Ligand-induced strain in hydrogen bonds of the c-Src SH3 domain detected by NMR

Changes in the molecular conformation of proteins can result from a variety of perturbations, and can play crucial roles in the regulation of biologic al activity. A new solution NMR method has been applied to monitor ligand-i nduced changes in hydrogen bond geometry in the chicken c-Src SH3 domain. T he structural response of this domain to ligand binding has been investigat ed by measuring trans-hydrogen bond N-15-C-13' scalar couplings in the free state and when bound to the high affinity class I ligand RLP2 containing r esidues RALPPLPRY. A comparison between hydrogen bonds in high resolution X -ray structures of this domain and those observed via (h3)J(NC') couplings in solution shows remarkable agreement. Two backbone-to-side-chain hydrogen bonds are observed in solution, and each appears to play a role in stabili zation of loop structure. Reproducible ligand-induced changes in trans-hydr ogen bond scalar couplings are observed across the domain that translate in to changes in hydrogen bond length ranging between 0.02 to 0.12 Angstrom. T he observed changes can be rationalized by an induced fit mechanism in whic h hydrogen bonds across the protein participate in a compensatory response to forces imparted at the protein-ligand interface. Upon ligand binding, mu tual intercalation of the two Leu-Pro segments of the ligand between three aromatic side-chains protruding from the SH3 surface wedges apart secondary structural elements within the SH3 domain. This disruption is transmitted in a domino-like effect across the domain through networks of hydrogen bond ed peptide planes. The unprecedented resolution obtained demonstrates the a bility to characterize subtle structural rearrangements within a protein up on perturbation, and represents a new step in the endeavor to understand ho w hydrogen bonds contribute to the stabilization and function of biological macromolecules. (C) 2000 Academic Press.