title: Purification and SAXS analysis of the integrin linked kinase, PINCH, parvin (IPP) heterotrimeric complex.
abstract: The heterotrimeric protein complex containing the integrin linked kinase (ILK), parvin, and PINCH proteins, termed the IPP complex, is an essential component of focal adhesions, where it interacts with many proteins to mediate signaling from integrin adhesion receptors. Here we conduct a biochemical and structural analysis of the minimal IPP complex, comprising full-length human ILK, the LIM1 domain of PINCH1, and the CH2 domain of α-parvin. We provide a detailed purification protocol for IPP and show that the purified IPP complex is stable and monodisperse in solution. Using small-angle X-ray scattering (SAXS), we also conduct the first structural characterization of IPP, which reveals an elongated shape with dimensions 120×60×40 Å. Flexibility analysis using the ensemble optimization method (EOM) is consistent with an IPP complex structure with limited flexibility, raising the possibility that inter-domain interactions exist. However, our studies suggest that the inter-domain linker in ILK is accessible and we detect no inter-domain contacts by gel filtration analysis. This study provides a structural foundation to understand the conformational restraints that govern the IPP complex.
authors: Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors: Amy L
authors: AL
authors: Stiegler
authors: Thomas D
authors: TD
authors: Grant
authors: Joseph R
authors: JR
authors: Luft
authors: David A
authors: DA
authors: Calderwood
authors: Edward H
authors: EH
authors: Snell
authors: Titus J
authors: TJ
authors: Boggon
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D060588:LIM Domain Proteins / Q000737:chemistry* / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000302:isolation & purification; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction; D000099200:Scaffold Protein ILK
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D003598:Cytoskeletal Proteins; D060588:LIM Domain Proteins; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D017346:Protein Serine-Threonine Kinases; D000099200:Scaffold Protein ILK; C118205:IPP protein, human; C088088:LIMS1 protein, human
references: Structure. 2003 Nov;11(11):1453-9
references: 14604535
references: J Mol Biol. 2008 Feb 15;376(2):492-505
references: 18164031
references: Genes Dev. 2003 Apr 1;17(7):926-40
references: 12670870
references: J Mol Biol. 1999 Sep 17;292(2):195-202
references: 10493868
references: Biophys J. 1999 Jun;76(6):2879-86
references: 10354416
references: J Cell Sci. 2009 Jan 15;122(Pt 2):159-63
references: 19118207
references: Mol Cell Biol. 1999 Mar;19(3):2425-34
references: 10022929
references: J Appl Crystallogr. 2009 Apr 1;42(Pt 2):342-346
references: 27630371
references: J Cell Sci. 2005 Dec 15;118(Pt 24):5899-910
references: 16317048
references: J Biol Chem. 2002 Oct 11;277(41):38328-38
references: 12167643
references: J Biol Chem. 2012 Sep 21;287(39):32566-77
references: 22869380
references: J Cell Sci. 2012 Jul 1;125(Pt 13):3221-32
references: 22454516
references: J Biol Chem. 2011 Jun 17;286(24):21886-95
references: 21524996
references: Nucleic Acids Res. 2007 Jul;35(Web Server issue):W460-4
references: 17567614
references: J Struct Biol. 2010 Apr;170(1):157-63
references: 19963065
references: Nat Rev Mol Cell Biol. 2006 Jan;7(1):20-31
references: 16493410
references: Biopolymers. 2011 Aug;95(8):559-71
references: 21509745
references: J Cell Biol. 2000 Dec 25;151(7):1435-48
references: 11134073
references: J Biol Chem. 2004 Oct 1;279(40):41695-705
references: 15284246
references: J Cell Sci. 2001 Feb;114(Pt 3):525-38
references: 11171322
references: J Biol Chem. 2009 Feb 27;284(9):5836-44
references: 19117955
references: J Mol Biol. 2004 Mar 26;337(3):635-45
references: 15019783
references: J Cell Sci. 2002 Dec 15;115(Pt 24):4777-86
references: 12432066
references: Proc Natl Acad Sci U S A. 2008 Dec 30;105(52):20677-82
references: 19074270
references: J Am Chem Soc. 2007 May 2;129(17):5656-64
references: 17411046
references: J Cell Sci. 2008 Oct 1;121(Pt 19):3121-32
references: 18799788
references: J Cell Biol. 2001 Apr 30;153(3):585-98
references: 11331308
references: Nat Rev Cancer. 2005 Jan;5(1):51-63
references: 15630415
references: Proc Natl Acad Sci U S A. 1998 Sep 15;95(19):11211-6
references: 9736715
references: J Immunol. 2006 Mar 15;176(6):3611-24
references: 16517730
references: Proc Natl Acad Sci U S A. 2007 Apr 17;104(16):6782-7
references: 17420447
references: Nature. 1996 Jan 4;379(6560):91-6
references: 8538749
references: EMBO J. 2009 Oct 21;28(20):3132-44
references: 19798050
references: Mol Cell. 2009 Dec 11;36(5):819-30
references: 20005845
references: Genes Dev. 2008 May 15;22(10):1325-30
references: 18483218
references: J Biol Chem. 2003 Dec 19;278(51):51324-33
references: 14551191
references: J Struct Biol. 2010 Jan;169(1):45-53
references: 19723583
references: Curr Biol. 2002 May 14;12(10):787-97
references: 12015115
references: J Cell Biol. 2001 Jun 11;153(6):1251-64
references: 11402068
references: J Cell Sci. 2005 Jul 1;118(Pt 13):2913-21
references: 15976450
references: Biophys J. 2005 Aug;89(2):1237-50
references: 15923225
references: J Cell Sci. 2009 Jun 1;122(Pt 11):1800-11
references: 19435803
references: Mol Cell Biol. 2005 Apr;25(8):3056-62
references: 15798193
references: J Biol Chem. 2001 Feb 16;276(7):4932-9
references: 11078733
references: Mol Biol Cell. 1998 Dec;9(12):3367-82
references: 9843575