Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases

Structural bioinformatics reveals autophosphorylation complexes hidden in published crystals of protein kinases. Autophosphorylation sites revealed Three-dimensional structural data from crystals of protein kinases have aided the development of drugs and provided insights into kinase regulation and substrate recognition. Many protein kinases trans-autophosphorylate; one kinase phosphorylates another molecule of the same kinase. Anticipating that published crystallographic data may include undescribed information, Xu et al. developed a bioinformatics method to analyze the crystals of kinases for the presence of complexes representing the conformation of kinases during autophosphorylation. The authors identified 15 autophosphorylation complexes in the Protein Data Bank, including five that had not been previously described. With this additional information, structural motifs involved in autophosphorylation become identifiable, which may aid in rational drug design and understanding disease-associated mutations. Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Crystal structures of several homomeric protein kinase complexes have a serine, threonine, or tyrosine autophosphorylation site of one kinase monomer located in the active site of another monomer, a structural complex that we call an “autophosphorylation complex.” We developed and applied a structural bioinformatics method to identify all such autophosphorylation complexes in x-ray crystallographic structures in the Protein Data Bank (PDB). We identified 15 autophosphorylation complexes in the PDB, of which five complexes had not previously been described in the publications describing the crystal structures. These five complexes consist of tyrosine residues in the N-terminal juxtamembrane regions of colony-stimulating factor 1 receptor (CSF1R, Tyr561) and ephrin receptor A2 (EPHA2, Tyr594), tyrosine residues in the activation loops of the SRC kinase family member LCK (Tyr394) and insulin-like growth factor 1 receptor (IGF1R, Tyr1166), and a serine in a nuclear localization signal region of CDC-like kinase 2 (CLK2, Ser142). Mutations in the complex interface may alter autophosphorylation activity and contribute to disease; therefore, we mutated residues in the autophosphorylation complex interface of LCK and found that two mutations impaired autophosphorylation (T445V and N446A) and mutation of Pro447 to Ala, Gly, or Leu increased autophosphorylation. The identified autophosphorylation sites are conserved in many kinases, suggesting that, by homology, these complexes may provide insight into autophosphorylation complex interfaces of kinases that are relevant drug targets.