B21-High Throughput SAXS
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Xiao-Han
Li
,
Conny W. H.
Yu
,
Natalia
Gomez-Navarro
,
Viktoriya
Stancheva
,
Hongni
Zhu
,
Andal
Murthy
,
Michael
Wozny
,
Ketan
Malhotra
,
Christopher M.
Johnson
,
Martin
Blackledge
,
Balaji
Santhanam
,
Wei
Liu
,
Jinqing
Huang
,
Stefan M. V.
Freund
,
Elizabeth A.
Miller
,
M.
Madan Babu
Diamond Proposal Number(s):
[24985]
Open Access
Abstract: A number of Intrinsically disordered proteins (IDPs) encoded in stress-tolerant organisms such as tardigrade, can confer fitness advantage and abiotic stress tolerance when heterologously expressed. Tardigrade-specific disordered proteins including the cytosolic abundant heat soluble (CAHS) proteins are proposed to confer stress tolerance through vitrification or gelation, whereas evolutionarily conserved IDPs in tardigrades may contribute to stress tolerance through other biophysical mechanisms. Here we characterized the mechanism of action of an evolutionarily conserved, tardigrade IDP, HeLEA1, which belongs to the group-3 late embryogenesis abundant (LEA) protein family. HeLEA1 homologs are found across different kingdoms of life. HeLEA1 is intrinsically disordered in solution but shows a propensity for helical structure across its entire sequence. HeLEA1 interacts with negatively charged membranes via dynamic disorder-to-helical transition, mainly driven by electrostatic interactions. Membrane interaction of HeLEA1 is shown to ameliorate excess surface tension and lipid packing defects. HeLEA1 localizes to the mitochondrial matrix when expressed in yeast and interacts with model membranes mimicking inner mitochondrial membrane. Yeast expressing HeLEA1 show enhanced tolerance to hyperosmotic stress under non-fermentative growth and increased mitochondrial membrane potential. Evolutionary analysis suggests that although HeLEA1 homologs have diverged their sequences to localize to different subcellular organelles, all homologs maintain a weak hydrophobic moment that is characteristic of weak and reversible membrane interaction. We suggest that such a dynamic and weak protein-membrane interaction buffering alterations in lipid packing could be a conserved strategy for regulating membrane properties and represents a general biophysical solution for stress tolerance across the domains of life.
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Jan 2024
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Laura
Piot
,
Christina
Heroven
,
Simon
Bossi
,
Joseph
Zamith
,
Tomas
Malinauskas
,
Chris
Johnson
,
Doris
Wennagel
,
David
Stroebel
,
Cécile
Charrier
,
A. Radu
Aricescu
,
Laetitia
Mony
,
Pierre
Paoletti
Abstract: Fast synaptic neurotransmission in the vertebrate central nervous system relies primarily on ionotropic glutamate receptors (iGluRs), that drive neuronal excitation, and type A γ-aminobutyric acid receptors (GABAARs), responsible for neuronal inhibition. However, the GluD1 receptor, an iGluR family member, is present at both excitatory and inhibitory synapses. Whether and how GluD1 activation may impact inhibitory neurotransmission is unknown. Here, using a combination of biochemical, structural and functional analyses, we demonstrate that GluD1 binds GABA, an unprecedented feature for iGluRs. GluD1 activation produces long-lasting enhancement of GABAergic synaptic currents in the adult mouse hippocampus through a non-ionotropic mechanism dependent on trans-synaptic anchoring. The identification of GluD1 as a GABA receptor that controls inhibitory synaptic plasticity challenges the classical dichotomy between glutamatergic and GABAergic receptors.
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Dec 2023
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15916, 21426]
Open Access
Abstract: Cilia formation is essential for human life. One of the earliest events in the ciliogenesis program is the recruitment of tau-tubulin kinase 2 (TTBK2) by the centriole distal appendage component CEP164. Due to the lack of high-resolution structural information on this complex, it is unclear how it is affected in human ciliopathies such as nephronophthisis. Furthermore, it is poorly understood if binding to CEP164 influences TTBK2 activities. Here, we present a detailed biochemical, structural, and functional analysis of the CEP164-TTBK2 complex and demonstrate how it is compromised by two ciliopathic mutations in CEP164. Moreover, we also provide insights into how binding to CEP164 is coordinated with TTBK2 activities. Together, our data deepen our understanding of a crucial step in cilia formation and will inform future studies aimed at restoring CEP164 functionality in a debilitating human ciliopathy.
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Sep 2021
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I04-Macromolecular Crystallography
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Gianni
Chessari
,
Ian R.
