A structural mechanism for spatial and temporal localization of phosphatidylinositol-4,5-bisphosphate into focal adhesions and NMR technique improvement

Phosphatidylinositol-4,5-bisphosphate (PIP2) controls a myriad of diverse cellular processes by recruiting and/or modulating cytosolic proteins. To ensure specificity in disparate cellular events, PIP2 must be localized to specific subcellular sites. At integrin-mediated focal adhesion (FA) sites, this localization is mediated, at least in part, via the recruitment and activation of tyrosine-phosphorylated PIP2-producing enzyme, type Igamma phosphatidylinositol phosphate kinase (PIPKIgamma) by a phosphortyrosine binding (PTB)-like domain of PIP2-binding protein, talin. We report the solution structure of talin-PTB domain in complex with a tyrosine-phosphorylated PIPKIgamma fragment providing atomic insight into the specific PIPKIgamma-talin interaction and a novel phosphotyrosine recognition mode for talin-PTB. We further show that PIPKIgamma and its product, PIP2, compete for binding to an overlapping site on talin-PTB indicating a self-limiting step for the temporal control of PIP2 level in FAs. Also, the competition of integrin-beta with PIP2 for the binding site on talin-PTB leads us to suggest the PIP2 role in talin assembling into FA and its possible role in further integrin activation. Based on competition experiments and mutagenesis studies, we have proposed a binding mode of PIP2 on talin-PTB. These findings define a structural mechanism for spatial and temporal generation of PIP2 that specifically targets talin and others for regulating integrin signaling and FA assembly/disassembly.; Intermolecular NOEs from free form ligand can be detected in intermolecular NOE experiments for macromolecule complexes under slow exchange condition with binding affinity about from several muM to 40nM. A detailed theoretical discussion has been given for the condition. Significant sensitivity could be gained when intermolecular NOEs from the free ligand are detected due to long transverse relaxation time of the free form ligand during filtering period. The lowest binding affinity to be able to detect intermolecular NOE is about in the same order of ligand concentration added. Experiment is demonstrated with a 13C/15N labeled talin-PTB bound with phosphorylated PIPKIgamma peptide.; The original experiment for measuring cross-correlation spin relaxation for the determination of protein backbone ϕ dihedral angle has been improved by the implementation of the TROSY approach. Resolution gain is achieved by splitting the four peaks into two sets of two peaks. Also significantly sensitivity gain could be achieved due to the TROSY effect during the long magnetization transfer period, especially when the experiment was applied to larger protein under higher magnetic field. The ratio measured with the proposed pulse scheme shows a good agreement with that obtained with the original experiment.