PDBsum entry 1y19

Structural protein, signaling protein

PDB id: 1y19

Contents

Protein chains

(+ 0 more) 11 a.a.

(+ 0 more) 192 a.a.

Waters ×466

References listed in PDB file

Key reference

Title

Structural basis for phosphatidylinositol phosphate kinase type igamma binding to talin at focal adhesions.

Authors

J.M.De pereda, K.L.Wegener, E.Santelli, N.Bate, M.H.Ginsberg, D.R.Critchley, I.D.Campbell, R.C.Liddington.

Ref.

J Biol Chem, 2005, 280, 8381-8386. [DOI no: 10.1074/jbc.M413180200]

PubMed id

15623515

Abstract

The cytoskeletal protein talin binds to a short C-terminal sequence in phosphatidylinositol phosphate kinase type Igamma (PIPKIgamma), activating the enzyme and promoting the local production of phosphatidylinositol 4,5 bisphosphate, which regulates focal adhesion dynamics as well as clathrin-mediated endocytosis in neuronal cells. Here we show by crystallographic, NMR, and calorimetric analysis that the phosphotyrosine binding (PTB)-like domain of talin engages the PIPKIgamma C terminus in a mode very similar to that of integrin binding. However, PIPKIgamma binds in the canonical PTB-peptide mode with an SPLH motif replacing the classic NPXY motif. The tighter packing of the SPLH motif against the hydrophobic core of talin may explain the stronger binding of PIPKIgamma. Two tyrosine residues flanking the SPLH motif (Tyr-644 and Tyr-649) have been implicated in the regulation of talin binding. We show that phosphorylation at Tyr-644, a Src phosphorylation site in vivo, has little effect on the binding mode or strength, which is consistent with modeling studies in which the phosphotyrosine makes surface-exposed salt bridges, and we suggest that its strong activating effect arises from the release of autoinhibitory restraints in the full-length PIPKIgamma. Modeling studies suggest that phosphorylation of Tyr-649 will likewise have little effect on talin binding, whereas phosphorylation of the SPLH serine is predicted to be strongly disruptive. Our data are consistent with the proposal that Src activity promotes a switch from integrin binding to PIPKIgamma binding that regulates focal adhesion turnover.

Figure 1.
FIG. 1. Crystal structure of the talin FERM F3-PIPKI interaction. A, surface representation of talin FERM F3 domain colored by electrostatic potential (red for -6 kT/e and blue for 6 kT/e) with the PIPKI ligand, residues 641-649, shown as sticks. Trp-642 (W642) in the PIPKI sequence (which is critical for binding) binds into a deep pocket on the surface of F3. The SPLH sequence adopts the same reverse-turn conformation as the classic NPXY motif. Thus, serine plays a similar role as that of asparagine, forming an N-cap to the reverse turn; the proline promotes the reverse turn, the leucine packs against the hydrophobic surface of F3, and the histidine takes the place of the tyrosine or phenylalanine found in the integrins, packing against a flat somewhat acidic surface. B, comparison of PIPKI (yellow) and integrin 3 (light green) sequences bound to talin FERM F3 (ribbon representation in blue). The 3 structure was superposed by aligning the conserved C atoms of the talin FERM F3 domain of the talin-PIPKI and talin- 3 complexes. PIPKI and 3 residues are labeled in red and green respectively. C, detailed stereo view of the talin-PIPKI interaction. Talin residues involved in the interaction are shown in gray, and the PIPKI ligand is shown in yellow. Intramolecular and intermolecular H-bonds are shown as dotted lines. Surface electrostatic potential was calculated with the program APBS (30). Molecular representation figures were generated with the programs MOLSCRIPT (31, 32), RASTER3D (32), and PyMol (http://www.pymol.org). Single letter amino acid abbreviations are used with position numbers.

Figure 2.
FIG. 2. Interaction of the talin F3 domain with PIPKI and -integrin peptides. A-C, chemical shift perturbation maps of the interaction of U-15N-labeled talin F3 with the 3-integrin tail peptide RAKWDTANNPLYKE (A), the PIPKPI peptide PTDERSWVYSPLHYSAR (B), and the same PIPKPI peptide phosphorylated at Tyr-644 (C). (HN,N) refers to the combined HN and N chemical shift changes, according to the equation (HN,N) = (( [HN]W[HN])2 + ( [N]W[N])2)0.5, where W[HN] and W[N] are weighting factors for the HN and N shifts respectively (W[HN] = 1, W[N] = 0.154) (33) and = [bound] - [free]. Full chemical shift data are given in the supplementary data available in the on-line version of this article. Secondary structure elements are indicated. D, NMR titration of 100 µM U-15N-labeled talin F3 with increasing concentrations of 3-integrin peptide. Overlays of the same region of successive 1H-15N HSQC spectra are shown (some titration points omitted for clarity). Black, magenta, blue, cyan, green, yellow, orange, and red peaks correspond to concentrations of 0, 0.8, 1.2, 1.6, 3.0, 6.5, 9, and 14 mM peptide. E, the location of the combined 1H and 15N chemical shift changes due to the unphosphorylated PIPKPI peptide mapped onto the crystal structure of the talin-PIPKI chimera. Small-to-large changes are indicated by the blue-to-red spectral gradation. The PIPKI peptide is shown in magenta.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2005, 280, 8381-8386) copyright 2005.