Structural Characterization of Proline-rich Tyrosine Kinase 2 (PYK2) Reveals a Unique (DFG-out) Conformation and Enables Inhibitor Design

Abstract

Proline-rich tyrosine kinase 2 (PYK2) is a cytoplasmic, non-receptor tyrosine kinase implicated in multiple signaling pathways. It is a negative regulator of osteogenesis and considered a viable drug target for osteoporosis treatment. The high-resolution structures of the human PYK2 kinase domain with different inhibitor complexes establish the conventional bilobal kinase architecture and show the conformational variability of the DFG loop. The basis for the lack of selectivity for the classical kinase inhibitor, PF-431396, within the FAK family is explained by our structural analyses. Importantly, the novel DFG-out conformation with two diarylurea inhibitors (BIRB796, PF-4618433) reveals a distinct subclass of non-receptor tyrosine kinases identifiable by the gatekeeper Met-502 and the unique hinge loop conformation of Leu-504. This is the first example of a leucine residue in the hinge loop that blocks the ATP binding site in the DFG-out conformation. Our structural, biophysical, and pharmacological studies suggest that the unique features of the DFG motif, including Leu-504 hinge-loop variability, can be exploited for the development of selective protein kinase Proline-rich tyrosine kinase 2 (PYK2) is a cytoplasmic, non-receptor tyrosine kinase implicated in multiple signaling pathways. It is a negative regulator of osteogenesis and considered a viable drug target for osteoporosis treatment. The high-resolution structures of the human PYK2 kinase domain with different inhibitor complexes establish the conventional bilobal kinase architecture and show the conformational variability of the DFG loop. The basis for the lack of selectivity for the classical kinase inhibitor, PF-431396, within the FAK family is explained by our structural analyses. Importantly, the novel DFG-out conformation with two diarylurea inhibitors (BIRB796, PF-4618433) reveals a distinct subclass of non-receptor tyrosine kinases identifiable by the gatekeeper Met-502 and the unique hinge loop conformation of Leu-504. This is the first example of a leucine residue in the hinge loop that blocks the ATP binding site in the DFG-out conformation. Our structural, biophysical, and pharmacological studies suggest that the unique features of the DFG motif, including Leu-504 hinge-loop variability, can be exploited for the development of selective protein kinase Proline-rich tyrosine kinase 2 (PYK2) tyrosine kinase protein tyrosine kinase protein and kinase the kinase of non-receptor tyrosine PYK2 and FAK a and a and a of FAK is PYK2 is with in and the PYK2 is PYK2 and of the of the of with and and signaling in studies the PYK2 and signaling and and the of a osteogenesis and and a PYK2 of PYK2 protein in Importantly, the of a PYK2 inhibitor, PF-431396, and in the in the and is of a family of with and structural within the ATP binding kinase inhibitors the ATP site and for classical inhibitors ATP binding the of for drug the of be by the of multiple kinase is is in unique for kinase inhibitors a including and inhibitors that conformation of the protein the example binding and binding with the ATP site and of the the for selectivity The of the loop motif, and is of the in the of the ATP and the kinase is a of kinases a DFG-out conformation be be in the of selective kinase the of PYK2 and and multiple structures of the PYK2 kinase structures of and a with PF-431396, a kinase a PYK2 kinase and studies that PYK2 a conformation. the a with a novel binding Our and structural the and the of a selective DFG-out inhibitor with PYK2 selectivity and The in and by a the domain of PYK2 a The with and and the with PYK2 domain the and for in and the domain protein in of inhibitor 2 of and with 2 a The by for in a The and a of with 2 The for with in a for and the The and the in of The a and with the with a with and multiple a the and a with multiple and a and for in and of PYK2 a with The of the PYK2 protein in and of the of the DFG a of PYK2 with the of the DFG loop and the a of PYK2 and of the protein with and the DFG loop the in the PYK2 kinase the of the DFG loop by the of in the a PYK2 domain with and a a by in and by 2 and and a of with the with PF-431396, and with the inhibitor a protein inhibitor a of a of of 2 2 of in and a that by of the multiple of and the inhibitor the in inhibitor the of the of PYK2 with a protein with 2 2 of and and of the of in and in for in for in for in for in for in for in a The of by with the of the FAK a the a with of of the and with and the with the The a for The of the by for the in with and complexes the in with and that for and for of by of the a and the of with a and with the for that This of with a for and The and that for and the and for PYK2 and FAK that human in of the kinase domain protein and that with for the of a of kinases by a and of of structures for human PYK2 kinase of and The is kinase a bilobal The a and a The is and the loop in and by the hinge the binding site for ATP and