Hardcastle
,
Jong Sook
Ahn
,
Burcu
Anil
,
Elizabeth
Anscombe
,
Ruth H.
Bawn
,
Luke D.
Bevan
,
Timothy J.
Blackburn
,
Ildiko
Buck
,
Celine
Cano
,
Benoit
Carbain
,
Juan
Castro
,
Ben
Cons
,
Sarah J.
Cully
,
Jane A.
Endicott
,
Lynsey
Fazal
,
Bernard T.
Golding
,
Roger J.
Griffin
,
Karen
Haggerty
,
Suzannah J.
Harnor
,
Keisha
Hearn
,
Stephen
Hobson
,
Rhian S.
Holvey
,
Steven
Howard
,
Claire E.
Jennings
,
Christopher N.
Johnson
,
John
Lunec
,
Duncan C.
Miller
,
David R.
Newell
,
Martin E. M.
Noble
,
Judith
Reeks
,
Charlotte H.
Revill
,
Christiane
Riedinger
,
Jeffrey D.
St. Denis
,
Emiliano
Tamanini
,
Huw
Thomas
,
Neil T.
Thompson
,
Mladen
Vinković
,
Stephen R.
Wedge
,
Pamela A.
Williams
,
Nicola E.
Wilsher
,
Bian
Zhang
,
Yan
Zhao
Abstract: Inhibition of murine double minute 2 (MDM2)-p53 protein–protein interaction with small molecules has been shown to reactivate p53 and inhibit tumor growth. Here, we describe rational, structure-guided, design of novel isoindolinone-based MDM2 inhibitors. MDM2 X-ray crystallography, quantum mechanics ligand-based design, and metabolite identification all contributed toward the discovery of potent in vitro and in vivo inhibitors of the MDM2-p53 interaction with representative compounds inducing cytostasis in an SJSA-1 osteosarcoma xenograft model following once-daily oral administration.
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Mar 2021
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I02-Macromolecular Crystallography
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Takashi
Ochi
,
Valentina
Quarantotti
,
Huawen
Lin
,
Jerome
Jullien
,
Ivan
Rosa E Silva
,
Francesco
Boselli
,
Deepak D.
Barnabas
,
Christopher M.
Johnson
,
Stephen H.
Mclaughlin
,
Stefan M. V.
Freund
,
Andrew N.
Blackford
,
Yuu
Kimata
,
Raymond E.
Goldstein
,
Stephen P.
Jackson
,
Tom L.
Blundell
,
Susan K.
Dutcher
,
Fanni
Gergely
,
Mark
Van Breugel
Diamond Proposal Number(s):
[9537]
Open Access
Abstract: Centrioles are cylindrical assemblies whose peripheral microtubule array displays a 9-fold rotational symmetry that is established by the scaffolding protein SAS6. Centriole symmetry can be broken by centriole-associated structures, such as the striated fibers in Chlamydomonas that are important for ciliary function. The conserved protein CCDC61/VFL3 is involved in this process, but its exact role is unclear. Here, we show that CCDC61 is a paralog of SAS6. Crystal structures of CCDC61 demonstrate that it contains two homodimerization interfaces that are similar to those found in SAS6, but result in the formation of linear filaments rather than rings. Furthermore, we show that CCDC61 binds microtubules and that residues involved in CCDC61 microtubule binding are important for ciliary function in Chlamydomonas. Together, our findings suggest that CCDC61 and SAS6 functionally diverged from a common ancestor while retaining the ability to scaffold the assembly of basal body-associated structures or centrioles, respectively.
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May 2020
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Krios I-Titan Krios I at Diamond
Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[23268]
Abstract: The Rag guanosine triphosphatases (GTPases) recruit the master kinase mTORC1 to lysosomes to regulate cell growth and proliferation in response to amino acid availability. The nucleotide state of Rag heterodimers is critical for their association with mTORC1. Our cryo–electron microscopy structure of RagA/RagC in complex with mTORC1 shows the details of RagA/RagC binding to the RAPTOR subunit of mTORC1 and explains why only the RagAGTP/RagCGDP nucleotide state binds mTORC1. Previous kinetic studies suggested that GTP binding to one Rag locks the heterodimer to prevent GTP binding to the other. Our crystal structures and dynamics of RagA/RagC show the mechanism for this locking and explain how oncogenic hotspot mutations disrupt this process. In contrast to allosteric activation by RHEB, Rag heterodimer binding does not change mTORC1 conformation and activates mTORC1 by targeting it to lysosomes.
