- Top of page
- Results and Discussion
- Materials and methods
Src tyrosine kinases are essential in numerous cell signaling pathways, and improper functioning of these enzymes has been implicated in many diseases. The activity of Src kinases is regulated by conformational activation, which involves several structural changes within the catalytic domain (CD): the orientation of two lobes of CD; rearrangement of the activation loop (A-loop); and movement of an α-helix (αC), which is located at the interface between the two lobes, into or away from the catalytic cleft. Conformational activation was investigated using biased molecular dynamics to explore the transition pathway between the active and the down-regulated conformation of CD for the Src-kinase family member Lyn kinase, and to gain insight into the interdependence of these changes. Lobe opening is observed to be a facile motion, whereas movement of the A-loop motion is more complex requiring secondary structure changes as well as communication with αC. A key result is that the conformational transition involves a switch in an electrostatic network of six polar residues between the active and the down-regulated conformations. The exchange between interactions links the three main motions of the CD. Kinetic experiments that would demonstrate the contribution of the switched electrostatic network to the enzyme mechanism are proposed. Possible implications for regulation conferred by interdomain interactions are also discussed.
Abbreviations: CD, catalytic domain; SH2, Src homology domain 2; SH3, Src homology domain 3; CDact, Lyn catalytic domain active conformation; CDdown, Lyn catalytic domain down-regulated conformation; N-lobe, N-terminal lobe; C-lobe, C-terminal lobe; MD, molecular dynamics; BMD, biased molecular dynamics; A-loop, activation loop; αC, α-helix C; RMSD, root mean squared deviation; α, pairwise force constant.
The Src tyrosine kinase family plays an essential role in numerous pathways of cellular signaling (Bolen and Brugge 1997; Thomas and Brugge 1997). Part of the regulatory mechanism in signaling mediated by Src kinases includes conformational activation and control of the equilibrium between active and down-regulated forms of Src. Tyrosine phosphorylation and intramolecular domain interactions are used in the cell to modulate this equilibrium, with maximum catalytic activity being achieved when the activation loop is phosphorylated and contacts between the catalytic domain (CD) and the regulatory SH2/SH3 domains are released (Sicheri and Kuriyan 1997; Boggon and Eck 2004). Nonetheless, some activity remains in the absence of loop phosphorylation (Porter et al. 2000; Adams 2003).
Normal cellular function requires tight control of Src kinase activity and the equilibrium between active and down-regulated conformations. Src kinases comprise SH3, SH2, and catalytic domains, flanked by N- and C-terminal tails. The end states of Src conformational activation are known from the considerable structural information accumulated for protein kinases and the Src family. The CD is highly conserved among all protein kinases, and the first view of this structure was reported for protein kinase A (Knighton et al. 1991). The structure of the activated form was determined for an isolated CD fragment from the Src family member Lck (Yamaguchi and Hendrickson 1996), and the down-regulated forms were first reported for constructs lacking only the N-terminal unique region of the two family members Hck (Sicheri et al. 1997) and Src (Williams et al. 1997; Xu et al. 1997). These studies not only revealed the key conformational differences between active and down-regulated forms of Src family CD but also defined for the first time how SH2 and SH3 contacts with CD influence activation.
The kinase catalytic domain has an N-terminal lobe (N-lobe) (Fig. 1, cyan and blue) and a C-terminal lobe (C-lobe) (Fig. 1, gold and red) separated by a deep cleft where ATP and substrate bind. The activation loop (A-loop) (Fig. 1, red) forms part of this cleft. Autophosphorylation of Tyr416 (chicken c-Src numbering is used throughout this report) on the A-loop activates Src in the cell (Barker et al. 1995). Several features distinguish this active CD conformation (CDact) (Yamaguchi and Hendrickson 1996; Breitenlechner et al. 2005) from the down-regulated conformation (CDdown) (Sicheri et al. 1997; Williams et al. 1997; Xu et al. 1997, 1999; Schindler et al. 1999; Cowan-Jacob et al. 2005). The primary difference is the alternative conformations of the A-loop; in CDact (Fig. 1A–C), the A-loop forms an extended structure that opens the cleft region to make the active site available for substrate binding. In contrast, the A-loop in CDdown (Fig. 1D–F) is more compact and forms two short α-helices that fill the cleft and thus occlude substrate. In addition, lobe–lobe orientation and the internal position of the α-helix (αC) differ. The orientation of the N- and C-lobes is more open in CDact relative to that in CDdown so that the size of the cleft opening is increased. The internal variation in αC is the position of this helix within the N-lobe; αC in CDact is displaced with respect to the β-sheet toward the interior of the protein.
