for immunisation policy with future variant vaccines in non-immune populations.
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Jun 2021
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I03-Macromolecular Crystallography
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Abstract: Synthetic peptides are attractive candidates to manipulate protein-protein interactions inside the cell as they mimic natural interactions to compete for binding. However, protein-peptide interactions are often dynamic and weak. A challenge is to design peptides that make improved interactions with the target. Here, we devise a fragment-linking strategy—“mash-up” design—to deliver a high-affinity ligand, KinTag, for the kinesin-1 motor. Using structural insights from natural micromolar-affinity cargo-adaptor ligands, we have identified and combined key binding features in a single, high-affinity ligand. An X-ray crystal structure demonstrates interactions as designed and reveals only a modest increase in interface area. Moreover, when genetically encoded, KinTag promotes transport of lysosomes with higher efficiency than natural sequences, revealing a direct link between motor-adaptor binding affinity and organelle transport. Together, these data demonstrate a fragment-linking strategy for peptide design and its application in a synthetic motor ligand to direct cellular cargo transport.
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Apr 2021
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Krios I-Titan Krios I at Diamond
Krios II-Titan Krios II at Diamond
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Open Access
Abstract: The ATPase Prp16 governs equilibrium between the branching (B∗/C) and exon ligation (C∗/P) conformations of the spliceosome. Here, we present the electron cryomicroscopy reconstruction of the Saccharomyces cerevisiae C-complex spliceosome at 2.8 Å resolution and identify a novel C-complex intermediate (Ci) that elucidates the molecular basis for this equilibrium. The exon-ligation factors Prp18 and Slu7 bind to Ci before ATP hydrolysis by Prp16 can destabilize the branching conformation. Biochemical assays suggest that these pre-bound factors prime the C complex for conversion to C∗ by Prp16. A complete model of the Prp19 complex (NTC) reveals how the branching factors Yju2 and Isy1 are recruited by the NTC before branching. Prp16 remodels Yju2 binding after branching, allowing Yju2 to remain tethered to the NTC in the C∗ complex to promote exon ligation. Our results explain how Prp16 action modulates the dynamic binding of step-specific factors to alternatively stabilize the C or C∗ conformation and establish equilibrium of the catalytic spliceosome.
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Mar 2021
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