Autophosphorylation mechanism of Mycobacterial PknB explored by molecular dynamics simulations.

Biochemistry

PubMedID: 24988180

Damle NP, Mohanty D. Autophosphorylation mechanism of Mycobacterial PknB explored by molecular dynamics simulations. Biochemistry. 2014;.
Mycobacterial Ser/Thr kinase, PknB is essential for the growth of the pathogen. Unphosphorylated PknB is catalytically inactive and its activation requires autophosphorylation of Thr residues on activation loop. Autophosphorylation can in principle take place via two distinct mechanisms. Intermolecular trans-autophosphorylation involves dimerization and phosphorylation of the activation loop of one chain in the catalytic pocket of the other chain. On the other hand, intramolecular cis-autophosphorylation involves phosphorylation of the activation loop of the kinases in its own catalytic pocket within a monomer. Based on the crystal structure of PknB in front-to-front dimeric form, it is currently believed that activation of PknB involves trans-autophosphorylation. However, due to lack of coordinates of the activation loop in the crystal structures, atomic details of the conformational changes associated with activation are yet to be deciphered. Therefore, to understand the conformational transitions associated with activation via autophosphorylation, a series of explicit solvent molecular dynamics simulations of 1┬Ás duration have been performed on each of the phosphorylated and nonphosphorylated forms of PknB catalytic domain in monomeric and dimeric states. Simulations on the phosphorylated PknB revealed differential network of crucial electrostatic and hydrophobic residues which stabilize the phosphorylated form in active conformation. Interestingly, in our simulations on non-phosphorylated monomers activation loop was observed to fold into its own active site thereby opening the novel possibility of activation through intramolecular cis-autophosphorylation. Thus our simulations suggest that autophosphorylation of PknB might also involve cis initiation followed by trans amplification as reported for other eukaryotic kinases based on recent reaction kinetics studies.