Figure Figure 1.. A comparison of the Lyn catalytic domain structures in the down-regulated (CDdown) (A–C) and active (CDact) (D–F) conformations. The SH3 and SH2 domains are not shown. The structures are from equilibrium molecular dynamics simulations initiated with coordinates obtained by homology with Lck (Yamaguchi and Hendrickson 1996) and Hck (Schindler et al. 1999) crystal structures. B and E are rotated 90° with respect to A and D. The N-lobe is shown in cyan, the C-lobe in gold, and αC in blue. The A-loop (red) extends away from the catalytic site in CDact and folds over in CDdown, occluding the substrate-binding site. Stereoview of the cleft region illustrates the important interactions, listed in Table 1, for CDdown (C) and CDact (F) conformations. ATP is shown in green; the C-lobe residues in gold; the A-loop in red; and N-lobe residues, including the helix αC, in blue. The structure figures were generated using VMD (Humphrey et al. 1996).
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Improper control of activation of different Src kinases has implicated members of the Src family in numerous disease states ranging from autoimmune diseases (Lowell et al. 1994; Lowell and Soriano 1996) to cancer (Verbeek et al. 1996; Lutz et al. 1998), with the result that Src kinases and other non–receptor protein tyrosine kinases are recognized to be key drug targets (Noble et al. 2004; Krause and Van Etten 2005). Inhibition by one effective treatment of chronic myelogenous leukemia, imatinib (Capdeville et al. 2002), is particularly noteworthy in that intermolecular recognition takes advantage of the structural differences associated with conformational activation (Schindler et al. 2000).
One approach to overcome problems with specificity, and to find more effective inhibitors, is to gain a better understanding of the physical behavior of kinases including the process of kinase conformational activation. To this end, computational studies of a Src-family member, Lyn kinase, were conducted and reported here. The sequence and structural similarities among Src kinases and protein kinases in general imply that deciphering the activation mechanism of one Src kinase will afford clues about the activation mechanism of the Src-family and other kinases. Lyn initiates (Burkhardt et al. 1991; Yamanashi et al. 1991) and down-regulates (Chan et al. 1997, 1998; Nishizumi et al. 1998; Smith et al. 1998; Hong et al. 2002) B-cell signaling. NMR studies to examine substrate recognition of Lyn for ITAM have been reported (Gaul et al. 2000).
The structures of CDdown (Sicheri et al. 1997; Xu et al. 1997) and CDact (Yamaguchi and Hendrickson 1996) provide the starting point for understanding the mechanism of kinase activation, although the static crystal structures give information on neither the order nor the interdependence of conformational changes that must occur for activation. Certain features of conformational activation have been inferred from these two end states. It has been suggested that intramolecular domain–domain contacts stabilize CDdown by either hindering αC motion, thus preventing the A-loop displacement required for activation (Schindler et al. 1999), or by stabilizing the closed conformation of the catalytic domain lobes and keeping the A-loop tyrosine inaccessible (Xu et al. 1999). The computational study reported here aims to begin to elucidate the conformational transition between CDdown and CDact and to define the interdependence between displacements in αC, the A-loop, and lobe–lobe orientation.
Biased molecular dynamics (BMD) was used to study the conformational transition between CDact and CDdown. The time scale for this transition has been measured for protein kinase A to be in the 5–6-msec range (Shaffer and Adams 1999). With BMD, a biasing potential is added to the molecular mechanics force field in order to study this transition within a computationally feasible time scale. The biasing potential moves the protein toward a target structure, and is added as a global sum over the system so that departures in local structure away from the target are allowed as long as the cumulative motion is in the desired direction. The consistent characteristics of the pathways from several trajectories can give information about possible energy barriers (Neria et al. 1996). Similar methods to study activated processes are targeted molecular dynamics (TMD) and steered molecular dynamics (SMD). For BMD, the potential is like a ratchet in that it is added only when the system moves away from the target, whereas in TMD and SMD the potential is present at all times. This feature of BMD is expected to be advantageous by allowing the system to follow the energy surface more closely. BMD has been previously used to study unfolding (Marchi and Ballone 1999; Paci and Karplus 1999; Morra et al. 2003) and dissociation (Paci et al. 2001; Li et al. 2005). TMD and SMD have been used to study the conformational changes of GroEL (Ma et al. 2000) and Ras (Diaz et al. 1997; Ma and Karplus 1997) and unbinding of avidin-biotin (Isralewitz et al. 2001a). TMD has also been used to examine certain features of the Src kinase conformational transition. These studies primarily focus on the role of the SH3–SH2 linker in coupling domains (Young et al. 2001) and the structural effects of the N-terminal residues of CD in interdomain communication (Banavali and Roux 2005).
