Regulation of the c-Abl (ABL1) tyrosine kinase is important because of its role in cellular signaling, and its relevance in the leukemiogenic counterpart (BCR-ABL). Both auto-inhibition and full activation of c-Abl are regulated by the interaction of the catalytic domain with the Src Homology 2 (SH2) domain. The mechanism by which this interaction enhances catalysis is not known. We combined computational simulations with mutagenesis and functional analysis to find that the SH2 domain conveys both local and global effects on the dynamics of the catalytic domain. Locally, it regulates the flexibility of the αC helix in a fashion reminiscent of cyclins in cyclin-dependent kinases, reorienting catalytically important motifs. At a more global level, SH2 binding redirects the hinge motion of the N and C lobes and changes the conformational equilibrium of the activation loop. The complex network of subtle structural shifts that link the SH2 domain with the activation loop and the active site may be partially conserved with other SH2-domain containing kinases and therefore offer additional parameters for the design of conformation-specific inhibitors.

The Abl kinase is a key player in many crucial cellular processes. It is also an important anti-cancer drug target, because a mutation leading to the fusion protein Bcr-Abl is the main cause for chronic myeloid leukemia (CML). Abl inhibitors are currently the only pharmaceutical treatment for CML. There are two main difficulties associated with the development of kinase inhibitors: the high similarity between active sites of different kinases, which makes selectivity a challenge, and mutations leading to resistance, which make it mandatory to search for alternative drugs. One important factor controlling Abl is the interplay between the catalytic domain and an SH2 domain. We used computer simulations to understand how the interactions between the domains modify the dynamic of the kinase and detected both local and global effects. Based on our computer model, we suggested mutations that should alter the domain-domain interplay. Consequently, we tested the mutants experimentally and found that they support our hypothesis. We propose that our findings can be of help for the development of new classes of Abl inhibitors, which would modify the domain-domain interplay instead of interfering directly with the active site.

Funding: We acknowledge partial financial support from the following sources: Spanish Ministry of Economy and Competitiveness ( http://www.idi.mineco.gob.es/ ) Grant BIO2010-20166. The PRACE Research Infrastructure resource (FP7/2007–2013, grant agreement nr. RI-283493). http://www.prace-ri.eu/ ERC grant i-FIVE. http://erc.europa.eu/ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

We used a multi-disciplinary approach combining elastic network models, extensive molecular dynamics simulations, free energy calculations and functional assays following mutagenesis to characterize the allosteric coupling of the CD of Abl with the SH2 domain as well as the modulation of the dynamic properties of the assembly by interactions in the “top-hat” conformation. Both the long atomistic simulations and the elastic network models indicate a significant change in the dynamics of key regulatory elements, providing a simple explanation of the mechanism of allosteric activation. Based on the computational results we designed a number of point mutants to validate the proposed model. These mutants were expressed in human cells and tested for kinase activity. We identified mutations, all distant from the active site, that were either activating the catalytic output of the kinase or were disrupting. Interestingly, we also identified a residue that when mutated lead to a decoupling of the activity of the CD from interaction with the SH2 domain. Collectively, the data suggested an effect of SH2 binding that results in changes of the properties of the αC helix, reminiscent of the effect that cyclins exert on cyclin-dependent kinases.

The SH3/linker region has also been shown to be involved in the regulation of Abl activation [31] . However, here we focus on the SH2 domain that, even in the absence of SH3 and the linker, has been shown to have strong activating effect.

While the molecular basis for the role of the SH2 and SH3 domains in Abl autoinhibition is well understood, the mechanism of their activating effect is less straightforward. Recent crystal structures and small angle X-ray scattering studies have revealed that the transition from the auto-inhibited to the fully activated form of Abl requires a complete reassembly of the complex formed by the CD, the SH2 and the SH3 domains, leading to migration of the SH2 domain from the C-lobe to the N-lobe of the CD (“top-hat” conformation) ( Figure 1C ) [24] . Importantly, the effect of this rearrangement is not reduced to merely revoking the autoinhibition of Abl by removing the SH2-SH3 domain clamp, but the SH2 domain, when bound to the N-lobe of the CD, enhances the activity of the kinase, although it bears no direct contact to the catalytic site. Recently, it was shown that the I164E mutation in the SH2 domain, which interrupts the hydrophobic interactions at the interface between SH2 and CD in the “top-hat” conformation, leads to deactivation of Abl [5] , [18] . A similar domain arrangement and activating effect has been observed in other kinases [19] , [29] , such as Fes [18] and Btk [30] . Hence, in multiple tyrosine kinases, the SH2 domain acts as an allosteric effector. Comparison of the crystal structures of the auto-inhibited and the activated forms of Abl does not reveal any marked conformational changes, particularly at the active site, that could explain the mechanism of activation by the SH2 domain, a finding that points towards a dynamic rather than static nature of the allosteric effect. The essential features of this allosteric effect and the molecular mechanism by which it is transferred from the N-lobe to the catalytic site still remain elusive.

