Molecular Recognition of Paxillin LD Motifs by the Focal Adhesion Targeting Domain

Abstract

Focal adhesions (FAs) are large submembrane signaling complexes formed at sites of cellular attachment to the extracellular matrix. The interaction of LD motifs with their targets plays an important role in the assembly of FAs. We have determined the molecular basis for the recognition of two paxillin LD motifs by the FA targeting (FAT) domain of FA kinase using a combination of X-ray crystallography, solution NMR, and homology modeling. The four-helix FAT domain displays two LD binding sites on opposite sites of the molecule that bind LD peptides in a helical conformation. Threading studies suggest that the LD-interacting domain of p95PKL shares a common four-helical core with the FAT domain and the tail of vinculin, defining a structural family of LD motif binding modules.

SLK-mediated phosphorylation of paxillin is required for focal adhesion turnover and cell migration

“…In addition to being required for SLK-mediated phosphorylation, the altered conformation of paxillin resulting from the mutation of serines 243 and 244 to glycines would suggest that LD3 is important in maintaining the integrity of the tertiary structure of the protein. Although the crystal structures of paxillin motifs LD2 and LD4 have been resolved, the crystal structure of the entire protein has yet to be determined [103,158,159]. Consequently, it is difficult to draw any definitive conclusions about the paxillin structure from these results alone.…”

GIT1 utilizes a focal adhesion targeting-homology domain to bind paxillin

“…In FAK, the extreme carboxyl terminal region is known as the "focal adhesion targeting" (FAT) domain, since this is the paxillin and talin binding site and this domain is required for focal adhesion localization [19]. The structure of the FAT domain of FAK, both alone or bound to paxillin LD motif peptides, has been determined [21][22][23][24][25]. The FAT sequence folds to form a single structural domain comprised of a bundle of four anti-parallel alpha helices.…”

“…The FAT domain contains two paxillin LD motif binding sites, one between helices 1 and 4, and a second on the opposite side between helices 2 and 3 [24]. In vitro, the paxillin LD2 and LD4 peptides each bind to either site, although a preference of LD2 for site 1/4 and LD4 for site 2/3 has been observed [24][25][26]. Thus, it is likely that one molecule of FAK binds to both the LD2 and LD4 motifs of one paxillin molecule at the same time.…”

“…While residues 643-679 had been proposed to form a paxillin binding sequence (PBS2) in chicken GIT2, and deletion of residues 643-679 in chicken GIT2 leads to a protein that no longer binds to a paxillin LD4-GST fusion protein in vitro [15], the ~75 residues beyond this PBS2 region to the end of the protein have no known function in any GIT family member. Based on the over 20 % sequence identity (50 % similarity) with the FAK focal adhesion targeting (FAT) domain, we have recently predicted that the entire 130 residue carboxyl terminal region of chicken GIT2 folds into a 4-helix bundle to form a paxillin binding domain analogous to the FAT domain of FAK [24].We reasoned that if the entire carboxyl terminal folds to form a compact 4-helix bundle FAT Homology (FAH) domain, the residues beyond the previously described PBS2 should be critical to form the functional paxillin binding site. On the other hand, if the predicted PBS2 region alone were sufficient for paxillin binding, removal of these final 75 residues may have little or no effect on paxillin binding.…”

FERM Domain Interaction Promotes FAK Signaling

“…The C-terminal domain can be further subdivided into the focal adhesion targeting (FAT) sequence, comprising the Cterminal 140 amino acids of the protein, and the region between the catalytic domain and the FAT sequence. Focal adhesion-associated FAT sequence binding partners have been identified, and insight into the molecular basis of FAT sequence function was recently obtained from crystal and nuclear magnetic resonance structure analyses (3,14,23,26,31). The sequence between the catalytic domain and the FAT sequence contains docking sites for SH3 domain-containing proteins and thus serves as a scaffold for the recruitment of signaling proteins.…”