PDBsum entry 1nyp

Cell adhesion

PDB id

1nyp

Contents

			Protein chain

					66 a.a. *

			Metals

					_ZN ×2

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Structural and functional insights into pinch lim4 domain-Mediated integrin signaling.

Authors

A.Velyvis, J.Vaynberg, Y.Yang, O.Vinogradova, Y.Zhang, C.Wu, J.Qin.

Ref.

Nat Struct Biol, 2003, 10, 558-564. [DOI no: 10.1038/nsb938]

PubMed id

12794636

Abstract

PINCH is an adaptor protein found in focal adhesions, large cellular complexes that link extracellular matrix to the actin cytoskeleton. PINCH, which contains an array of five LIM domains, has been implicated as a platform for multiple protein-protein interactions that mediate integrin signaling within focal adhesions. We had previously characterized the LIM1 domain of PINCH, which functions in focal adhesions by binding specifically to integrin-linked kinase. Using NMR spectroscopy, we show here that the PINCH LIM4 domain, while maintaining the conserved LIM scaffold, recognizes the third SH3 domain of another adaptor protein, Nck2 (also called Nckbeta or Grb4), in a manner distinct from that of the LIM1 domain. Point mutation of LIM residues in the SH3-binding interface disrupted LIM-SH3 interaction and substantially impaired localization of PINCH to focal adhesions. These data provide novel structural insight into LIM domain-mediated protein-protein recognition and demonstrate that the PINCH-Nck2 interaction is an important component of the focal adhesion assembly during integrin signaling.



	Figure 1. Figure 1. Structural properties of LIM4 and its sequence and structural comparisons with other LIM domains. (a) Primary sequence comparison of PINCH LIM domains. Residues in LIM1 and LIM4 that are perturbed in HSQC spectra of free versus bound protein (>100 Hz in LIM1 and >10 Hz in LIM4) are highlighted in green. Zinc-coordinating residues are shown in red. Symbols at the top indicate secondary structure elements in LIM4. (b) Stereo view of best-fit superposition of 20 structures of LIM4 with the least NOE violations. (c) Zn2+ coordination. In the top, Cys193, Cys196, His213 and His216 coordinate Zn2+ in the N-terminal finger. In the bottom, Cys219, Cys222, Cys240 and His243 coordinate Zn2+ in the C-terminal finger. (d) C 2 planes for His213, His216 and His243 (left to right) in 3D 15N,13C-edited NOESY spectrum showing NOE peaks (labels at right) of imidazole ring H 2 atoms that unambiguously establish ring orientations for Zn2+-coordinating histidines. (e) Best-fit superposition of minimized average LIM4 (blue) with CRIP (red), showing the relative twist between N- and C-terminal Zn-fingers in CRIP and absence thereof in LIM4. Red spheres represent Zn2+ ions in LIM4. The orientation is a 90� rotation about vertical axis compared with b.		Figure 4. Figure 4. R197A-R198A double mutation reduces the efficiency of PINCH localization to focal adhesions. ILK binding as analyzed by western blotting with (a) HRP-conjugated anti-GFP and (b) mouse monoclonal anti-ILK 65.1. Lysates (8 g per lane) of C2C12 cells expressing GFP-FLAG -tagged PINCH, the GFP-FLAG -tagged PINCH mutant with the R197A-R198A mutation (PINCHm) or GFP alone (control) were mixed with rabbit polyclonal antibodies to GFP (Clontech) and then immunoprecipitated as indicated. (c -f) Subcellular localization. C2C12 cells expressing GFP-FLAG-PINCHm (c and d) or GFP-FLAG-PINCH (e and f) were plated on fibronectin-coated coverslips and stained with mouse monoclonal antibody to paxillin (a marker of focal adhesions; clone 349, Transduction Laboratories) and rhodamine-conjugated anti-mouse IgG. GFP-FLAG-PINCHm (c), GFP-FLAG-PINCH (e) and paxillin (d and f) were visualized under a fluorescence microscope equipped with GFP (c and e) and rhodamine (d and f) filters. Bar, 10 m. Although the GFP-FLAG-PINCH mutant and GFP-FLAG-PINCH proteins bind to ILK equally well, the GFP-FLAG-PINCH mutant localized to focal adhesions much less efficiently than GFP-FLAG-PINCH.

PROCHECK

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