A The c-Abl isoform Ib is characterized by myristoylation (Myr) on Gly-2 of the N-terminal capping region (cap). The tyrosine kinase domain is preceded by the SH3 and SH2 domains and a connecting linker. The last exon region contains nuclear localization signals and a C-terminal actin binding domain (ABD). B In the down-regulated state (PDB entry 2FO0), the SH2 domain binds the C-lobe of the kinase domain, the myristate is bound in its cognate pocket and the SH3 domain binds the SH2-CD linker. C In the active “top-hat” conformation (PDB entry 1OPL), the SH2 domain moves to interact with the N-lobe of the kinase domain. The αC helix and the activation loop are highlighted in red and pink, respectively. D Positions of the most important point mutations at the SH2-CD interface and in the β3-αC loop.

In most non receptor-type tyrosine kinases, the catalytic domain is preceded by a Src homology 2 (SH2) domain [11] ( Figure 1A ). The importance of the SH2 domain in the auto-inhibition and/or activation of the catalytic domain has been shown in c-Src [6] , [8] , [12] – [14] , Hck [15] – [17] , Fes [18] , [19] and c-Abl, among others. The role of the SH2 domain in c-Abl is of special interest, because it is involved both in auto-inhibition and activation of the CD [18] , [20] , [21] , and mutations in the SH2 domain have been related to imatinib-resistance in CML patients [18] , [19] , [22] . In the auto-inhibited state, the SH3 and SH2 domains and the SH2-kinase linker form a rigid clamp around the CD, which is locked in place by an N-terminal myristoyl modification of the N-terminal cap region inserted deeply into the CD [7] , [23] ( Figure 1B ). This grip reduces the flexibility of the CD and, in particular, dampens the opening and closing of its N- and C-termini around the active site [16] , . This so-called hinge or breathing motion of the CD is required for catalysis, and its impairment is associated with low catalytic output [26] – [28] .

In physiological conditions the catalytic activity of tyrosine kinases is tightly regulated through the interplay between various protein domains, phosphorylation events and associated conformational states of the catalytic domain (CD) [10] . During the catalytic cycle, its high intrinsic flexibility allows the CD to react to the regulatory elements by switching reversibly between a number of distinct active and inactive states.

In the light of the above it is not surprising that the mechanisms regulating the activation and deactivation of both the wild type c-Abl and BCR-ABL tyrosine kinases have attracted a considerable interest [4] – [9] .

The expression of the constitutively active BCR-ABL fusion tyrosine kinase is sufficient for the initiation and maintenance of chronic myelogenous leukemia (CML) in humans [1] . BCR-ABL is the result of the t(9;22) chromosomal translocation that leads to the fusion of the Abelson tyrosine kinase (ABL1) and the breakpoint cluster region (BCR) gene [2] , [3] . The dysregulated fusion protein activates a number of signaling pathways associated with inhibition of apoptosis and uncontrolled proliferation.

