BMB/Bi/Ch170 Proteins Lecture 7, Oct 17
• Microfilaments – Ac • Microtubules - green • Microfilaments - red • Intermediate filaments - yellow • Fibroblast cell Collagen Microtubule Microfilament Cellular movement driven by microfilaments Amoeba pseudopod driven by ac • Provide mechanical support for the cell – Shape – Movement – Junc Ocerbein et al Science (2001) 293:708-11 Looks like!? Ac disulfide crosslinking X-ray fiber diffr from F-ac Holmes et al Nature (1990) 347:44-9 Kudryashov et al PNAS (2005) 102:13105-10 pdb101 Near atomic F-ac Fujii, Iwane, Yanagida & Namba Nature (2010) 467:724-8 (3mfp) F-ac • Actomyosin – Rabbit F-ac Schröder et al Nature (1993) 364:171-4 Structural model of ac Myosin Head Mendelson & Morris PNAS (1997) 94:8533:8 Myosin in ac • Model from tomograms of insect flight muscle • In rigor (nucleo Example of class averages Chen et al J Struct Biol (2002) 138:92-104 (1m8q) DNaseI/Ac • First ac Holmes/Kabsch lab: Kabsch et al Nature (1990) 347:37-44 Ac Complex network branched at Arp junc Robinson et al Science (2001) 294:1679-84 (1k8k) Arp2/3 complex Arp3 p21 p34 p20 p16 • Bovine Arp2/3 Complex Arp2 (par6al) • 7 proteins (220 kD) • Arp 2 & 3 have ac Robinson et al Science (2001) 294:1679-84 (1k8k) Branching models • Side branching or Barbed-end branching Side model • Difference maps of labeled in vitro complex and EM • 10Å resolu Egile et al PLoS Biology (2005) 3:1902-9 Ac Waterman Lab, NIH Svitkina & Borisy J Cell Bio (1999) 145:1009-26 Gelsolin/Ac Domain 1 McLaughlin et al Nature (1993) 364:685-92 (1eqy) Gelsolin - an S6 S4 S2 ac S1 S5 S3 • Involved in F-ac Ac • Co-crystal structure with ac • PIP2 causes disassocia Schuc et al Nature (1993) 365:810-6 (2by) Listeria and ac • Listeria monocytogenes – causa Theriot lab, Stanford Listeria in Xenopus extract ActA s Theriot lab, Stanford MreB • mre (murein cluster e) operon • Locus determines cell shape • First demonstrated in B. sub7lis • Not found in round cells Jones et al Cell (2001) 104:913-22 Löwe lab; van den Ent et al Nature (2001) 413:39-44 MreB structure MreB1 from T. mari7ma Löwe lab; van den Ent et al Nature (2001) 413:39-44 (1jce) F-ac • MreB crystals contain proto-filaments (seen by EM) • Similar filament sta Löwe lab; Moeller-Jensen et al EMBO J (2002) 21:3119-27 Ent et al EMBO J (2002) 21:6935-43 ParM bacterial ac • Apo (1mwk), ADP (1mwm) • Forms similar structures to ac Review: Shih & Rothfield Micro Mol Bio Rev (2006) 70:729-54 Cordell & Löwe FEBS lecers (2001) 492:160-5 (1hyq) Microtubules • Tubulin-GFP • Early embryonic Drosophila cells • 24 nm in diameter Rosalind Silverman-Gavrila (U of Toronto) Greider et al Development (2000) 127:4253-64 MT provide cellular framework • “Dynamic instability” • Grow from centrosomes • Tubules fill cell to create cellular network • Motor proteins carry organelles – Kinesins to the plus end – Dyneins moves to the minus end Waterman lab (NIH) Review: Howard & Hyman Nature (2003) 4222:753-8 (+) β-tubulin α/β tubulin (1tub) • 55kD proteins • Both bind GTP - only β can hydrolyze • GTP form polymerizes, GDP falls apart • Dimer solved by electron crystallography of Zn stabilized sheets • Taxol – an<-cancer drug – 1963 from Pacific Yew – $1.6 bln in 2000 (-) • (+) and (-) ends α-tubulin Downing lab: Nogales et al Nature (1998) 393:191-202 Microtubule structure • 8Å cryo-EM model • Side contacts create extremely stable structure • GTP hydrolysis induces curve Downing lab: Li et al Structure (2002) 10:1317-28 Model for tubule forma • Dynamic instability comprises cycles of growth and polymeriza Review: Howard & Hyman Nature (2003) 4222:753-8 Motor Proteins • Kinesins (+) end motors – KIF1A - Monomeric kinesin motor – ATP state primed for (+) end mo Kikkawa et al Nature (2001) 411:439-45 Dyneins Motor Domains • Move along microtubules • Roles – cilia/flagella Myosin V – cargo and mito • Processive Kinesin-1 Dynein Carter (2013) J Cell Sci 126:705 (Review) Dynein structure Andrew Carter lab, LMB Cambridge Schmidt, Gleave & Carter (2012) NSMB 19:492 Many proteins involved in regula • RB3-SLD (stathmin like domain) sequesters pairs of dimers • Prevents addi Ravelli et al Nature (2004) 428:198-202 (1sa0) γ−tubulin Nuclea Agard lab: Aldaz et al Nature (2005) 435:523-7 (1z5w) Moritz et al Nature Cell Bio (2000) 2:365-70 Bacterial homologues - FtsZ • Localizes to septa of dividing cell • Forms rings that contract • In vitro forms asters – Dependent on Ca2+ and GTP Sun & Margolin J Bacteriology (1998) 180:2050-6 Yu & Margolin EMBO J (1997) 16:5455-63 FtsZ structure • First solved as monomer from M. jannaschii • FtsZ dimer with MgGTP complex Löwe & Amos Nature (1998) 391:203-6 (monomer 1fsz) Oliva et al Nat Struc Mol Biol (2004) 11:1243-50 (1w5a) Bacterial tubulin • BtubA/B • Found in Prosthecobacter only • Horizontal gene transfer? • B contains GDP/ A is empty • 2btq Löwe lab: Schlieper et al PNAS (2005) 102:9170-5 1tub 2btq 1w5a Tubulin Mitchie & Löwe Ann Rev Biochemistry (2006) 75:467-92 family Intermediate filaments • Eukaryo • MT (red) • IFs (blue) • Plec • Bacterial IF homologue from Caulobacter crescentus • Needed for crescent shape Ausmees et al Cell (2003) 705-13 Clathrin coated vesicles • Endocytosis of receptors • Binding of adaptor proteins to cellular receptors causes clathrin binding • Series of EM stained images Clathrin lattice Trimers form icosahedral lattice Kirchhausen lab, HMS Clathrin structure • Complete lattice from cryo- EM (8Å) • Smallest lattice from 28, 36, 60 triskelions • Light and heavy chains Fotin et al Nature (2004) 432:573-9 (1xi4) Auxilin uncoating • Auxilin (a J-domain protein) localizes Hsc70 • Hsc70 catalyzes global disassembly • 12Å cryo-EM model Fotin et al Nature (2004) 432:649-53 (1xi5)