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Structural Basis of Actin Nucleation
 

Structural Basis of Actin Nucleation

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    Structural Basis of Actin Nucleation Structural Basis of Actin Nucleation Presentation Transcript

    • Structural Basis of Actin Nucleations :Lessons from bacterial actin nucleator
    • Actin 101Actin is one of the most abundant, highly conserved and well characterized ofall eukaryotic proteins Monomeric Actin (42kDa) Filamentous Actin (G-Actin) (F-Actin)
    • Actin Filament is the main „building block‟ of cytoskeleton Borish & Svitskina
    • Dynamic actin filament remodeling is the key for theregulation of various cellular process Cytokinesis Developments Cell Motility Transport Phagocytosis Vesicle
    • Spatial and temporal regulation of Actin filament remodeling When? How long? Where? Type of actin filaments
    • Remodeling of actin filaments New Filament Generations Depolymerizations/Severing Capping/DecappingVarious Actin Binding Proteins (ABP) mediate actin filament remodeling
    • Nucleation Seed Formation is the rate limiting step for actin polymerization Rapid Polymerization Rate Limiting Slow Slow FastStabilization of actin nucleation seed is critical for theRapid actin polymerizationsActin nucleators : Family of proteins stabilize actin nucleations
    • Each actin nucleator determine specific type of:59 Structure actin Annual Reviews AR131-19 REGULATION OF ACTIN FILAMENT NETWORK 651 Arp2/3 complex Formins Spire Branched actin filamentsure 1 Electron micrographs of branched actin filaments. (a) Electron micrograph of Straight actin bundlesleading edge of a migrating Xenopus keratocyte prepared by detergent extraction and in Lamellipodiary shadowing. (b) Detail of boxed region from (a), with a branched filament network In Stress fibers and filopodiaed in blue. (c) Gold-labeled antibody to ARPC2 localized to a branch point in thement network at the leading edge. (d ) Branch formed in vitro by pure actin filaments Acanthamoeba Arp2/3 complex. (a–c) modified from 14. (d ) Modified from 13.p2/3 complex (hereafter referred to as nucleation promoting factors), are being
    • Arp2/3 complex : 6 Protein complex makes branched networks Boczkowska et al., Structure 2008 Requires activator proteins called nucleation promoting factor (NPF)
    • Formin Otomo et al., Nature 2005Responsible for the straight bundles of filaments (Stress fiber, Fillopodia)Caps fast growing ends (barbed end) of actin filament and enhance elongation rates
    • New Class of Actin Nucleators:Tandem W domain containing actin nucleatorsOocyte maturation, Endocytosis (Drosophila, mouse, human)Brain specific actin nucleator (mouse, human)Vibrio parahemolyticus / Vibrio choleraRicketssia sp.
    • WASP-homology domain 2 (WH2):Common Actin binding motifs in actin nucleators and NPF ~25aa long conserved sequences Present in many actin Nucleation Promoting Factor (WASP, N-WASP, WAVE) and actin nucleators Binds hydrophobic cleft of actin Present in various Nucleation Promoting Factors And tandem WH2 domain containing nucleatorsActin -crosslinked first WH2 domain structureRebowski and Namgoong, J. Mol. Biol. 2010
    • Mechanism of tandem W domain containing nucleators Initial model of actin nucleation of spire (2005, Quinlan et al.,)Spire KIND WWW W S FYVE zinc-finger ‘Actin Template Model’ ubiquitin-L Pro-rich CC-? W W WCobl 152 229 325 425 560 600 1181 1337VopL W W W VCD 99 484Sca2 FH2-like ? P W W W CD 34 671 700 872 1020 1086 1544Similar Organization: More or less similar mechanism?
    • W domain containing nucleators is not created as equal..Nucleation mechanism of each protein will be different..
    • QuestionsMechanism of tandom W domain nucleators- Huge differences in nucleation activities suggest theDifferences innucleation mechanismIs Tandom WH2 domain sufficient for nucleationactivity?- Involvement of other domains in nucleation?
    • Vibrio parahemolyticus VopL as model system for actinNucleation mechanism- Encoded by bacteria Vibrio parahemolyticus (main agents for food poisoning)- It injects ~20 effector proteins into host cell using Type III secretion machinary (VopL is one of them)- Robust actin nucleation activity in vitro & in vivo- Actin Cytoskeleton is usual target for various effector proteins
    • Bacterial Type III Secretion System and EffectorsAnd host cell actin cytoskeleton
    • Listeria monocytogenesActin-based motility
    • Mapping of minimal actin nucleaton requirement in VopL 3W domains and C-terminal domain are both essential for actin nucleation
    • Actin polymerization assay using pyrene labeled actins(“Industry Standard” actin polymerization assay) Higher F-actin Activity Fluorescence Lower Activity Actin only G-actin Pyrene labeled actin at Cys-374 Time
