On the importance of a funneled energy landscape for the assembly and regulation of multidomain Src tyrosine kinases

Abstract

Regulation of signaling pathways in the cell often involves multidomain allosteric enzymes that are able to adopt alternate active or inactive conformations in response to specific stimuli. It is therefore of great interest to elucidate the energetic and structural determinants that govern the conformational plasticity of these proteins. In this study, free-energy computations have been used to address this fundamental question, focusing on one important family of signaling enzymes, the Src tyrosine kinases. Inactivation of these enzymes depends on the formation of an assembly comprising a tandem of SH3 and SH2 modules alongside a catalytic domain. Activation results from the release of the SH3 and SH2 domains, which are then believed to be structurally uncoupled by virtue of a flexible peptide link. In contrast to this view, this analysis shows that inactivation depends critically on the intrinsic propensity of the SH3-SH2 tandem to adopt conformations that are conducive to the assembled inactive state, even when no interactions with the rest of the kinase are possible. This funneling of the available conformational space is encoded within the SH3-SH2 connector, which appears to have evolved to modulate the flexibility of the tandem in solution. To further substantiate this notion, we show how constitutively activating mutations in the SH3-SH2 connector shift the assembly equilibrium toward the disassembled, active state. Based on a similar analysis of several constructs of the kinase complex, we propose that assembly is characterized by the progressive optimization of the protein's conformational energy, with little or no energetic frustration.

Fig. 1.

Modular assembly of Src tyrosine kinases. (A) Crystal structure of the Src-type tyrosine kinase Hck in the inactive, assembled state (7); the PP1 inhibitor has been replaced by an ATP molecule, as in entry 1AD5 (8). Molecular graphics throughout this work were rendered with Pymol. (B) Virtual thermodynamic cycle used for estimating the shift in the assembly equilibrium induced by mutations in the SH3–SH2 connector (see Results for description).

Fig. 2.

Potential-of-mean-force surfaces for the SH3–SH2 tandem, in the following constructs: wild-type, assembled state (A); wild-type, disassembled state (B); mutant, assembled state (C); and mutant, disassembled state (D). The mutant constructs include glycine substitutions of residues S138, E140, T141, and E142, in the SH3–SH2 connector. See Methods for definition of the reaction coordinates and further details. Contours (black lines) are plotted for 0.5-kcal/mol increments. The white box in A indicates the location of the four crystal structures in entries 2HCK and 1AD5 of the PDB (8). In B and D, the small circle locates the structure of the SH3–SH2 tandem from the Lck kinase in entry 1LCK (16). Note the change in the scale between graphs; to aid the comparison, the location of regions I, II, and III on the free-energy surfaces is indicated, and, for B and D, the 3.5-kcal/mol isocontour from the surfaces in A and C, respectively, is also shown (dashed white lines). These free-energy surfaces correspond to averages over 10 independent calculations. Standard deviations about these averages (<1 kcal/mol) are given in supporting information (SI) Fig. 4.

Fig. 3.

Diagrammatic representation of a putative assembly pathway. The conformational free-energy surface of the SH3–SH2 tandem is shown for each step, alongside representative snapshots of the molecular structure (constructs I through IV from bottom to top; see Table 1). The methodological details for the computation of the PMF surfaces of constructs II and III (in the middle) are analogous to those of constructs I and IV, shown in Fig. 2.