Abstract
Nck proteins are essential Src homology (SH) 2 and SH3 domain-bearing adapters that modulate actin cytoskeleton dynamics by linking proline-rich effector molecules to tyrosine kinases or phosphorylated signaling intermediates. Two mammalian pathogens, enteropathogenic Escherichia coli and vaccinia virus, exploit Nck as part of their infection strategy. Conflicting data indicate potential differences in the recognition specificities of the SH2 domains of the isoproteins Nck1 (Nckalpha) and Nck2 (Nckbeta and Grb4). We have characterized the binding specificities of both SH2 domains and find them to be essentially indistinguishable. Crystal structures of both domains in complex with phosphopeptides derived from the enteropathogenic E. coli protein Tir concur in identifying highly conserved, specific recognition of the phosphopeptide. Differential peptide recognition can therefore not account for the preference of either Nck in particular signaling pathways. Binding studies using sequentially mutated, high affinity phosphopeptides establish the sequence variability tolerated in peptide recognition. Based on this binding motif, we identify potential new binding partners of Nck1 and Nck2 and confirm this experimentally for the Arf-GAP GIT1.
Highlights
(1) indicate that the function of the proteins may substantially overlap
Nck1 and Nck2 have both been implicated in the infection process of enteropathogenic Escherichia coli (EPEC) [11], a frequent cause of severe infant diarrhea [12]
By surface plasmon resonance (SPR) spectroscopy, we identify a translocated intimin receptor” (Tir)-derived phosphopeptide as the strongest natural ligand of both Nck1 and Nck2 SH2 domains
Summary
Production of GST Fusion Proteins—The cDNA for Nck and Grb proteins was a kind gift from Michael Way (Cancer Research UK, Lincoln’s Inn Fields Laboratories, London, UK) and Ottmar Janssen (Institute of Immunology, Kiel, Germany). The GST/Nck-SH2 fusion protein was eluted using 10 mM glutathione in PBS (Nck1) or 20 mM Tris/HCl, pH 8.0, 200 mM NaCl (Nck2) and either used as such in peptide overlay studies or cleaved using PreScission protease (Amersham Biosciences). Nck1-SH2:Tir, where Tir is a 12-residue, chemically synthesized peptide derived from the EPEC-protein Tir. The complex was crystallized in 2.4 M (NH4)2HPO4, 0.1 M Tris, pH 8.5; Nck2-SH2/Tir in 50% MPD, 15% ethanol, and 0.01 M Na acetate. Structures were solved by molecular replacement using EPMR [26] and Grb as a search model for Nck1-SH2 and the latter for both protein-peptide complexes. The sensorgram data were quantified by plotting the resonance units at equilibrium (Req) against the protein concentration.
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