GIT1 paxillin-binding domain is a four-helix bundle, and it binds to both paxillin LD2 and LD4 motifs

Abstract

The G protein-coupled receptor kinase-interacting protein 1 (GIT1) is a multidomain protein that plays an important role in cell adhesion, motility, cytoskeletal remodeling, and membrane trafficking. GIT1 mediates the localization of the p21-activated kinase (PAK) and PAK-interactive exchange factor to focal adhesions, and its activation is regulated by the interaction between its C-terminal paxillin-binding domain (PBD) and the LD motifs of paxillin. In this study, we determined the solution structure of rat GIT1 PBD by NMR spectroscopy. The PBD folds into a four-helix bundle, which is structurally similar to the focal adhesion targeting and vinculin tail domains. Previous studies showed that GIT1 interacts with paxillin through the LD4 motif. Here, we demonstrated that in addition to the LD4 motif, the GIT1 PBD can also bind to the paxillin LD2 motif, and both LD2 and LD4 motifs competitively target the same site on the PBD surface. We also revealed that paxillin Ser(272) phosphorylation does not influence GIT1 PBD binding in vitro. These results are in agreement with the notion that phosphorylation of paxillin Ser(272) plays an essential role in regulating focal adhesion turnover.

FIGURE 1.

Structure of the PBD of rat GIT1. a, sequence alignment based on structures of the rat GIT1 PBD, the chicken FAK FAT domain (Protein Data Bank code 1KTM), and H2 to H5 of the chicken Vt domain (code 1QKR). The sequences of human GIT2, FAK2/PYK2, and vinculin are also aligned based on the structure-based sequence alignment. Residues that are significantly perturbed by the binding of LD peptides are indicated with red dots. The hydrophobic core is composed of residues marked by filled diamonds. Hydrophobic residues are marked by filled diamonds, and the two polar Ts are marked by open diamonds. The absolutely conserved residues are highlighted in yellow. b, stereo view of the backbone trace of the ensemble of the 20 best structures. The side chain of the hydrophobic core is shown. c, ribbon representation of the GIT1 PBD structure. H1 to H4 are shown in blue, green, yellow, and red, respectively. Side chains of the interlaced core are also presented. d, representation of the H/D exchange experiments. The unambiguously observed non-proline amide proton atoms are represented as small balls in the structure. After the first 15 min, the solvent-exchanged amide proton atoms are colored in blue, whereas the protected amide protons are colored in yellow. The atoms that remain protected after 380 min are colored in red.

FIGURE 2.

Electrostatic surfaces of PBD and FAT domains. Positive charge is shown in blue and negative charge in red. Equivalent residues are marked in both structures. The residues that exhibited the most significant perturbations during LD peptide titrations are labeled on the GIT1 H1/H4 surface. The figure was generated by Pymol, based on the electrostatic potential generated by GRASP.

FIGURE 3.

The interaction between the PBD and LD peptides. a and b, overlay of GIT1 PBD spectra titrated with LD4 and LD2 peptides, respectively. Blue, GIT1 PBD alone; cyan, adding 0.5 eq of ligand; green, adding 1 eq of ligand; gold, adding 2 eq of ligand; red, adding 5 eq of ligand. Arrows mark significantly perturbed resonance, and dashed arrows mark perturbation with intermediate exchange rate. c, sequence alignment of human LD2 and LD4 peptides used in this study. Identical residues are marked with asterisks, and the LDXLLXXL motif is shown in bold. Conventional nomenclature is used; the first Leu is Φ0, and other residues are named as in the figure. d and e, composite chemical shift perturbation plotted against residue number for LD4 and LD2, respectively. Composite chemical shift perturbation was calculated by the equation Δδ_composite = (Δδ_NH² + Δδ_N²/25)^1/2. Lys⁶⁶³, Lys⁷⁵⁸, Gln, and Tyr⁷⁵¹ are labeled, and they are among those dramatically shifted peaks.