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.
Highlights
Integrin adhesion receptors are an essential class of cell surface glycoproteins that mediate cell adhesion, migration and spreading by linking the extracellular matrix with the actin cytoskeleton
His-tagged PINCH1-LIM1, which is soluble in E. coli, was expressed alone, and cells mixed with the integrin linked kinase (ILK)/a-parvin-CH2 expressing cells prior to co-sonication
Remaining GST protein was removed by incubation with glutathione-agarose beads and size-exclusion chromatography to achieve purity of crystallization quality, and immunoblot analysis confirms the presence of ILK in the complex (Figure 1D)
Summary
Integrin adhesion receptors are an essential class of cell surface glycoproteins that mediate cell adhesion, migration and spreading by linking the extracellular matrix with the actin cytoskeleton. In part, by the binding of adaptor and signaling proteins to the short integrin cytoplasmic tails. Once recruited, these proteins convert integrins to their high-affinity/ active conformations, which in turn triggers cellular responses to cell adhesion such as cell migration, differentiation and survival [1]. An important cytoplasmic component localized to integrin receptors at focal adhesions is the heterotrimeric protein complex comprised of the integrin linked kinase (ILK), parvin, and PINCH, termed the IPP complex for its member proteins. ILK was first identified as an integrin b1 cytoplasmic tail binding protein [3], and is the central member of the IPP complex. It was originally reported that ILK contains a short pleckstrin homology (PH) domain (residues 180–212) between the ARD and pKD regions [14]; subsequent structural studies revealed that the majority of this segment (residues 185–212) is integral to the pseudokinase fold [9]
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