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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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Stelios
Chrysostomou
,
Rajat
Roy
,
Filippo
Prischi
,
Lucksamon
Thamlikitkul
,
Kathryn L.
Chapman
,
Uwais
Mufti
,
Robert
Peach
,
Laifeng
Ding
,
David
Hancock
,
Christopher
Moore
,
Miriam
Molina-Arcas
,
Francesco
Mauri
,
David J.
Pinato
,
Joel M.
Abrahams
,
Silvia
Ottaviani
,
Leandro
Castellano
,
Georgios
Giamas
,
Jennifer
Pascoe
,
Devmini
Moonamale
,
Sarah
Pirrie
,
Claire
Gaunt
,
Lucinda
Billingham
,
Neil M.
Steven
,
Michael
Cullen
,
David
Hrouda
,
Mathias
Winkler
,
John
Post
,
Philip
Cohen
,
Seth J.
Salpeter
,
Vered
Bar
,
Adi
Zundelevich
,
Shay
Golan
,
Dan
Leibovici
,
Romain
Lara
,
David R.
Klug
,
Sophia N.
Yaliraki
,
Mauricio
Barahona
,
Yulan
Wang
,
Julian
Downward
,
J. Mark
Skehel
,
Maruf M. U.
Ali
,
Michael J.
Seckl
,
Olivier E.
Pardo
Diamond Proposal Number(s):
[9424]
Abstract: Lung and bladder cancers are mostly incurable due to early development of drug resistance and metastatic dissemination. Hence, better therapies that tackle these two processes are urgently needed to improve clinical outcome. We have identified RSK4 as a promoter of drug resistance and metastasis in lung and bladder cancer cells. Silencing this kinase, either through RNA interference or CRISPR, sensitised tumor cells to chemotherapy and hindered metastasis in vitro and in vivo in a tail- vein injection model. Drug screening revealed several floxacin antibiotics as potent RSK4 activation inhibitors and trovafloxacin reproduced all effects of RSK4 silencing in vitro and in/ex vivo using lung cancer xenograft and genetically-engineered mouse models and bladder tumour explants. Through X-ray structure determination and Markov transient and Deuterium exchange analyses, we identified the allosteric binding site and revealed how this compound blocks RSK4 kinase activation through binding to an allosteric site and mimicking a kinase auto-inhibitory mechanism involving the RSK4’s hydrophobic motif. Last, we show that patients undergoing chemotherapy and adhering to prophylactic levofloxacin in the large placebo-controlled randomised phase 3 SIGNIFICANT Trial had significantly (p =0.048) increased long-term overall survival times. Hence, we suggest that RSK4 inhibition may represent an effective therapeutic strategy for treating lung and bladder cancer.
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Jul 2021
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Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[17434]
Abstract: A key step in translational initiation is the recruitment of the 43S preinitiation complex by the cap-binding complex [eukaryotic initiation factor 4F (eIF4F)] at the 5′ end of messenger RNA (mRNA) to form the 48S initiation complex (i.e., the 48S). The 48S then scans along the mRNA to locate a start codon. To understand the mechanisms involved, we used cryo–electron microscopy to determine the structure of a reconstituted human 48S. The structure reveals insights into early events of translation initiation complex assembly, as well as how eIF4F interacts with subunits of eIF3 near the mRNA exit channel in the 43S. The location of eIF4F is consistent with a slotting model of mRNA recruitment and suggests that downstream mRNA is unwound at least in part by being “pulled” through the 40S subunit during scanning.
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Sep 2020
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Krios I-Titan Krios I at Diamond
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Shabih
Shakeel
,
Eeson
Rajendra
,
Pablo
Alcon
,
Francis
O'Reilly
,
Dror S.
Chorev
,
Sarah
Maslen
,
Gianluca
Degliesposti
,
Christopher J.
Russo
,
Shaoda
He
,
Chris H.
Hill
,
J. Mark
Skehel
,
Sjors H. W.
Scheres
,
Ketan J.
Patel
,
Juri
Rappsilber
,
Carol V.
Robinson
,
Lori A.