Results

The SH2 is allosterically coupled to important catalytic motifs in the kinase domain of Abl To elucidate the mechanism by which the SH2 domain stimulates the catalytic activity of the Abl kinase we first characterized the dynamics of the CD alone and with the SH2 domain bound in the activating conformation using elastic network models. In the free CD, the two predominant modes emerging from the normal mode analysis (NMA) corresponded to the well-described hinge motion [26], [28], [32] and to a twist of N- and C-lobe against each other (Supplemental Figure S1A). When the SH2 domain was included in the elastic network model, the hinge motion continued to be the principal motion, but the corresponding normal mode included a sliding of the SH2 domain along the binding interface, while the amplitude of the movement of the N-lobe of the CD was reduced (Supplemental Figure S1B). This finding suggests that one role of the SH2 domain may be to regulate the hinge motion while restricting lobe twists during catalysis. We analyzed the allosteric coupling of local conformational fluctuations along these normal modes [33] (see Methods and Text S1). Figure 2A and Supplemental Figure S1C show that local distortions in the SH2 domain are associated with conformational changes in both lobes of the CD. Important couplings are detected between the SH2 domain and specific, spatially separate motifs in the C-lobe, including the catalytically important activation loop (A-loop), the αD-αE loop, the αF-αG loop and the αG helix. The αD-αE loop forms part of the myristoyl binding pocket. The αG helix is known to have an important role in the catalytic mechanisms of many kinases. In some of them, c-Abl among them, it forms part of a platform for substrate binding [34]. Furthermore, it has been proposed that in Abl and EGFR the αF helix and the αF-αG loop are allosterically coupled to the αC helix and are involved in the dynamically enhanced stabilization of active conformations [20], [29]. Virtually all other motifs coupled to the SH2 domain in turn also couple to the crucial A-loop. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 2. Allosteric coupling and flexibility of c-Abl. A Allosteric couplings of residues in the CD to the SH2 domain. High values (yellow and red) indicate strong allosteric interactions (See also Supplemental Figure S1 A–C). B Free CD colored by RMSF from MD simulations. C SH2-CD construct colored by RMSF. (See also Figure S2). https://doi.org/10.1371/journal.pcbi.1003863.g002 The allosteric coupling analysis thus suggests that the SH2 domain acts primarily on the N-lobe loops and the A-loop. All these motifs are coupled among them through a dense network of allosteric interactions, which affect also the P-loop and the αC helix, two structural motifs that, together with the A-loop, participate actively in the catalytic process.

The SH2 domain modifies the inactive to active conformational equilibrium To investigate the effects of the SH2 domain on the A-loop conformation propensities we computed the inactive-to-active free energy landscape. To that aim we used a multiple-replica free energy algorithm (parallel-tempering Metadynamics) and a structure based hybrid force field. A similar computational strategy has been successfully used in the case of the CD of the highly homologous Src kinase to study the A-loop opening, where it was able to provide an accurate reconstruction of the free energy surface underlying the transition [42]. This force field reproduces fairly well the flexibility patterns observed with long all-atom explicit solvent simulations, apart from a small discrepancy in the region corresponding to the αG-helix in the CD. This region appears to be somewhat more rigid that it should be in the CD, but recovers the correct flexibility in the complex (Supplemental Figure S4). In absence of SH2, the A-loop of the CD is mostly closed, as expected from its in-vitro low catalytic activity (Figure 4, left). The A-loop active-like, or “open”, conformation is still a local minimum of the free energy but at a much higher value (ΔG O-C ≈6 kcal/mol). Moreover, the large free energy barrier separating the closed to the open A-loop state (ΔG ‡-O ≈14 kcal/mol) disfavors the transition. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 4. The free energy of conformational changes of the A-loop. The free energy surfaces of the A-loop transition from open (active-like) to closed (inactive-like) conformation in case of the ABL catalytic domain alone (left) and in presence of the SH2 regulatory domain in the “top-hat” conformation as a function of the contact map distances to the respective reference structures. For the deepest minima a representative structure is also shown below with the CD colored in blue, the SH2 in green, the A-loop in yellow and the aC-helix in red. https://doi.org/10.1371/journal.pcbi.1003863.g004 In contrast, the effect of the SH2 regulatory domain is to shift the equilibrium towards an active-like conformation of the A-loop (Figure 4, right) rendering it as stable as the closed conformation. The free energy barrier between the two states is greatly reduced (∼6 kcal/mol lower than without SH2) and the open basin is widened increasing the A-loop flexibility.

