Our data indicate that the ability
Our data indicate that the ability of these promiscuous kinases to bind chemically diverse inhibitors is defined by the hydrophobic pocket formed by the activation loop, which is only accessible in the DFG-Asp-out conformation. Inhibitors do not artificially induce the DFG-Asp-out conformation as was once surmised, but it is a stable, accessible conformation of kinases. Analysis of the available apo structures of the promiscuous kinases PDGFRA, c-Kit, and CSF1R and our MD analysis of DDR1 show that the DFG-Asp-out inactive conformation is the preferred conformation of this unusual set of kinases (Table S2). This analysis of a large chemical genomic dataset identified a phenotypically distinct class of medically important signaling enzymes. Further analysis revealed a shared structural mechanism underlying this characteristic that would not have been obtained by sequence comparison alone. We speculate that future analysis of similar datasets will yield further insight into the function, regulation, and druggability of enzymes.
Significance In this study we have analyzed a publicly available dataset of protein kinase inhibitors. Rather than analyzing the specificity of kinase inhibitors as is typically done to identify which off-target kinases an inhibitor will affect, we analyzed how kinases differ in their ability to bind a set of fairly specific inhibitors. We were able to group the 400 kinases into PYR-41 with similar inhibitor binding properties. To our surprise, a group of eight kinases was significantly more promiscuous than the rest. We found that the stability of the DFG-Asp-out inactive conformation underlies the promiscuity of these kinases. As more large-scale functional datasets become available, we expect that grouping of proteins by functional conservation (e.g., with respect to inhibitor binding) will complement the insight from grouping by sequence conservation to reveal how conserved structural features underlie the function of proteins. In the case of kinases, such studies may aid the development of better therapeutics (e.g., by identifying clusters of commonly co-inhibited kinases) and understanding of the mechanism of resistance mutations.
Acknowledgments We acknowledge support for this work by NIH R35 GM119437 (M.A.S.), R01 GM108904 (J.S.R.), P30 CA008748, R01 GM121505 (J.D.C.), R01 CA58530 (W.T.M.), SKI and Cycle for Survival (J.D.C.). We acknowledge the generosity of donated CPU time of [email protected] donors and statistical consulting by the Stony Brook University Biostatistical Consulting Core. X-ray diffraction data were collected at the National Synchrotron Light Source at Brookhaven National Laboratories beamline X29. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Introduction Discoidin domain receptors (DDR1 and DDR2) are widely expressed receptor tyrosine kinases that regulate a variety of cellular processes including cell adhesion, differentiation, proliferation and migration , collagen fibrillogenesis , , , and remodeling of the extracellular matrix . Collagen(s) is the only known ligand for DDRs . Both the collagen binding domains of the receptors , , ,  and their binding site on the collagen triple helix , , ,  have been elucidated in recent years. In addition, it has been established that DDRs exist as constitutive homodimers on the cell membrane prior to collagen binding and receptor activation , , . DDRs undergo slow and sustained receptor activation upon ligand binding. However, the reasons for the delayed kinetics of DDR phosphorylation upon ligand binding remain poorly defined. Receptor clustering or higher order receptor oligomerization has been postulated by us ,  and others , , ,  as important modulators of both DDR–collagen interaction and receptor phosphorylation.