We examined the unphosphorylated, isolated catalytic domain of Lyn for this initial computational analysis of activation. The Src-family catalytic domain is constitutively active (Yamaguchi and Hendrickson 1996) and inhibited by small molecules to a similar extent as the full-length form (Zhu et al. 1999). In addition, Src kinase activity is observed in the absence of Tyr416 phosphorylation (Porter et al. 2000; Adams 2003). Therefore, the catalytic domain captures significant features of conformational activation, and a description of the transition behavior in the absence of phosphorylation on Tyr416 provides information on the underlying physical processes involving the overall structure. Computational investigations should also give insight into the structural basis for conformationally selective ligand binding (Schindler et al. 2000; Kwak et al. 2005), and thus assist in the development of new kinase inhibitors. The key result from the analysis of the BMD simulations is that the network of electrostatic interactions in CDdown switches to an alternative network in CDactso that the transition may be described as a switched electrostatic network. A set of six polar or charged residues consistently switch in a temporally short fashion between binding partners over the course of the transition. These residues are highly conserved, and some are already assigned important roles in catalysis or binding. We propose that these residues also serve central roles in the transition process by functioning as a switched electrostatic network to guide the transition as anchor points along the pathway.
- Top of page
- Results and Discussion
- Materials and methods
Biased molecular dynamics simulations were used to obtain an initial description of the conformational transition between the active and the down-regulated states of unphosphorylated Src kinase catalytic domain, and thus provide the groundwork for more detailed study of the energetics of the activation pathway.
BMD and other similar methods (TMD [Schlitter et al. 1993] and SMD [Isralewitz et al. 2001b]) that use an additional potential to overcome energy barriers and move from one state to the other in nanoseconds are useful in studying conformational transitions that may take place in microseconds to seconds in the cell. Such nonequilibrium methods make it possible to determine pathways and energy barriers for these conformational transitions. The underlying energy landscape guides the transition; however, the sequence or relative timescales of events in the pathway is dependent on the magnitude of the potential as applied to a selection of atoms, with higher forces resulting in greater loss of temporal detail (Bryant et al. 2000). We used different force constants to study the forward and the reverse transition for activation in order to obtain the consistent characteristics. Our results describe conformational features for CD activation and certain aspects of interdependence among them, rather than the specific sequence with which they take place.
We find that the activation and deactivation transition of CD progresses nonuniformly. The opening of the two lobes occurs early in the activation transition and is unimpeded. An intrinsic flexibility in the lobe–lobe orientation has also been deduced from crystallographic analyses of Src kinase structures whereby the B-factors associated with the N-lobe were twice as high as those of the C-lobe (Breitenlechner et al. 2005). On the other hand, a helix–coil transition in the A-loop is slower and appears to be hindered by an energy barrier. The reverse deactivation transition is complicated by the interdependence of the outward rotation of αC and the folding of the A-loop over the active site.
Elucidation of the pathway at the detailed atomic level allowed only by computation is valuable for understanding the enzymatic activity of protein kinases without A-loop phosphorylation (Porter et al. 2000; Adams 2003) and inhibition by conformationally selective compounds such as imatinib (Capdeville et al. 2002) and gefitinib (Kwak et al. 2005). Intramolecular domain–domain interactions are thought to deactivate Src kinase by locking the lobe orientation or by stabilizing the inactive conformation of αC. It has been suggested that when the lobes open, Tyr416 becomes accessible for phosphorylation (Xu et al. 1999). The fast and facile opening of the lobes observed in this study of CD without the SH2/SH3 regulatory domains indicates that this suggestion is indeed reasonable. The ease of lobe reorientation and the electrostatic switch mechanism that functions in the transition in the absence of phosphorylation suggest that the active conformation is transiently populated by kinase absent of A-loop phosphorylation and bound inhibitor. Thus, we propose that the activity of Src unphosphorylated on the A-loop (Porter et al. 2000; Adams 2003) arises from the transient formation of CDact rather than another conformation of the kinase of lower catalytic power. Stabilization of CDact relative to CDdown likely requires phosphorylation of the A-loop tyrosine. A similar behavior was observed for NtrC in which phosphorylation shifts the equilibrium rather than inducing a new structure (Volkman et al. 2001). From the interdependence of the displacements in the A-loop and αC and the time dependence of the Glu310 exchange between Arg409 and Lys295 in BMD, we conclude that phosphorylation of Tyr416 would stabilize not only the A-loop but also αC conformations in CDact.
