Multidomain assembled states of Hck tyrosine kinase in solution

Abstract

An approach combining small-angle X-ray solution scattering (SAXS) data with coarse-grained (CG) simulations is developed to characterize the assembly states of Hck, a member of the Srcfamily kinases, under various conditions in solution. First, a basis set comprising a small number of assembly states is generated from extensive CG simulations. Second, a theoretical SAXS profile for each state in the basis set is computed by using the Fast-SAXS method. Finally, the relative population of the different assembly states is determined via a Bayesian-based Monte Carlo procedure seeking to optimize the theoretical scattering profiles against experimental SAXS data. The study establishes the concept of basis-set supported SAXS (BSS-SAXS) reconstruction combining computational and experimental techniques. Here, BSS-SAXS reconstruction is used to reveal the structural organization of Hck in solution and the different shifts in the equilibrium population of assembly states upon the binding of different signaling peptides.T yrosine kinases of the Src-family are large multidomain allosteric enzymes implicated in the signaling pathways regulating cell growth and proliferation (1). The key role that Src kinases play in the onset of many human diseases, particularly cancer, makes them important targets for therapeutic intervention (2).All Src kinases share a common structural organization comprising the SH3 and SH2 binding domains followed by a highly conserved catalytic domain connected by flexible linkers (1). Crystal structures have offered a detailed view of the down-regulated forms of Hck and c-Src (3, 4), characterized by a compact assembled form stabilized by auto-inhibitory intramolecular interactions between the N and C terminus of the catalytic domain with the SH3 and SH2 modules, respectively. The SH2 and SH3 modules of the Src-family tyrosine kinases play dual roles: both are involved in the auto-inhibitory intramolecular interactions down-regulating kinase activity, but can also serve as possible binding receptors for cellular signals leading to kinase activation.In a broadly accepted paradigm, Src inactivation/activation is pictured in terms of a simple two-state process involving an assembled (inactive) and a disassembled (active) conformation, i.e., once any of the intramolecular interaction is released, the kinase domain switches to a disassembled catalytically active state targeting down-stream cellular substrates (5-8). Crystal structures (3, 4) and mutational studies complemented by molecular dynamics (MD) simulations (9) are consistent with this view. However, one crystal structure of c-Src in a partially activated state in which the SH2-SH3 modules are reassembled in a different orientation with respect to the catalytic domain (10), suggests that the situation could be considerably more complex. In this regard, it seems likely that different signals could promote different assembly states, leading to differently configured activated kinases. While it is understood that Src increases its activity in respon...

Multisteric Regulation by Structural Disorder in Modular Signaling Proteins: An Extension of the Concept of Allostery

“…Activation of the enzyme is induced by Tyr dephosphorylation and other modifications, which release intramolecular contacts and lead to a largely disassembled and more disordered active state. Recent SAXS experiments 26 showed an ensemble of distinct closed and open structures in both the inactive and active states ( Figure 2D), therefore signaling due to dephosphorylation is best described as a shift in the conformational ensemble. 26 In a way similar to the case of interdomain allostery, Hck also represents a combination of traditional and novel types of allostery, because shifts in the population are converted to structure-based signal propagation in the catalytic domain (Table 2).…”

“…24 It also became clear that allosteric coupling might occur without a discernible conformational change, by way of altering the dynamics of the protein only 6,21,25 ( Figure 1C) to favor a particular downstream binding/catalytic event. By combining elements of classical cooperativity (subunit interactions) and conformational coupling augmented by structural disorder (novel allostery), recently it was also reported that domaindomain interactions enabled by a disordered linker 13,26 might permit the formation of an energetic pathway traversing separate domains through their interface (Figure1D).…”

“…It is rather common that even in the naïve state, besides the inactive conformation (e.g. the autoinhibited closed conformation of Hck, 26 and Vav 106 ) the activated state must also be present, as demanded by local accessibility to the input signal (e.g. a post-translational modification or an incoming binding partner).…”

“…26 Like all kinases in the Src-family, Hck is a multidomain allosteric enzyme with and N-terminal SH4 domain, a unique domain, SH3 and SH2 domains, and a catalytic domain, all connected by flexible linkers. Inactive Hck is dominated by a closed (assembled) conformation maintained by intramolecular interactions between a Cterminal pTyr residue and the SH2 domain, and the Pro-rich linker and the SH3 domain ( Figure 2D).…”

Equilibrium simulations of proteins using molecular fragment replacement and NMR chemical shifts

“…These developments have generated an interest in developing new methods to make optimal use of the available experimental data. From the initial emphasis on the determination of the native structures of proteins, the focus is increasingly shifting toward generating conformational ensembles representing their conformational fluctuations (2)(3)(4). In this context, problems with overfitting are systematically addressed (5), and in general efforts are made to implement the structural restraints as conservatively as possible, for instance by using the maximum entropy principle (6)(7)(8)(9).…”