Determination of the solution structure of the SH3 domain of human p56 Lck tyrosine kinase

Summary

The solution structure of the SH3 domain of human p56 Lck tyrosine kinase (Lck-SH3) has been determined by multidimensional heteronuclear NMR spectroscopy. The structure was calculated from a total of 935 experimental restraints comprising 785 distance restraints derived from 1017 assigned NOE cross peaks and 150 dihedral angle restraints derived from 160 vicinal coupling constants. A novel combination of the constant-time ¹H−¹³C NMR correlation experiment recorded with various delays of the constant-time refocusing delays and a fractionally ¹³C-labelled sample was exploited for the stereo-specific assignment of prochiral methyl groups. Additionally, 28 restraints of 14 identified hydrogen bonds were included. A family of 25 conformers was selected to characterize the solution structure. The average root-mean-square deviations of the backbone atoms (N, C^α, C′, O) among the 25 conformers is 0.42 Å for residues 7 to 63. The N- and C-terminal residues, 1 to 6 and 64 to 81, are disordered, while the well-converged residues 7 to 63 correspond to the conserved sequences of other SH3 domains. The topology of the SH3 structure comprises five anti-parallel β-strands arranged to form two perpendicular β-sheets, which are concave and twisted in the middle part. The overall secondary structure and the backbone conformation of the core β-strands are almost identical to the X-ray structure of the fragment containing the SH2-SH3 domains of p56 Lck [Eck et al. (1994) Nature, 368, 764–769]. The X-ray structure of the SH3 domain in the tandem SH2-SH3 fragment is spatially included within the ensemble of the 25 NMR conformers, except for the segment of residues 14 to 18, which makes intermolecular contacts with an adjacent SH2 molecule and the phosphopeptide ligand in the crystal lattice. Local structural differences from other known SH3 domains are also observed, the most prominent of which is the absence in Lck-SH3 of the two additional short β-strands in the regions Ser¹⁵ to Glu¹⁷ and Gly²⁵ to Glu²⁷ flanking the so-called ‘RT-Src’ loop. This loop (residues Glu¹⁷ to Leu²⁴), together with the ‘n-Src’ loop (residues Gln³⁷ to Ser⁴⁶) confines the ligand interaction site which is formed by a shallow patch of hydrophobic amino acids (His¹⁴, Tyr¹⁶, Trp⁴¹, Phe⁵⁴ and Phe⁵⁹). Both loops are flexible and belong to the most mobile regions of the protein, which is assessed by the heteronuclear ¹⁵N,¹H-NOE values characterizing the degree of internal backbone motions. The aromatic residues of the ligand binding site are arranged such that they form three pockets for interactions with the polyproline ligand.