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Instruments / Technical Area	Publication Details	Published
Krios II-Titan Krios II at Diamond	CryoEM of RUVBL1–RUVBL2–ZNHIT2, a complex that interacts with pre-mRNA-processing-splicing factor 8 Marina Serna , Ana González-Corpas , Sofía Cabezudo , Andres Lopez-Perrote , Gianluca Degliesposti , Eduardo Zarzuela , J. mark Skehel , Javier Muñoz , Oscar Llorca Nucleic Acids Research 493 DOI: 10.1093/nar/gkab1267 Diamond Proposal Number(s): [20135] Open Access Show Abstract ▼ Abstract: Biogenesis of the U5 small nuclear ribonucleoprotein (snRNP) is an essential and highly regulated process. In particular, PRPF8, one of U5 snRNP main components, requires HSP90 working in concert with R2TP, a cochaperone complex containing RUVBL1 and RUVBL2 AAA-ATPases, and additional factors that are still poorly characterized. Here, we use biochemistry, interaction mapping, mass spectrometry and cryoEM to study the role of ZNHIT2 in the regulation of the R2TP chaperone during the biogenesis of PRPF8. ZNHIT2 forms a complex with R2TP which depends exclusively on the direct interaction of ZNHIT2 with the RUVBL1–RUVBL2 ATPases. The cryoEM analysis of this complex reveals that ZNHIT2 alters the conformation and nucleotide state of RUVBL1–RUVBL2, affecting its ATPase activity. We characterized the interactions between R2TP, PRPF8, ZNHIT2, ECD and AAR2 proteins. Interestingly, PRPF8 makes a direct interaction with R2TP and this complex can incorporate ZNHIT2 and other proteins involved in the biogenesis of PRPF8 such as ECD and AAR2. Together, these results show that ZNHIT2 participates in the assembly of the U5 snRNP as part of a network of contacts between assembly factors required for PRPF8 biogenesis and the R2TP-HSP90 chaperone, while concomitantly regulating the structure and nucleotide state of R2TP.	Dec 2021
Krios IV-Titan Krios IV at Diamond	Structural basis for substrate specificity of heteromeric transporters of neutral amino acids Carlos F. Rodriguez , Paloma Escudero-Bravo , Lucía Díaz , Paola Bartoccioni , Carmen Garcia-Martin , Joan G. Gilabert , Jasminka Boskovic , Víctor Guallar , Ekaitz Errasti-Murugarren , Oscar Llorca , Manuel Palacín Proceedings Of The National Academy Of Sciences 118 DOI: 10.1073/pnas.2113573118 Diamond Proposal Number(s): [20135] Show Abstract ▼ Abstract: Despite having similar structures, each member of the heteromeric amino acid transporter (HAT) family shows exquisite preference for the exchange of certain amino acids. Substrate specificity determines the physiological function of each HAT and their role in human diseases. However, HAT transport preference for some amino acids over others is not yet fully understood. Using cryo–electron microscopy of apo human LAT2/CD98hc and a multidisciplinary approach, we elucidate key molecular determinants governing neutral amino acid specificity in HATs. A few residues in the substrate-binding pocket determine substrate preference. Here, we describe mutations that interconvert the substrate profiles of LAT2/CD98hc, LAT1/CD98hc, and Asc1/CD98hc. In addition, a region far from the substrate-binding pocket critically influences the conformation of the substrate-binding site and substrate preference. This region accumulates mutations that alter substrate specificity and cause hearing loss and cataracts. Here, we uncover molecular mechanisms governing substrate specificity within the HAT family of neutral amino acid transporters and provide the structural bases for mutations in LAT2/CD98hc that alter substrate specificity and that are associated with several pathologies.	Dec 2021
Krios I-Titan Krios I at Diamond	Cryo-EM visualises dynamic molecular machines Oscar Llorca Diamond Proposal Number(s): [20135] Show Abstract ▼ Abstract: iology essenger RNA (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene. Inside a cell, mRNA is used as a template to build a protein. However, mRNA molecules are prone to errors, and cells use quality-control processes called mRNA surveillance mechanisms to ensure the mRNA molecules (and hence proteins) are correct. Nonsense-mediated mRNA decay (NMD) is one of these quality-control mechanisms. Its role is to degrade mRNAs with premature termination codons (PTCs), which might otherwise produce truncated proteins. AAA-ATPases are proteins found in all organisms. They are essential for many cellular functions, including DNA replication, protein degradation and the regulation of gene expression. RUVBL1 and RUVBL2 are two closely related AAA-ATPases required for the initiation of the NMD pathway. However, how RUVBL1 and RUVBL2 regulate NMD and how interacting partners regulate the ATPase activity of RUVBL1 and RUVBL2 remain poorly understood. Researchers from the Spanish National Cancer Research Centre (CNIO) and the University of Edinburgh investigated which of the core NMD factors could form a direct complex with RUVBL1-RUVBL2 to determine the structure of the complex and study if the interaction affected the ATPase activity of the chaperone. To image these dynamic molecular machines to this level of detail, they used cryo-electron microscopy (cryo-EM). Access to the high-end instrumentation at the Electron Bio-Imaging Centre (eBIC) at Diamond Light Source was essential to solve these structures. Their results revealed the regulation of RUVBL1-RUVBL2 by a factor participating in the NMD pathway, and the core elements of their model may apply to other processes where these ATPases participate.	Jul 2021