kinase inhibitors structures inhibitors and and conformation in the DFG the structures in the of inhibitors and PF-4618433) binding distinct the ATP The loop DFG in structures and in 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the in a of and the of with a This a PYK2 inhibitor with of the osteogenesis with of PYK2 in a in be the of PF-431396, the be and the kinase selectivity that the two DFG-out a of PYK2 and selectivity the for pharmacological in the osteogenesis with of and a in and and The of the DFG-out PYK2 inhibitor a of DFG-out and and with the kinase selectivity with classical inhibitors the of PYK2 with the the in a with the the Leu-504 the binding site in the Leu-504 in a conformation that in the a the a with the of in the hinge loop. in the the DFG motif, in the and the of is by a with the of the and the a and the of the site of PYK2 in with The is for the loop the is in and two and in in the structures of PYK2 with different inhibitor complexes show the conformational variability of the DFG The DFG-out conformation in two diarylurea inhibitors reveals a distinct subclass of non-receptor tyrosine kinases identifiable in by gatekeeper Met-502 and the unique hinge loop conformation of Leu-504. Our structural and studies suggest that the features of the DFG including Leu-504 variability in the hinge loop can be in the and development of selective inhibitors protein Proline-rich tyrosine kinase 2 (PYK2) tyrosine kinase protein tyrosine kinase protein and kinase the kinase of non-receptor tyrosine PYK2 and FAK a and a and a of FAK is PYK2 is with in and the PYK2 is PYK2 and of the of the of with and and signaling in studies the PYK2 and signaling and and the of a osteogenesis and and a PYK2 of PYK2 protein in Importantly, the of a PYK2 inhibitor, PF-431396, and in the in the and PYK2 is of a family of with and structural within the ATP binding kinase inhibitors the ATP site and for classical inhibitors ATP binding the of for drug the of be by the of multiple kinase is is in unique for kinase inhibitors a including and inhibitors that conformation of the protein the example binding and binding with the ATP site and of the the for selectivity The of the loop motif, and is of the in the of the ATP and the kinase is a of kinases a DFG-out conformation be be in the of selective kinase the of PYK2 and and multiple structures of the PYK2 kinase structures of and a with PF-431396, a kinase a PYK2 kinase and studies that PYK2 a conformation. the a with a novel binding Our and structural the and the of a selective DFG-out inhibitor with PYK2 selectivity and The in and by a the domain of PYK2 a The with and and the with PYK2 domain the and for in and the domain protein in of inhibitor 2 of and with 2 a The by for in a The and a of with 2 The for with in a for and the The and the in of The a and with the with a with and multiple a the and a with multiple and a and for in and of PYK2 a with The of the PYK2 protein in and of the of the DFG a of PYK2 with the of the DFG loop and the a of PYK2 and of the protein with and the DFG loop the in the PYK2 kinase the of the DFG loop by the of in the a PYK2 domain with and a a by in and by 2 and and a of with the with PF-431396, and with the inhibitor a protein inhibitor a of a of of 2 2 of in and a that by of the multiple of and the inhibitor the in inhibitor the of the of PYK2 with a protein with 2 2 of and and of the of in and in for in for in for in for in for in for in a The of by with the of the FAK a the a with of of the and with and the with the The a for The of the by for the in with and complexes the in with and that for and for of by of the a and the of with a and with the for that This of with a for and The and that for and the and for PYK2 and FAK that human in of the kinase domain protein and that with for the of a of kinases by a and of and by a the domain of PYK2 a The with and and the with PYK2 domain the and for in and the domain protein in of inhibitor 2 of and with 2 a The by for in a The and a of with 2 The for with in a for and the The and the in of The a and with the with a with and multiple a the and a with multiple and a and for in and of PYK2 a with The of the PYK2 protein in and of the of the DFG a of PYK2 with the of the DFG loop and the a of PYK2 and of the protein with and the DFG loop the in the PYK2 kinase the of the DFG loop by the of in the a PYK2 domain with and a a by in and by 2 and and a of with the with PF-431396, and with the inhibitor a protein inhibitor a of a of of 2 2 of in and a that by of the multiple of and the inhibitor the in inhibitor the of the of PYK2 with a protein with 2 2 of and and of the of in The of by with the of the FAK a the a with of of the and with and the with the The a for The of the by for the in with and complexes the in with and that for and for of by of the a and the of with a and with the for that This of with a for and The and that for and the and for PYK2 and FAK that human in of the kinase domain protein and that with for the of a of kinases by a and of of structures for human PYK2 kinase of and The is kinase a