Passmore
Diamond Proposal Number(s):
[18091, 17434]
Abstract: The Fanconi anaemia (FA) pathway repairs DNA damage caused by endogenous and chemotherapy-induced DNA crosslinks, and responds to replication stress. Genetic inactivation of this pathway by mutation of genes encoding FA complementation group (FANC) proteins impairs development, prevents blood production and promotes cancer1,3. The key molecular step in the FA pathway is the monoubiquitination of a pseudosymmetric heterodimer of FANCD2–FANCI4,5 by the FA core complex—a megadalton multiprotein E3 ubiquitin ligase6,7. Monoubiquitinated FANCD2 then recruits additional protein factors to remove the DNA crosslink or to stabilize the stalled replication fork. A molecular structure of the FA core complex would explain how it acts to maintain genome stability. Here we reconstituted an active, recombinant FA core complex, and used cryo-electron microscopy and mass spectrometry to determine its structure. The FA core complex comprises two central dimers of the FANCB and FA-associated protein of 100 kDa (FAAP100) subunits, flanked by two copies of the RING finger subunit, FANCL. These two heterotrimers act as a scaffold to assemble the remaining five subunits, resulting in an extended asymmetric structure. Destabilization of the scaffold would disrupt the entire complex, resulting in a non-functional FA pathway. Thus, the structure provides a mechanistic basis for the low numbers of patients with mutations in FANCB, FANCL and FAAP100. Despite a lack of sequence homology, FANCB and FAAP100 adopt similar structures. The two FANCL subunits are in different conformations at opposite ends of the complex, suggesting that each FANCL has a distinct role. This structural and functional asymmetry of dimeric RING finger domains may be a general feature of E3 ligases. The cryo-electron microscopy structure of the FA core complex provides a foundation for a detailed understanding of its E3 ubiquitin ligase activity and DNA interstrand crosslink repair.
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Nov 2019
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[15916]
Abstract: Mutations in the E3 ubiquitin ligase parkin (PARK2) and the protein kinase PINK1 (PARK6) are linked to autosomal-recessive juvenile Parkinsonism (AR-JP)1,2, and at the cellular level cause defects in mitophagy, the cellular process that organises destruction of damaged mitochondria3,4. Parkin is autoinhibited, and requires activation by PINK1, which phosphorylates Ser65 in ubiquitin and in the parkin ubiquitin-like (Ubl) domain. Parkin binds phospho-ubiquitin, which enables efficient Parkin phosphorylation; however, the enzyme remains autoinhibited with an inaccessible active site5,6. It is unclear how phosphorylation of parkin activates the molecule. Here we follow the activation of full-length human parkin by hydrogen deuterium exchange mass spectrometry, and reveal large-scale domain rearrangement in the activation process, in which the phospho-Ubl rebinds to the parkin core, and releases the catalytic RING2 domain. A 1.8 Å crystal structure of phosphorylated human parkin reveals the binding site of the phosphorylated Ubl on the unique parkin domain (UPD), involving a phosphate-binding pocket lined by AR-JP mutations. Strikingly, a conserved linker region between Ubl and UPD acts as an activating element (ACT) that contributes to RING2 release by mimicking RING2 interactions on the UPD, explaining further AR-JP mutations. Our data unveil how autoinhibition in parkin is resolved, and suggest how parkin ubiquitinates its substrates via an untethered RING2 domain. This opens exciting new avenues to design parkin activators for clinical use.
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Jun 2018
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Krios I-Titan Krios I at Diamond
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Fabrizio
Martino
,
Mohinder
Pal
,
Hugo
Muñoz-Hernández
,
Carlos F.
Rodríguez
,
Rafael
Núñez-Ramírez
,
David
Gil-Carton
,
Gianluca
Degliesposti
,
J. Mark
Skehel
,
Mark
Roe
,
Chrisostomos
Prodromou
,
Laurence H.
Pearl
,
Oscar
Llorca
Diamond Proposal Number(s):
[13312, 13520, 15997]
Open Access
Abstract: The R2TP/Prefoldin-like co-chaperone, in concert with HSP90, facilitates assembly and cellular stability of RNA polymerase II, and complexes of PI3-kinase-like kinases such as mTOR. However, the mechanism by which this occurs is poorly understood. Here we use cryo-EM and biochemical studies on the human R2TP core (RUVBL1–RUVBL2–RPAP3–PIH1D1) which reveal the distinctive role of RPAP3, distinguishing metazoan R2TP from the smaller yeast equivalent. RPAP3 spans both faces of a single RUVBL ring, providing an extended scaffold that recruits clients and provides a flexible tether for HSP90. A 3.6 Å cryo-EM structure reveals direct interaction of a C-terminal domain of RPAP3 and the ATPase domain of RUVBL2, necessary for human R2TP assembly but absent from yeast. The mobile TPR domains of RPAP3 map to the opposite face of the ring, associating with PIH1D1, which mediates client protein recruitment. Thus, RPAP3 provides a flexible platform for bringing HSP90 into proximity with diverse client proteins.
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Apr 2018
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B21-High Throughput SAXS
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Laura C.
Lehmann
,
Graeme
Hewitt
,
Shintaro
Aibara
,
Alexander
Leitner
,
Emil
Marklund
,
Sarah L.
