Abstract
Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is regulated by integrins. Upon activation, FAK generates signals that modulate crucial cell functions, including cell proliferation, migration, and survival. The C-terminal focal adhesion targeting (FAT) sequence mediates localization of FAK to discrete regions in the cell called focal adhesions. Several binding partners for the FAT domain of FAK have been identified, including paxillin. We have determined the solution structure of the avian FAT domain in complex with a peptide mimicking the LD2 motif of paxillin by NMR spectroscopy. The FAT domain retains a similar fold to that found in the unliganded form when complexed to the paxillin-derived LD2 peptide, an antiparallel four-helix bundle. However, noticeable conformational changes were observed upon the LD2 peptide binding, especially the position of helix 4. Multiple lines of evidence, including the results obtained from isothermal titration calorimetry, intermolecular nuclear Overhauser effects, mutagenesis, and protection from paramagnetic line broadening, support the existence of two distinct paxillin-binding sites on the opposite faces of the FAT domain. The structure of the FAT domain-LD2 complex was modeled using the program HADDOCK based on our solution structure of the LD2-bound FAT domain and mutagenesis data. Our model of the FAT domain-LD2 complex provides insight into the molecular basis of FAK-paxillin binding interactions, which will aid in understanding the role of paxillin in FAK targeting and signaling.
Highlights
Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is regulated by integrins
Multiple lines of evidence, including the results obtained from isothermal titration calorimetry, intermolecular nuclear Overhauser effects, mutagenesis, and protection from paramagnetic line broadening, support the existence of two distinct paxillin-binding sites on the opposite faces of the focal adhesion targeting (FAT) domain
The sequences within FAK responsible for localization have been mapped to ϳ140 residues near the C terminus, a region defined as the focal adhesion targeting (FAT) sequence or domain [19, 20]
Summary
A mutant of ␣4 that is defective for paxillin binding fails to promote cell motility This interaction is required for ␣41-dependent tyrosine phosphorylation of FAK and Pyk, and these two tyrosine kinases were implicated in regulating motility downstream of ␣41 [30, 31]. Evidence that the LD motif peptide was ␣-helical in solution was presented, other aspects of the interaction were different Their results indicated that the FAT domain contains a single LD motif-binding site at HP2 [40]. The peptide docked in the opposite orientation relative to the model of Hayashi et al [39], which was based on the crystal structure of the FAT domain These studies have provided important insight into the structure of the FAT domain, there are a number of unresolved issues, including the details of the interaction with paxillin. Our experimentally derived models of FAT domain-LD2 complexes provide important insight into the structural basis of paxillin recognition
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