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Oct 2019
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[16192]
Open Access
Abstract: McrBC is one of the three modification-dependent restriction enzymes encoded by the Escherichia coli K12 chromosome. Amongst restriction enzymes, McrBC and its close homologues are unique in employing the AAA+ domain for GTP hydrolysis-dependent activation of DNA cleavage. The GTPase activity of McrB is stimulated by the endonuclease subunit McrC. It had been reported previously that McrB and McrC subunits oligomerise together into a high molecular weight species. Here we conclusively demonstrate using size exclusion chromatography coupled multi-angle light scattering (SEC-MALS) and images obtained by electron cryomicroscopy that McrB exists as a hexamer in solution. Furthermore, based on SEC-MALS and SAXS analyses of McrBC and the structure of McrB, we propose that McrBC is a complex of two McrB hexamers bridged by two subunits of McrC, and that the complete assembly of this complex is integral to its enzymatic activity. We show that the nucleotide-dependent oligomerisation of McrB precedes GTP hydrolysis. Mutational studies show that, unlike other AAA+ proteins, the catalytic Walker B aspartate is required for oligomerisation.
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Dec 2018
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Leonardo
Almeida-Souza
,
Rene A. W.
Frank
,
Javier
Garcia-Nafria
,
Adeline
Colussi
,
Nushan
Gunawardana
,
Christopher M.
Johnson
,
Minmin
Yu
,
Gillian
Howard
,
Byron
Andrews
,
Yvonne
Vallis
,
Harvey T.
Mcmahon
Diamond Proposal Number(s):
[11235]
Open Access
Abstract: Multiple proteins act co-operatively in mammalian clathrin-mediated endocytosis (CME) to generate endocytic vesicles from the plasma membrane. The principles controlling the activation and organization of the actin cytoskeleton during mammalian CME are, however, not fully understood. Here, we show that the protein FCHSD2 is a major activator of actin polymerization during CME. FCHSD2 deletion leads to decreased ligand uptake caused by slowed pit maturation. FCHSD2 is recruited to endocytic pits by the scaffold protein intersectin via an unusual SH3-SH3 interaction. Here, its flat F-BAR domain binds to the planar region of the plasma membrane surrounding the developing pit forming an annulus. When bound to the membrane, FCHSD2 activates actin polymerization by a mechanism that combines oligomerization and recruitment of N-WASP to PI(4,5)P2, thus promoting pit maturation. Our data therefore describe a molecular mechanism for linking spatiotemporally the plasma membrane to a force-generating actin platform guiding endocytic vesicle maturation.
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Jun 2018
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8547, 11235]
Open Access
Abstract: Ciliopathies are a group of genetic disorders caused by a failure to form functional cilia. Due to a lack of structural information, it is currently poorly understood how ciliopathic mutations affect protein functionality to give rise to the underlying disease. Using X-ray crystallography, we show that the ciliopathy-associated centriolar protein CEP120 contains three C2 domains. The point mutations V194A and A199P, which cause Joubert syndrome (JS) and Jeune asphyxiating thoracic dystrophy (JATD), respectively, both reduce the thermostability of the second C2 domain by targeting residues that point toward its hydrophobic core. Genome-engineered cells homozygous for these mutations have largely normal centriole numbers but show reduced CEP120 levels, compromised recruitment of distal centriole markers, and deficient cilia formation. Our results provide insight into the disease mechanism of two ciliopathic mutations in CEP120, identify putative binding partners of CEP120 C2B, and suggest a complex genotype-phenotype relation of the CEP120 ciliopathy alleles.
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May 2018
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[11235]
Open Access
Abstract: Cilia are thin cell projections with essential roles in cell motility, fluid movement, sensing, and signaling. They are templated from centrioles that dock against the plasma membrane and subsequently extend their peripheral microtubule array. The molecular mechanisms underpinning cilia assembly are incompletely understood. Cep104 is a key factor involved in cilia formation and length regulation that rides on the ends of elongating and shrinking cilia. It is mutated in Joubert syndrome, a genetically heterogeneous ciliopathy. Here we provide structural and biochemical data that Cep104 contains a tubulin-binding TOG (tumor overexpressed gene) domain and a novel C2HC zinc finger array. Furthermore, we identify the kinase Nek1, another ciliopathy-associated protein, as a potential binding partner of this array. Finally, we show that Nek1 competes for binding to Cep104 with the distal centriole-capping protein CP110. Our data suggest a model for Cep104 activity during ciliogenesis and provide a novel link between Cep104 and Nek1.
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Dec 2016
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