Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[26876]
Abstract: To organize microtubules, cells tightly control the activity of the microtubule nucleator γ-tubulin ring complex (γTuRC). The open ring-shaped γTuRC was proposed to nucleate microtubules by a template mechanism. However, its splayed structure does not match microtubule symmetry, leaving it unclear how γTuRC becomes an efficient nucleator. Here, we identify the mechanism of γTuRC activation by CDK5RAP2 centrosomin motif 1 (CM1). Using cryoelectron microscopy (cryo-EM), we find that activation involves binding of multiple CM1 dimers to five distinct sites around the outside of the γTuRC cone, which crucially depends on regulatory modules formed by MZT2 and the N-terminal extensions of GCP2 subunits. CM1 binding promotes lateral interactions between GCP subunits to facilitate microtubule-like conformations and release of luminal actin that is integral to non-activated γTuRC. We propose a model where generation of γTuRC with an expanded conformational range, rather than perfect symmetry, is sufficient to boost nucleation activity.
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Sep 2024
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Krios I-Titan Krios I at Diamond
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Josep
Rullo-Tubau
,
Maria
Martinez-Molledo
,
Paola
Bartoccioni
,
Ignasi
Puch-Giner
,
Ángela
Arias
,
Suwipa
Saen-Oon
,
Camille
Stephan-Otto Attolini
,
Rafael
Artuch
,
Lucía
Díaz
,
Víctor
Guallar
,
Ekaitz
Errasti-Murugarren
,
Manuel
Palacin
,
Oscar
Llorca
Diamond Proposal Number(s):
[26876]
Open Access
Abstract: Recent cryoEM studies elucidated details of the structural basis for the substrate selectivity and translocation of heteromeric amino acid transporters. However, Asc1/CD98hc is the only neutral heteromeric amino acid transporter that can function through facilitated diffusion, and the only one that efficiently transports glycine and D-serine, and thus has a regulatory role in the central nervous system. Here we use cryoEM, ligand-binding simulations, mutagenesis, transport assays, and molecular dynamics to define human Asc1/CD98hc determinants for substrate specificity and gain insights into the mechanisms that govern substrate translocation by exchange and facilitated diffusion. The cryoEM structure of Asc1/CD98hc is determined at 3.4–3.8 Å resolution, revealing an inward-facing semi-occluded conformation. We find that Ser 246 and Tyr 333 are essential for Asc1/CD98hc substrate selectivity and for the exchange and facilitated diffusion modes of transport. Taken together, these results reveal the structural bases for ligand binding and transport features specific to human Asc1.
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Apr 2024
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Krios IV-Titan Krios IV at Diamond
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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
Diamond Proposal Number(s):
[20135]
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.
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Dec 2021
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Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[20135]
Open Access
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.
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Dec 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):
[20135]
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.
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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):
[20135]
Open Access
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.
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Nov 2020
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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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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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I24-Microfocus Macromolecular Crystallography
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Shuo
Chen
,
Doryen
Bubeck
,
Bryan T.
Macdonald
,
Wen-Xue
Liang
,
Jian-Hua
Mao
,
Tomas
Malinauskas
,
Oscar
Llorca
,
Alexandru
Aricescu
,
Christian
Siebold
,
Xi
He
,
E. Yvonne
Jones
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.
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Dec 2011
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