Specific point mutations affect the CD-SH2 interplay Based on these computational results, we proposed a number of point mutations, both in the SH2 and in the catalytic domains, that we expect to modify the dynamic interaction between the domains by interrupting essential interactions or altering the flexibility of important motifs (Table 1 and Text S1). We mainly focused on mutations in the β3-αC loop and the hinge region, as in the simulations these motifs were most strongly affected by SH2 binding. Both motifs are known to be relevant for catalysis, so mutations in these regions can be expected to affect kinase activation in general. However, based on the simulations, we predicted that for some of them the effect should be different in the free CD and in the SH2-CD construct and therefore strengthen our proposal of the how the SH2 domain interferes with c-Abl dynamics. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. List of c-Abl point mutants investigated in this study with summary of the effect of mutations. https://doi.org/10.1371/journal.pcbi.1003863.t001 The effect of the mutations was characterized by assays of c-Abl kinase activity in vivo and in vitro. None of the candidate mutations have been described previously as clinically relevant and could in principle have either a moderate activating, neutral, or disruptive effect. The effect of the mutations on the catalytic activity of c-Abl was assessed both in the context of the SH2-CD module and in the isolated CD, in order to discern whether the mutation generally affects the fold or activation state of the kinase domain, or if it specifically targets the SH2-mediated regulation mechanism. The mutations were introduced in HA-tagged Abl SH2-CD or CD-only constructs which were transiently expressed in human embryonic kidney 293 (HEK293) cells, and the level of their in vivo activity was assessed by phospho-Y412-Abl and total phosphotyrosine immunoblotting of the crude lysates. The mutated proteins mostly accumulated to levels comparable to the wild-type constructs, indicating that they did not affect protein stability. The impact on c-Abl enzymatic activity using an in vitro assay with an optimal peptide substrate was assessed for selected mutations (Figure 5). As observed previously [5], under the conditions of this assay, the wild-type SH2-CD module exhibits at least 2-fold higher activity than the wt CD construct. Since the peptide substrate contains a single phosphorylation site, the observed difference in activity could be ascribed to the SH2 domain-mediated activation that is independent of phosphotyrosine binding and processive phosphorylation events that may ensue. The comparison of SH2-CD wt and SH2-CD S173N (a FLVRES motif mutant which abolishes pTyr binding by SH2) using this assay shows no difference in kinase activity, hence pTyr binding contributions can be excluded [5]. Thus, this system should be well suited to study the effects resulting from SH2-kinase domain interactions. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 5. Effect of point mutations on the kinase activity of c-Abl. Abl proteins were immunoprecipitated and assayed for phosphorylation of an optimal Abl substrate peptide. The kinase activity was normalized to the amount of protein and to the activity of the wild-type CD or SH2-CD construct. A M297L was found to decrease kinase activity, whereas M297G had an activating effect in the absence of the SH2 domain. The kinase-dead mutant D382N presented no activity, and the known inactivating (I164E) and activating (T231R) mutations also show, accordingly, a decreased and increased activity. B The M297G mutant is significantly more active in the context of the isolated kinase domain, but not in the presence of the SH2 domain. The error bars are standard deviations from biological quadruplicates (n = 4, **P<0.01, Student t test). C Mutations E294P and E294P V299P have an activating effect. The reactions were performed at 37°C. D The activity of wild-type and E294P V299P Abl proteins was measured at increasing substrate concentrations and 25 µM ATP. E Y339G substitution is neutral, whereas Y339P diminishes Abl activity. The effect is seen both at 24°C and at an elevated temperature. F, G The ratio of the kinase activity at the elevated and room temperatures. Except for Y339P, the SH2-CD proteins retain their activity at the elevated temperature, whereas the activity of the CD constructs is reduced at least 2-fold. The error bars are standard deviations from technical triplicates except for (B). (See also Figures S4 and S5). https://doi.org/10.1371/journal.pcbi.1003863.g005 Some of the tested mutations were neutral, such as N165A and E187K in the SH2 domain (Supplemental Figure S5), or F330A in the β5 sheet. Others abolished kinase activity and could not be rescued by the SH2 domain, like K266E in the β1-β2 loop, P328G P329G in the β4-β5 loop, F330A in the β5 sheet, or G340P in the hinge. In these cases, the effect of the mutation goes beyond interruption of the CD-SH2 interaction and interferes directly with the catalytic mechanism. All