Src Kinase Conformational Activation: Thermodynamics, Pathways, and Mechanisms

Citation: Yang S, Roux B ( Src Kinase Conformational Activation: Thermodynamics, Pathways, and Mechanisms Sichun Yang 0 Benot Roux 0 Gennady Verkhivker, University of California San Diego, United States of America 0 Department of Biochemistry and Molecular Biology, Gordon Center for Integrative Science, The University of Chicago , Chicago, Illinois , United States of America Tyrosine kinases of the Src-family are large allosteric enzymes that play a key role in cellular signaling. Conversion of the kinase from an inactive to an active state is accompanied by substantial structural changes. Here, we construct a coarsegrained model of the catalytic domain incorporating experimental structures for the two stable states, and simulate the dynamics of conformational transitions in kinase activation. We explore the transition energy landscapes by constructing a structural network among clusters of conformations from the simulations. From the structural network, two major ensembles of pathways for the activation are identified. In the first transition pathway, we find a coordinated switching mechanism of interactions among the aC helix, the activation-loop, and the b strands in the N-lobe of the catalytic domain. In a second pathway, the conformational change is coupled to a partial unfolding of the N-lobe region of the catalytic domain. We also characterize the switching mechanism for the aC helix and the activation-loop in detail. Finally, we test the performance of a Markov model and its ability to account for the structural kinetics in the context of Src conformational changes. Taken together, these results provide a broad framework for understanding the main features of the conformational transition taking place upon Src activation. - Funding: This work was supported by NIH grant CA-093577. Computational support was provided in part by the San Diego Supercomputer Center and the Teragrid project. Competing Interests: The authors have declared that no competing interests exist. The nonreceptor tyrosine kinases of the Src-family are large allosteric enzymes involved in signaling pathways, regulating cell growth and proliferation [14]. These enzymes have the ability to undergo large conformational changes, thereby switching between different inactive and active states in response to either intracellular or extracellular signals. The key role that these kinases play in the onset of many human diseases, particularly cancer, makes them important targets for therapeutic intervention [5]. The nine members of the Src kinase family share a common structural organization, which consists of two regulatory SH3 and SH2 binding modules, followed by the catalytic domain [69]. A number of high-resolution crystal structures from three members of the Src-family (Hck, Lck, and c-Src) in different conformations have been captured, offering a great opportunity for a detailed view of the mechanism of allosteric regulation [1015]. In its down-regulated inactive form, the three domains are assembled into an auto-inhibitory complex [1012]. In its up-regulated active form, the complex is disassembled. The kinase catalytic domain is highly conserved among all protein kinases and its overall architecture resembles very closely that of other kinases such as protein kinase A [1618] and Csk [1921]. The catalytic domain comprises an N-terminal lobe (N-lobe) and a C-terminal lobe (Clobe) (Figure 1). The active site is located between these two lobes, where the c-phosphoryl group of ATP can be transferred to tyrosine residues of substrate peptides during the phosphorylation process [6,22]. One important difference between the inactive and active form is the alternative conformations of the central activation-loop (A-loop), which controls accessibility to the active site [13,15,23]. In the down-regulated form, the A-loop is compact and blocks the active site to the substrate [11,14]. Additional differences lie in the internal rotation of the aC helix and the relative orientation between the N- and C-lobes [24]. Structural studies of Src kinases by many groups have suggested some mechanisms for the regulation of the catalytic activity inferred from two end-point structures, although picturing how the protein dynamically switches from one state to another has remained elusive. One challenge for experiments to obtain the dynamic information is that the conformational switching process is inherently transient. Computer simulations based on physical models could provide a complementary approach to addressing these issues. To relate these static structures to the function, the dynamics of protein motion is required to fully monitor the conformational change process [2528]. Theoretical studies based on standard all-atom simulations are prohibitive because the timescale of the transition is on the order of msec [29]. A possible strategy to overcome timescale difficulties is to carry targeted or steered simulations [24,3032], though there is always the concern that the presence of nonphysical restraints may bias the transition pathway during the conformational change. This might be especially true when the transition involves multiple competing pathways. To overcome the timescale limitation of allatom simulations and also avoid the nonphysical restraints used in biased simulations, we employ a coarse-grained model of Src kinase Hck. The model incorporates two individual experimental structures Src tyrosine kinases are large protein molecules that play an important role in the regulation of cellular growth and proliferation. In doing so, Src kinases have the ability to affect the activity of other proteins inside the cell by turning them on or off. Dysfunctional Src kinase activity has been associated with many human diseases, most importantly cancer, which makes them important targets for therapeutic intervention. To understand how a Src kinase molecule is able to change its shape (conformation) and switch between its active or inactive states, we constructed a computer model. The results from the model provide a broad conceptual framework for interpreting the main features of the change of protein conformation taking place upon Src activation. It is our hope that these results will help design new experiments to refine our understanding of the activation of Src kinases. and allows switching between them. This is accomplished by using the recently developed multi-state model, or two-state Go model, in which both experimental end-point structures are explicitly encoded in the energy function [3341]. The present model differs from the symmetrized-Go model used previously for studying domain swapping, in which the alternative conformation was implicitly in the monomeric conformation [4244]. In the present study, we use this simplified model to explore the conformational activation of the Src catalytic domain. Notably, the regulatory modules SH2 and SH3 are not included in the present model. While the comple (...truncated)