Krios I-Titan Krios I at Diamond	Structure of the TELO2-TTI1-TTI2 complex and its function in TOR recruitment to the R2TP chaperone 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 Cell Reports 36 DOI: 10.1016/j.celrep.2021.109317 Open Access Show Abstract ▼ 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.	Jul 2021
Krios I-Titan Krios I at Diamond	Regulation of RUVBL1-RUVBL2 AAA-ATPases by the nonsense-mediated mRNA decay factor DHX34, as evidenced by Cryo-EM Andrés López-Perrote , Nele Hug , Ana González-Corpas , Carlos F. Rodríguez , Marina Serna , Carmen Garcia-Martin , Jasminka Boskovic , Rafael Fernandez-Leiro , Javier F Caceres , Oscar Llorca Elife 9 DOI: 10.7554/eLife.63042 Diamond Proposal Number(s): [20135] Open Access Show Abstract ▼ Abstract: Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that degrades aberrant mRNAs and also regulates the expression of a wide range of physiological transcripts. RUVBL1 and RUVBL2 AAA-ATPases form an hetero-hexameric ring that is part of several macromolecular complexes such as INO80, SWR1 and R2TP. Interestingly, RUVBL1-RUVBL2 ATPase activity is required for NMD activation by an unknown mechanism. Here, we show that DHX34, an RNA helicase regulating NMD initiation, directly interacts with RUVBL1-RUVBL2 in vitro and in cells. Cryo-EM reveals that DHX34 induces extensive changes in the N-termini of every RUVBL2 subunit in the complex, stabilizing a conformation that does not bind nucleotide and thereby down-regulates ATP hydrolysis of the complex. Using ATPase-deficient mutants, we find that DHX34 acts exclusively on the RUVBL2 subunits. We propose a model, where DHX34 acts to couple RUVBL1-RUVBL2 ATPase activity to the assembly of factors required to initiate the NMD response.	Nov 2020
Krios I-Titan Krios I at Diamond	RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex 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 Nature Communications 9 DOI: 10.1038/s41467-018-03942-1 Diamond Proposal Number(s): [13312, 13520, 15997] Open Access Show Abstract ▼ 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.	Apr 2018
Krios I-Titan Krios I at Diamond	The Structure of the R2TP Complex Defines a Platform for Recruiting Diverse Client Proteins to the HSP90 Molecular Chaperone System Angel Rivera-calzada , Mohinder Pal , Hugo Muñoz-hernández , Juan R. Luque-ortega , David Gil-carton , Gianluca Degliesposti , J. Mark Skehel , Chrisostomos Prodromou , Laurence H. Pearl , Oscar Llorca Structure 25, 1145 - 1152.e4 DOI: 10.1016/j.str.2017.05.016 Diamond Proposal Number(s): [13520, 14507] Open Access Show Abstract ▼ 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.	Jul 2017
I24-Microfocus Macromolecular Crystallography	Structural and functional studies of LRP6 ectodomain reveal a platform for Wnt signaling. Shuo Chen , Doryen Bubeck , Bryan T Macdonald , Wen-xue Liang , Jian-hua Mao , Tomas Malinauskas , Oscar Llorca , Alexandru Aricescu , Christian Siebold , Xi He , Edith Jones Developmental Cell 21, 848 - 861 DOI: 10.1016/j.devcel.2011.09.007 PMID: 3564486 Show Abstract ▼ Abstract: LDL-receptor-related protein 6 (LRP6), alongside Frizzled receptors, transduces Wnt signaling across the plasma membrane. The LRP6 ectodomain comprises four tandem β-propeller–EGF-like domain (PE) pairs which harbor binding sites for Wnt morphogens and their antagonists including Dickkopf 1 (Dkk1). To understand how these multiple interactions are integrated, we combined crystallographic analysis of the third and fourth PE pairs with electron microscopy (EM) to determine the complete ectodomain structure. An extensive inter-pair interface, conserved for the first-to-second and third-to-fourth PE interactions, contributes to a compact platform-like architecture, which is disrupted by mutations implicated in developmental diseases. EM reconstruction of the LRP6 platform bound to chaperone Mesd exemplifies a binding mode spanning PE pairs. Cellular and binding assays identify overlapping Wnt3a- and Dkk1-binding surfaces on the third PE pair, consistent with steric competition, but also suggest a model in which the platform structure supports an interplay of ligands through multiple interaction sites.	Dec 2011

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