bilobal The a and a The is and the loop in and by the hinge the binding site for ATP and kinase inhibitors structures inhibitors and and conformation in the DFG the structures in the of inhibitors and PF-4618433) binding distinct the ATP The loop DFG in structures and in the and of PYK2 and FAK of the PYK2 and FAK kinase domain reveals and of for in the in the and structural in the of in the FAK the and in FAK a the of the the PYK2 is and the structural is of and ATP binding of protein kinases the for the of and of inhibitors a the of the ATP binding site kinase structural and the ATP binding of kinases exploited selectivity and the the a by the of and The and the of the the of and This is the of the of ATP with and and ATP inhibitors of the the binding with the in the the of and of the with the and of The is by with the of The is in with and The the binding and is the The and a with of the DFG The the is the loop by with and for PYK2 and FAK with of and 2 of the binding in FAK with conformational in the structural of for the lack of selectivity two FAK family of the kinases with in a and with for and for the FAK is be the of in a the PYK2 kinase be a for a DFG-out inhibitor a that with selectivity The DFG-out kinase inhibitor, in a PYK2 and with of the for the the kinases and for a of kinases osteogenesis of with with and and with for by of and the by for The the is in and in by with the kinase inhibitors that DFG-out inhibitors of kinase two of of show that is the for PYK2 the classical inhibitor, with of and with of It is of that and PYK2 with the and with DFG-out inhibitors of the kinase for and the of and the of DFG-out It is structures that the kinase in the conformation. the that the for the kinase be for kinase the conformational The that the that of the DFG-out conformation that can the conformational of of PYK2 binding binding PYK2 binding PYK2 and binding of site and DFG-out inhibitors binding PYK2 of site of is that DFG-out inhibitors of This that PYK2 the DFG-out conformation for binding in a of the for This the of and for the development of DFG-out PYK2 a in in the for kinase that binding of DFG-out inhibitors kinase of the DFG-out conformation the a DFG-out inhibitor for the of the PYK2 kinase domain with and the the of PYK2 a with the of the The the of by conformational in the of the binding of a classical ATP site inhibitor of the the of the that the is the of a in the The of the with the in the of the and the in the DFG loop that the DFG loop with a DFG-out in The by is with the of DFG inhibitors by This that the of DFG-out inhibitors is by the of of the DFG-out conformation. of DFG-out inhibitors in the of the of the of the PYK2 kinase PYK2 PYK2 in the of of a classical ATP site PYK2 PYK2 in the of The in the is the of the DFG of PYK2 the the loop conformation with the DFG in a DFG-out The in the DFG by and the of the with the gatekeeper Met-502 the of in the PYK2 is that of in the the distinct inhibitors and the DFG-out conformation show different of the in the structures of and The different binding of the in PYK2 with the in and is in the in the gatekeeper the a gatekeeper the domain and the hinge loop structural and is in with and the structures of and the gatekeeper a the of the with in conformation in conformational for the DFG binding of in is in and the DFG-out conformation in of the structures of the by the DFG-out binding of and PYK2 with gatekeeper conformation in the hinge with in and the with the of the of the with of the The is in a of and The DFG-out conformation is by of the with the of of the and the of with the The is by with and the of the in a the hinge loop. the the the the by a with the of is the by the of the of binding in the in the hinge loop by the and of and hinge of the and the Leu-504 the a with and and This of the leucine with in the hinge loop and the and of the ATP binding the DFG-out conformation with Leu-504 is with ATP binding of of and Leu-504 with the and of our is the first example that a leucine residue in the hinge loop blocks the ATP binding site in the DFG-out of PYK2 in with and a in a DFG-out binding for and selectivity for of the of is that is a selectivity of the in a of and the of with a This a PYK2 inhibitor with of the osteogenesis with of PYK2 in a in be the of PF-431396, the be and the kinase selectivity that the two DFG-out a of PYK2 and selectivity the for pharmacological in the osteogenesis with of and a in and and The of the DFG-out PYK2 inhibitor a of DFG-out and and with the kinase selectivity with classical inhibitors the of PYK2 with the the in a with the the Leu-504 the binding site in the Leu-504 in a conformation that in the a the a with the of in the hinge loop. in the the DFG motif, in the and the of is by a with the of the and the a and the of the site of PYK2 in with The is for the loop the is in and two and in in the structures of PYK2 with different inhibitor complexes show the conformational variability of the DFG The DFG-out conformation in two diarylurea inhibitors reveals a distinct subclass of non-receptor tyrosine kinases identifiable