Binding, Domain Orientation, and Dynamics of the Lck SH3−SH2 Domain Pair and Comparison with Other Src-Family Kinases

Abstract

The catalytic activity of Src-family kinases is regulated by association with its SH3 and SH2 domains. Activation requires displacement of intermolecular contacts by SH3/SH2 binding ligands resulting in dissociation of the SH3 and SH2 domains from the kinase domain. To understand the contribution of the SH3-SH2 domain pair to this regulatory process, the binding of peptides derived from physiologically relevant SH2 and SH3 interaction partners was studied for Lck and its relative Fyn by NMR spectroscopy. In contrast to Fyn, activating ligands do not induce communication between SH2 and SH3 domains in Lck. This can be attributed to the particular properties of the Lck SH3-SH2 linker which is shown to be extremely flexible thus effectively decoupling the behavior of the SH3 and SH2 domains. Measurements on the SH32 tandem from Lck further revealed a relative domain orientation that is distinctly different from that found in the Lck SH32 crystal structure and in other Src kinases. These data suggest that flexibility between SH2 and SH3 domains contributes to the adaptation of Src-family kinases to specific environments and distinct functions.

Crystal Structure of the Src Family Kinase Hck SH3-SH2 Linker Regulatory Region Supports an SH3-dominant Activation Mechanism

“…However, the Lck SH3 domain orientation significantly differs from Hck and is rotated away from the SH2 domain by 100°relative to the Hck SH3 domain. This comparison demonstrates inter-domain flexibility across SFKs even in the crystal state and is consistent with 15 N NMR relaxation data that showed differences in correlation times between the SH3-SH2 domains of Fyn, Lck, and c-Abl (27,34,35).…”

Interaction with the Src Homology (SH3-SH2) Region of the Src-family Kinase Hck Structures the HIV-1 Nef Dimer for Kinase Activation and Effector Recruitment

“…Interestingly, the relative orientations of the SH3 and SH2 domains in our new structure are unique compared with those observed in these earlier SH3-SH2 structures (37,46,47), highlighting the variability in interdomain positioning among the Src-family kinases. In addition, 15 N NMR relaxation data show differences in correlation times between the SH3 and SH2 domains of Fyn, Lck, and c-Abl when these domains are joined (46,(52)(53)(54). Finally, NMR studies of Nef interaction with a Hck SH3-SH2-linker protein demonstrated significant chemical shift perturbations not only for residues in the Hck SH3 domain but also in the connector and N-terminal region of the SH2 domain upon Nef binding (55).…”

Structure and dynamic regulation of Src-family kinases

“…In these experiments, chemical shift changes were monitored in the domain not involved in the direct binding of the peptide ligand. NMR studies of Fyn, c-Src, and Lck SH32 proteins revealed that peptide ligand binding to the SH2 domain resulted in either small (c-Src, Fyn) [75 -77] or undetectable (Lck) [78] chemical shift changes in the SH3 domain, again suggesting that SH2 ligation is not structurally communicated to the SH3 domain. Together, the HXMS and NMR results are consistent with the idea that SFK SH3 and SH2 domains have independent folding and binding activity which is not grossly influenced by being next to one another in sequence.…”