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  • JNJ 5207852 dihydrochloride Consistent with previous simulat


    Consistent with previous simulations on the DFG-Asp-out/-in interconversion of Abl kinase, we only observe the DFG flip with protonated Asp747 (Shan et al., 2009). We showed previously that the pKa for the DFG-Asp in Abl is elevated at 6.5. Further “constant pH” simulations whereby protonation events can happen freely would be required to predict the pKa of Asp747 in DDR1 and the absolute populations of DFG-in/-out more precisely (Radak et al., 2017). Importantly, our simulations predict the change in DFG populations (5-fold), in good agreement with the 7- to 10-fold increase in biochemical kinase activity. Interestingly, we observed that during the DFG-Asp-out/-in interconversion in the DDR1 simulations the αC helix remained in the “in” conformation. This is different from other kinases, e.g., Abl, in which the αC helix transiently moves out of the way for the DFG flip to occur (Shan et al., 2009). We speculate that differences in the mechanism of the DFG flip could lead to differences in the kinetics of the DFG flip. For Abl kinase, the transition of the protonated DFG aspartate through a JNJ 5207852 dihydrochloride pocket accessible upon αC helix movement can become the rate-limiting step for the DFG flip (Shan et al., 2009). Since the DFG flip in DDR1 appears to occur without movement of the αC helix, this might indicate the absence of this rate-limiting step. To examine the relationship of the relative stability of the DFG-Asp-out conformation to the promiscuous property of DDR1 and other kinases, we compared the conformations of DDR1⋅VX-680 and Abl⋅VX-680. As stated earlier, Abl binds VX-680 in the active conformation, where the activation loop extends outward, but the P loop of the kinase folds over to shield the inhibitor from the solvent. In DDR1 the kinase is in the inactive conformation, and instead of the P loop the activation loop provides the hydrophobic contact VX-680 needs to bind (Figure 4C). Similarly, in structures of KIT, DDR1, and CSF1R bound to imatinib, the activation loop provides the hydrophobic shield that the kinked P loop of Abl provides. To test our hypothesis that the relative stability of the DFG-Asp-out conformation is related to promiscuity, we looked at data available in the PKIS2 set for a kinase that was tested in a state stabilized in DFG-Asp-in and the DFG-Asp-out conformation. We found data for activation loop phosphorylated and non-phosphorylated Abl kinase. Phosphorylated Abl is stabilized in the DFG-Asp-in active conformation (Hari et al., 2013). The number of compounds Abl binds with high affinity increases in the non-phosphorylated state, in which the DFG-Asp-out conformation is favored (Figure S2C). This implies that also for Abl kinase, the relative stability of the DFG-Asp-out conformation correlates with promiscuity. How could disease-related mutations affect the stability of the DFG-Asp-out conformation and the ability of kinases to bind ligands with high affinity? The Two Sample Logo analysis identified residues across the entire kinase domain that are specific to the group of promiscuous kinases. These residues could affect the overall stability of the DFG-Asp-out conformation, and in turn affect the ability of the kinase to bind ligands (Figure 4A). We found that seven of them correspond to sites of clinical mutations that confer imatinib resistance in Abl (Azam et al., 2003) (Figure S5B). Two of these residues (Abl resistance mutants M370T/I and M491I) are distant from the imatinib binding site and the mechanism of their resistance is unclear. Our model suggests that mutations at these sites destabilize the DFG-Asp-out conformation and thereby weaken the affinity for imatinib. In addition, the D681N/Y/G mutations in PDGFRA (Asp671 of the salt bridge in DDR1) are activating mutations that confer resistance to imatinib and sunitinib (COSMIC Study: COSU419, COSU375) (Zehir et al., 2017). This is consistent with our previous finding that a distributed network of residues stabilizes the DFG-Asp-out conformation in kinases (Seeliger et al., 2007). Mutating these residues reduces the stability of the DFG-Asp-out conformation, conferring resistance to ligands such as imatinib that favor this conformation. Similarly, when we compare the sequences of DDR1 and the less promiscuous DDR2, we find that no amino acids within 5 Å of the inhibitors differ between DDR1 and DDR2. This indicates that in fact differences in secondary shell or even more remote residues underlie the difference in promiscuity between DDR1 and DDR2 (Figures S5C–S5E).