Purification and SAXS analysis of the integrin linked kinase, PINCH, parvin (IPP) heterotrimeric complex

• Published in: PloS one 2013-01, Vol.8 (1), p.e55591-e55591
• Main Authors: Stiegler, Amy L, Grant, Thomas D, Luft, Joseph R, Calderwood, David A, Snell, Edward H, Boggon, Titus J
• Format: Article
• Language: English
• Subjects: Adaptor Proteins, Signal Transducing - chemistry; Adaptor Proteins, Signal Transducing - genetics; Analysis; BASIC BIOLOGICAL SCIENCES; Biology; Cell adhesion & migration; crystal structure; Crystallography; Cytoskeletal Proteins - chemistry; Cytoskeletal Proteins - genetics; E coli; Elongated structure; elution; Flexibility; focal adhesions; Gel filtration; Growth factors; Humans; ILK protein; Integrins; Kinases; LIM Domain Proteins - chemistry; LIM Domain Proteins - genetics; Localization; Medical research; Medicine; Membrane Proteins - chemistry; Membrane Proteins - genetics; Models, Molecular; Molecular biology; Multiprotein Complexes - chemistry; Multiprotein Complexes - isolation & purification; Optimization; Pharmacology; Polypropylene; Protein Binding; protein complexes; Protein Conformation; Protein Interaction Domains and Motifs; Protein-Serine-Threonine Kinases - chemistry; Protein-Serine-Threonine Kinases - genetics; Proteins; proteolysis; Purification; Receptors; Scattering, Small Angle; serine proteases; Signaling; Small angle X ray scattering; small-angle scattering; Structural analysis; X-Ray Diffraction; X-ray scattering
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0055591

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.