of the tested mutations at the T291 position in the β3-αC loop exerted a moderately disrupting effect both in the context of the SH2-CD module, as well as the CD alone, suggesting that the effect of the mutation is not likely to be explainable solely by the abolishment of the interaction with the SH2 domain (Supplemental Figure S6). It is possible that T291 plays an important role due to its localization in the key β3-αC loop, where it might be required to confer or sustain an active conformation of the kinase domain. A number of mutants (Figure 1D), however, were more revealing in terms of understanding the SH2 activation mechanism. M297 in the β3-αC loop turned out to be very sensitive to mutations. Based on the MD simulations, we hypothesized that the β3-αC loop may act as a lever, transmitting the signal from the SH2 domain to the catalytic site and positioning the αC helix correctly. The relatively conservative M297L mutation led to a several-fold decrease in kinase activity (Figure 5A). Surprisingly, the more drastic change of the M297G mutation did not impair kinase activity, but had a slightly activating effect on the isolated CD, while it was neutral in the context of the SH2-CD construct (Figure 5A, B). The slight increase in CD activity had not been anticipated from the simulations of wt c-Abl, and suggests a de novo effect, which underlines the importance of this region for modulating c-Abl activity. In the presence of SH2, the activating effect of M297G was either suppressed, masked by similar changes, or compensated for by a corresponding drop in c-Abl activity. This suggests that SH2 indeed uses the β3-αC region as a key lever to efficiently redirect the conformational changes of CD. Changing the side chain and therefore the highly sensitive interaction network (M297L) severely decreases c-Abl activity, while introduction of additional flexibility (M297G) has a slightly activating effect and makes c-Abl activation less dependent on SH2 domain binding. The pivotal role of the β3-αC loop is further confirmed by the effect of mutating E294 and V299 to prolines, which are the equivalent residues in c-Src, a protein closely related to c-Abl but not known to be activated by SH2. We would expect the E294P V299P double mutant to stiffen the β3-αC loop lever and, consequently, the αC helix. In turn, this should activate the CD and enhance the effect of the SH2 domain. The double mutant was indeed found to be markedly activating, both in the context of the catalytic domain alone as well as within the SH2-CD construct (Figure 5C). The introduction of E294P and V299P resulted in a substantial increase in enzyme velocity compared to wt SH2-CD (Figure 5D). The single mutation E294P also exerted an activating effect on Abl activity, however the effect was more pronounced in the presence of V299P, suggesting that the two mutations might act synergistically (Figure 5C). Lastly, we have investigated the effect of changes in the flexibility of the hinge region. In the MD simulations, Y339 in the hinge region showed the highest fluctuations. Mutation of the tyrosine to glycine, which should make the hinge even more flexible, has no effect on c-Abl kinase activity (Figure 5E). In contrast, the Y339P mutation, which should rigidify the hinge region, was indeed found to be disruptive to c-Abl activity (Figure 5E). Mutation of another flexible hinge residue, G340, to proline also abrogated c-Abl activity, as observed by a decrease in phosphorylation of cellular proteins on tyrosine. Finally, another striking evidence of the stabilizing effect of the SH2 domain on the kinase domain comes from the changes of in vitro measured c-Abl activity upon temperature increase (Figure 5F, G). At higher temperature, the activity of the c-Abl CD constructs decreased substantially, most likely due to an increase in mobility that is undirected and unproductive. However, under the same conditions, the activity of c-Abl SH2-CD increased, suggesting that the SH2 domain is able to direct the enhanced movements of the kinase towards more productive states, as had been indicated by the changes in the PCA eigenvalue spectrum due to SH2 binding. Interestingly, the Y339P mutation which should rigidify the hinge region, could not be rescued by an increase in temperature, and the activity of the mutant SH2-CD was only slightly raised at 35°C as compared to CD Y339P (Figure 5F). Contrary to the Y339P mutant in the hinge region, the M297G and E294P V299P mutants in the β3-αC loop preserve the effect of the temperature increase (Figure 5G), suggesting that it is, in fact, the hinge motion that is responsible for the effect. We suggest that the hinge region always has to maintain an important degree of flexibility in order for the kinase to be functional. In the free CD, conformations with a large opening of the hinge dominate, and additional twists and distortions render the hinge motion largely ineffective. Upon SH2 binding, the non-catalytic motions are strongly restricted and the hinge motion is directed to the optimal amplitude and opening needed for catalysis.