Molecular Dynamics Simulations of the Catalytic Subunit of Protein Kinase a Reveal New Insight into the Catalytic Process

Abstract

Protein kinases chemically add phosphate groups to target proteins. Phosphorylation usually results in a functional change of the target protein, and serves as a regulatory mechanism to elicit a response to a stimulus at the cellular or organism level. