The SH3 domain of nebulin binds selectively to type II peptides: theoretical prediction and experimental validation.

Nebulin, a giant modular protein from muscle, is thought to act as a molecular ruler in sarcomere assembly. The C terminus of nebulin, located in the sarcomere Z-disk, comprises an SH3 domain, a module well known for its roà le in protein/protein interactions. SH3 domains are known to recognize proline-rich ligands, which have been classi®ed as type I or type II, depending on their relative orientation with respect to the SH3 domain in the complex formed. Type I ligands are bound with their N terminus at the RT loop of the SH3 domain, while type II ligands are bound with their C terminus at the RT loop. Many SH3 domains can bind peptides of either class. Despite the potential importance of the SH3 domain for the function of nebulin as an integral part of a complex network of interactions, no in vivo partner has been identi®ed so far. We have adopted an integrated approach, which combines bioinformatic tools with experimental validation to identify possible partners of nebulin SH3. Using the program SPOT, we performed an exhaustive screening of the muscle sequence databases. This search identi®ed a number of potential nebulin SH3 partners, which were then tested experimentally for their binding af®nity. Synthetic peptides were studied by both ¯uorescence and NMR spectroscopy. Our results show that nebulin SH3 domain binds selectively to type II peptides. The af®nity for a type II peptide, 12 residues long, spanning the sequence of a stretch of titin known to colocalise with nebulin in the Z-disk is in the submicromolar range (0.7 mM). This af®nity is among the highest found for SH3/peptide complexes, suggesting that the identi®ed stretch could have signi®cance in vivo. The strategy outlined here is of more general applicability and may provide a valuable tool to identify potential partners of SH3 domains and of other peptide-binding modules