    • Limitation of pyrene labeled actin assayGeneration of more nucleation seeds Enhancement of elongation speeds (Example : Arp2/3 Complex) (Example : Formins) Traditional fluorescence-based bulk assay cannot distinguish the difference between two
    • Single-Molecule Actin Polymerization Assay usingTotal Internal Reflection Fluorescence Microscopy (TIRFM) Oregon-Green Labeled Actin Myosin S1 head Laser excitation High resolution imaging sufficient for visualizing single actin filaments is possible..
    • Single molecule TIRFM assay confirms that VopL is genuine actin nucleators (more filament seeds, no change in elongation speed) Actin only 1.0 P-3W-VCD 31.0 W W W VCD 3W-VCD 27.0 W W W VCD VCD 1.0 VCDNamgoong et al., Nature Struct. Mol. Biol. 2011
    • Generation of more nucleation seeds Enhancement of elongation speeds
    • • C-terminal domain of VopL (VCD) is essential for the actin nucleation. (By its own it has negligible nucleation activity)• What is the functional roles of VCD in actin nucleation of VopL ?
    • Leucine-Zipper like coiled coil is predicted in C-terminalDoes VopL exist as a dimer?
    • VopL is dimerize by VCD and dimerization is essential for robust nucleation activity Multi angle light scattering-Size Pyrene-actin Polymerization Assay Exclusion chromatogrphy (SEC-MALS)But dimerization difficient mutants (3W-VCD*) still retains trace ofnucleation activityVCD is doing more than dimerization? Namgoong et al., Nature Struct. Mol. Biol. 2011
    • VCD can bind F-actin and 1W-VCD+Actin forms complex of 2:2
    • Two different binding mode of Actin nucleatorsArp2/3 complex stay on the „pointed end‟ (slow growing end) of actin filamentsFormins remains processively bound on „barbed end‟ (fast growing end)
    • QD FH2 P PYeast Formin (Bni1) labeled with Quantum dotFormin moves along with fast-growing end of actin filamentsPaul and Pollard, J.Biol. Chem. 2009
    • Bound and Stay Rapid Dissociation at the pointed end Nucleation Detachment Q Q D D W W W W W W W W W W W P PP W PActin Nucleations by VopL is initiated from the pointed end of actinFormed filaments is rapidly dissociated from VopL Namgoong et al., Nature Struct. Mol. Biol. 2011
    • VCD is globular domain Poly-Pro W-1 W-2 PP W-3 DD• No homologs in PDB Predicte d As Coiled-• Mostly alpha-helical Coil• C-terminal is predicted as coiled-coil
    • Crystallization of VCD VopL VopFCrystal is readily obtained, but poor diffractions..Crystal is very sensitive for the cryoprotection (weak diffraction 6-7Å)- Without any cryoprotections, diffraction is even poorer..- Most cryoprotectants (Glycerol, PEG400, Oil, Ethylene glycol, Sucrose etc) are not effective- Stepwise increase of cryoprotectant- Dihydrations somewhat improve diffraction in Native, but not in SeMet Crystal..- Room temp diffraction did not works..‘Flash dipping’ of crystal (less than 1 sec) in mother liquid + 15% PEG400
    • Tails Base ArmsArms Namgoong et al., Nature Struct. Mol. Biol. 2011
    • Namgoong et al., Nature Struct. Mol. Biol. 2011
    • Namgoong et al., Nature Struct. Mol. Biol. 2011
    • SAXS reconstitution of Dimerization Domains and actin+VCD complex Namgoong et al., Nature Struct. Mol. Biol. 2011
    • Model of actin binding with VopL VCD Namgoong et al., Nature Struct. Mol. Biol. 2011
    • Namgoong et al., Nature Struct. Mol. Biol. 2011
    • Dimerization of other W domain contains nucleators enhance nucleations Namgoong et al., Nature Struct. Mol. Biol. 2011
    • Dimerization of W containing nucleators are common strategies in different nucleators
    • ConclusionVopL utilizes unique VCD domain as a ‘platform’ foractin nucleationUnique ‘catalytic’ nature of actin nucleatorDimerization of actin nucleators are universal activationMechanism : actin filament has two strands, so dimericNucleator is needed. - Spire - Formin - arp2/3 complex
    • AcknowledgementsDominguez Lab in University of PennsylvaniaRoberto DominguezGrzegorz RebowskiBoczkowska MalgorzataUniversity of ChicagoMicheal GlistaDavid KovarAdvanced Photon Source (IMCA-CAT, 17-BM / Bio-CAT)CHESS A1
    • • SAD data collections at CHESS A1 - Fixed wavelength at 0.9772 - Relatively week diffractions (3.1-3.2A) - Radiation damages• Two datasets are collected from the Same crystal with different orientations (360 frames eachs)• merged and scaled using first 240 frames of two datasets
    • • Locate 6 Se atoms using SHELXD• Solvent flattenned experimental map• Identification of C-terminal coiled-coil• Build a initial model
    • Identification ofNoncrystallographicsymmetry from initialmodel and operators andmask generationsNCS averaged mapRefine with PHENIX usingsecondary structure/ NCSrestrainsFinal Refined 2Fo-Fc map