Maslen
,
Varun
Maturi
,
Yang
Chen
,
David
Van Der Spoel
,
J. Mark
Skehel
,
Aristidis
Moustakas
,
Simon J.
Boulton
,
Sebastian
Deindl
Diamond Proposal Number(s):
[11171]
Abstract: Human ALC1 is an oncogene-encoded chromatin-remodeling enzyme required for DNA repair that possesses a poly(ADP-ribose) (PAR)-binding macro domain. Its engagement with PARylated PARP1 activates ALC1 at sites of DNA damage, but the underlying mechanism remains unclear. Here, we establish a dual role for the macro domain in autoinhibition of ALC1 ATPase activity and coupling to nucleosome mobilization. In the absence of DNA damage, an inactive conformation of the ATPase is maintained by juxtaposition of the macro domain against predominantly the C-terminal ATPase lobe through conserved electrostatic interactions. Mutations within this interface displace the macro domain, constitutively activate the ALC1 ATPase independent of PARylated PARP1, and alter the dynamics of ALC1 recruitment at DNA damage sites. Upon DNA damage, binding of PARylated PARP1 by the macro domain induces a conformational change that relieves autoinhibitory interactions with the ATPase motor, which selectively activates ALC1 remodeling upon recruitment to sites of DNA damage.
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Dec 2017
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: High-fidelity DNA replication depends on a proofreading 3′–5′ exonuclease that is associated with the replicative DNA polymerase. The replicative DNA polymerase DnaE1 from the major pathogen Mycobacterium tuberculosis (Mtb) uses its intrinsic PHP-exonuclease that is distinct from the canonical DEDD exonucleases found in the Escherichia coli and eukaryotic replisomes. The mechanism of the PHP-exonuclease is not known. Here, we present the crystal structure of the Mtb DnaE1 polymerase. The PHP-exonuclease has a trinuclear zinc center, coordinated by nine conserved residues. Cryo-EM analysis reveals the entry path of the primer strand in the PHP-exonuclease active site. Furthermore, the PHP-exonuclease shows a striking similarity to E. coli endonuclease IV, which provides clues regarding the mechanism of action. Altogether, this work provides important insights into the PHP-exonuclease and reveals unique properties that make it an attractive target for novel anti-mycobacterial drugs.
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Oct 2017
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[13520, 14507]
Open Access
Abstract: The R2TP complex, comprising the Rvb1p-Rvb2p AAA-ATPases, Tah1p, and Pih1p in yeast, is a specialized Hsp90 co-chaperone required for the assembly and maturation of multi-subunit complexes. These include the small nucleolar ribonucleoproteins, RNA polymerase II, and complexes containing phosphatidylinositol-3-kinase-like kinases. The structure and stoichiometry of yeast R2TP and how it couples to Hsp90 are currently unknown. Here, we determine the 3D organization of yeast R2TP using sedimentation velocity analysis and cryo-electron microscopy. The 359-kDa complex comprises one Rvb1p/Rvb2p hetero-hexamer with domains II (DIIs) forming an open basket that accommodates a single copy of Tah1p-Pih1p. Tah1p-Pih1p binding to multiple DII domains regulates Rvb1p/Rvb2p ATPase activity. Using domain dissection and cross-linking mass spectrometry, we identified a unique region of Pih1p that is essential for interaction with Rvb1p/Rvb2p. These data provide a structural basis for understanding how R2TP couples an Hsp90 dimer to a diverse set of client proteins and complexes.
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Jul 2017
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I02-Macromolecular Crystallography
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Open Access
Abstract: The R2TP cochaperone complex plays a critical role in the assembly of multisubunit machines, including small nucleolar ribonucleoproteins (snoRNPs), RNA polymerase II, and the mTORC1 and SMG1 kinase complexes, but the molecular basis of substrate recognition remains unclear. Here, we describe a phosphopeptide binding domain (PIH-N) in the PIH1D1 subunit of the R2TP complex that preferentially binds to highly acidic phosphorylated proteins. A cocrystal structure of a PIH-N domain/TEL2 phosphopeptide complex reveals a highly specific phosphopeptide recognition mechanism in which Lys57 and 64 in PIH1D1, along with a conserved DpSDD phosphopeptide motif within TEL2, are essential and sufficient for binding. Proteomic analysis of PIH1D1 interactors identified R2TP complex substrates that are recruited by the PIH-N domain in a sequence-specific and phosphorylation-dependent manner suggestive of a common mechanism of substrate recognition. We propose that protein complexes assembled by the R2TP complex are defined by phosphorylation of a specific motif and recognition by the PIH1D1 subunit.
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Apr 2014
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