Intramolecular Co-evolving Network Analysis Of Catalytic Domains Of PKs

Protein phosphorylation is the most widespread and important type of post-translational modification used in cellular regulation. It effects every basic cellular process, including metabolism, growth, differentiation, division, motility, and muscle contraction, immunity, learning and memory. It has been estimated that 30% of all cellular proteins are phosphorylated on at least one residue in a typical eukaryotic cell. Therefore, protein kinases (PKs) are one of the largest protein families, comprising～2% of all eukaryotic genes. Protein kinases catalyse the transfer of theγ-phosphate from ATP to specific amino acids in proteins; in eukaryotes, these are usually Ser, Thr and Tyr residues.In eukaryotes, almost all PKs have a structurally well conserved two-lobe structure of the catalytic domains. The peptide substrate is held in the groove between the two lobes. When PKs are in the active forms, the catalytic domains of these PKs have a similar 'closed' conformation. In fact, protein substrate binding can be divided into two components: binding interactions at the active site and binding interactions at a site distal to the active site, usually at the C-terminal of catalytic subunit. Previous studies suggested that there are communication pathways between the active and distal binding sites in PKs. This coupling pathway remains to be determined.How can we identify this coupling pathway in PKs? We used sequence-based statistical methods for estimating covariant residues in the multiple sequence alignments (MSA) of catalytic subunit families of Serine/Threonine and Tyrosine PKs, respectively. These methods include the Statistical Coupling, Residue Correlated, and Mutual Information analyses. The basis of these statistical methods is that the coupling of two sites in a protein, whether for structural or functional reasons, should cause those two positions to co-evolve. And then we performed the structural alignment for these two familes. At last, we made molecular dynamic simulations and residue network analysis for catalytic domain of cAMP-dependent protein kinase (PKAc) with and without its peptide substrate. Based on these studies, we got the following conclusions on catalytic subunit of PK:1) We identified two distinct co-evolving networks (i.e. the 9-shaped and y-shaped networks) in the catalytic subunits family of Serine/Threonine PKs (Ser/Thr PKc family) by using three statistical analysis methods. Theθ-shaped network links the protein substrate binding and active sites, which might participate in the coupling between substrate binding and catalysis.θ-shaped network also participates in determinants of the substrate specificity of PKs. Theγ-shaped network is mainly located the one side of substrate binding region, linking the activation loop and protein substrate binding region. It might play important role in supporting substrate binding region and the activation loop before catalysis, and mediating product releasing after catalysis. Our studies of molecular dynamics simulations and residue network analysis for interactions between PKAc and its peptide substrate provide some support for our speculation on the function of these two co-evolving networks2) We showed both differences and similaries between the sequences of Ser/Thr PKc and TyrKc families by using structural alignment and sequence analysis. These results are helpful to extensively understand the difference of substrate specificity for these two families.3) We designed, implemented and tested the new programs for residue network analysis based on the Dijkstra's algorithm of the shortest pathway. In addition, we made some new conclusions on the application of Statistical Coupling Analysis method.