A major outcome of this investigation is the proposal that the transition is conformationally controlled by a switched electrostatic network involving the exchange between a three-way and a two-way network of interactions among Asp386, Tyr416, Arg409, Glu310, Lys295, and Asp404. The switch residues are highly conserved in tyrosine kinases, suggesting that this switched network may play an important role in other kinases as well. The ATP-binding Lys295, metal-binding Asp404, and catalytic Asp386 are absolutely conserved. Arg409 is absolutely conserved across the Src kinase family and functionally conserved across the kinase family. Helix αC Glu310 is highly conserved if not strictly conserved across all kinases. Our analysis suggests that the same residues essential for either structural stabilization (Yamaguchi and Hendrickson 1996; Schindler et al. 1999; Xu et al. 1999) or for catalysis (Kamps and Sefton 1986; Gibbs and Zoller 1991; Porter et al. 2000) also play important roles in the transition between the two end states.
The six residues of the network exchange from one group to another when the A-loop main chain extends out and refolds, and thus serve as anchor points to guide the conformational transition. Interactions in CDdown of Tyr416 and Arg409 on the A-loop connect to active-site residues and to αC, and link the A-loop conformational change to the reorientation of the N- and C-lobes and to the displacement of αC (Fig. 9). Further, the switch centered on Glu310 is likely to be energetically significant to the conformational change of the A-loop and αC. The Glu310–Arg409 interaction impedes rotation of αC and the inward positioning of Glu310 for activation. This interaction seems particularly stable since it breaks late in the activation process where αC rotates in, and forms early in the reverse deactivation process (Fig. 8). Phosphorylation of Tyr416 would likely shift the equilibrium in favor of the Glu310–Lys295 position of the switch in CDact by competing to form the Try416–Arg409 interaction and thus release Glu310 to interact with Lys295. The electrostatic network, therefore, offers a mechanism for conformational activation, whereby phosphorylated Tyr416 on the A-loop could influence not only the change in the A-loop conformation but also the conformational transition throughout the catalytic domain. It is of interest to know how concerted the exchanges are for the four central residues, but the concertedness could not be determined from the current sampling in the biased simulations.
The switched electrostatic network mechanism proposed here concerns the conformational activation of protein kinases in contrast to earlier discussions of electrostatic switches for equilibrium states. Previous studies on Src kinases (Xu et al. 1999) recognized the switch in interactions between the two crystallographic structures of active and down-regulated kinases and their role in stabilization of two conformational states. The current study expands the roles proposed for these critical residues to include stabilization of the conformational transition. The term “electrostatic switch” has also been used to describe a change in the binding equilibrium of Src kinases (McLaughlin and Aderem 1995), whereby phosphorylation of the N terminus was shown to enhance membrane-binding affinity and reverse the relative binding affinity between membrane-bound or cytoplasmic states of Src kinase.
Given the high conservation in the switch residues in the kinase family, the exchange pathway between active and down-regulated forms could be generally conserved in kinases. Conservation of conformational transition pathways has been suggested from evolutionary relationships for other proteins (Süel et al. 2003; Zheng et al. 2005). Slight variations in kinase catalytic domain structure, such as the A-loop conformation or relative lobe–lobe orientations, may result in differences in the order of events.
The presence of a switched electrostatic network in conformational activation can be tested experimentally providing conditions are established where conformational activation is a component of the rate-limiting step (Adams 2003). Kinetic assays on Src (Barker et al. 1995) and the Src-family kinase Hck (Moarefi et al. 1997) show that enzymatic activity exhibits an enzyme concentration-dependent lag phase, which can be eliminated by preincubation with MgATP. As the lag phase is likely dependent on conformational activation, dependence of the lag phase on salt concentration can provide information about the effect of the electrostatic switch on the enzyme mechanism. Similar kinetic studies to those reported are currently under way in the laboratory of our collaborator.