in by gatekeeper Met-502 and the unique hinge loop conformation of Leu-504. Our structural and studies suggest that the features of the DFG including Leu-504 variability in the hinge loop can be in the and development of selective inhibitors protein of structures for human PYK2 kinase of and The is kinase a bilobal The a and a The is and the loop in and by the hinge the binding site for ATP and kinase inhibitors structures inhibitors and and conformation in the DFG the structures in the of inhibitors and PF-4618433) binding distinct the ATP The loop DFG in structures and in the and The of PYK2 and FAK of the PYK2 and FAK kinase domain reveals and of for in the in the and structural in the of in the FAK the and in FAK a the of the the PYK2 is and the structural is of and ATP binding of protein kinases the for the of and of inhibitors a the of the ATP binding site kinase structural and the ATP binding of kinases exploited selectivity and the the a by the of and The and the of the the of and This is the of the of ATP with and and ATP inhibitors of the the binding with the in the the of and of the with the and of The is by with the of The is in with and The the binding and is the The and a with of the DFG The the is the loop by with and for PYK2 and FAK with of and 2 of the binding in FAK with conformational in the loop. The structural of for the lack of selectivity two FAK family of the kinases with in a and with for and for the FAK is be the of in a the PYK2 kinase be a for a DFG-out inhibitor a that with selectivity The DFG-out kinase inhibitor, in a PYK2 and with of kinase inhibitors that DFG-out inhibitors of kinase two of of show that is the for PYK2 the classical inhibitor, with of and with of It is of that and PYK2 with the and with DFG-out inhibitors of the kinase for and the of and the of DFG-out It is structures that the kinase in the conformation. the that the for the kinase be for kinase the conformational The that the that of the DFG-out conformation that can the conformational of of is that DFG-out inhibitors of This that PYK2 the DFG-out conformation for binding in a of the for This the of and for the development of DFG-out PYK2 a in in the for kinase that binding of DFG-out inhibitors kinase of the DFG-out conformation the a DFG-out inhibitor for the of the PYK2 kinase domain with and the the of PYK2 a with the of the The the of by conformational in the of the binding of a classical ATP site inhibitor of the the of the that the is the of a in the The of the with the in the of the and the in the DFG loop that the DFG loop with a DFG-out in The by is with the of DFG inhibitors by This that the of DFG-out inhibitors is by the of of the DFG-out conformation. of DFG-out inhibitors in the of the DFG-out of PYK2 the the loop conformation with the DFG in a DFG-out The in the DFG by and the of the with the gatekeeper Met-502 the of in the PYK2 is that of in the the distinct inhibitors and the DFG-out conformation show different of the in the structures of and The different binding of the in PYK2 with the in and is in the in the gatekeeper the a gatekeeper the domain and the hinge loop structural and is in with and the structures of and the gatekeeper a the of the with in The the with the of the of the with of the The is in a of and The DFG-out conformation is by of the with the of of the and the of with the The is by with and the of Importantly, the in a the hinge loop. the the the the by a with the of is the by the of the of binding in the in the hinge loop by the and of and hinge of the and the Leu-504 the a with and and This of the leucine with in the hinge loop and the and of the ATP binding the DFG-out conformation with Leu-504 is with ATP binding of of and Leu-504 with the and of our is the first example that a leucine residue in the hinge loop blocks the ATP binding site in the DFG-out conformation. of PYK2 in with and a in a DFG-out binding for and selectivity for of the of is that is a selectivity of the in a of and the of with a This a PYK2 inhibitor with of the osteogenesis with of PYK2 in a in be the of PF-431396, the be and the kinase selectivity that the two DFG-out a of PYK2 and selectivity the for pharmacological in the osteogenesis with of and a in and and The of the DFG-out PYK2 inhibitor a of DFG-out and and with the kinase selectivity with classical inhibitors the of PYK2 with the the in a with the the Leu-504 the binding site in the Leu-504 in a conformation that in the a the a with the of in the hinge loop. in the the DFG motif, in the and the of is by a with the of the and the a and the of the the structures of PYK2 with different inhibitor complexes show the conformational variability of the DFG The DFG-out conformation in two diarylurea inhibitors reveals a distinct subclass of non-receptor tyrosine kinases identifiable in by gatekeeper Met-502 and the unique hinge loop conformation of Leu-504. Our structural and studies suggest that the features of the DFG including Leu-504 variability in the hinge loop can be in the and development of selective inhibitors protein for of for of and for of in