Late Abstracts LA1 Theoretical studies on conformational changes imposed by phosphorylation and ATP binding in serine/threonine kinase from Bacillus subtilis, PrkC

The PrkC is a serine/threonine kinase, from Hanks super family [1]. The activity of the enzyme increases considerably after phosphorylation of four threonine residues, located on the activation loop (T162, T163, T165, T167) and one serine in the 214 position, located in the C-terminal lobe of PrkC [2]. The purpose of this work was to investigate the structural changes imposed by the phosphorylation and ATP binding of the PrkC intracellular domain (PrkCc) using molecular dynamics (MD) simulations. Four models were built, two unphosphorylated: PrkCc, PrkCc-ATP and two phosphorylated: pPrkCc, pPrkCc-ATP and subjected to 5ns long MD simulations. Sequence alignment of the PrkC shows surprisingly high similarities in the catalytic domain, and in the activation loop, with the PknB kinase from Mycobacterium tuberculosis [3]. Analysis of the ATP binding pocket (see Fig. 1) within phosphorylated and unphosphorylated complexes revealed that phosphorylation did not affect nucleotide contact residues, which are highly conserved. The overall flexibility of the activation loop, calculated by the RMSd, as well as visualized, suggests that the ATP binds after phosphorylation of the activation loop, and does not bind to the unphosphorylated kinase, since we find the PrkCcATP complex to be unstable. Residues closer than 4.0 Å from ATP (orange) are presented as sticks (black) and the PrkCc interface as a cartoon representation. The structural parts, characteristic for the activity of the enzyme, were studied: DFG-motif, HRD-motif and αC helix. It was shown that the presence of ATP is responsible for most changes of their spatial arrangement. DFGin conformation was seen only when the nucleotide was placed in the binding pocket. At the same time the presence of strong salt-bridge stabilized the position of the αC helix. The overall position of the HRD triad remains unchanged for most of the models (besides PrkCc-ATP) and indicates the active state of the enzyme. Studies on the conformation of the phosphorylated residues provided some ideas about their role. The phosphorylation loop, by interaction with the positively charged arginine cluster, found in the αC helix, stabilizes the position of the catalytically important residue E59. The conformational diversity of the activation segment increases in the presence of ATP or after phosphorylation. We speculate that the phosphorylated threonine residues do not induce critical conformational changes of the kinase, such as opening/closing conformations of the protein. Their role probably correlates with substrate recognition or dimerization, likewise the phosphorylated serine residue (pS214). Based on our study, we find the phosphorylated catalytic domain of PrkC, complexed with ATP (pPrkCc-ATP), as fully active in the closed conformation. The mechanism of the activation of PrkC is still unknown, but we hope this research brings us a little closer to its understanding.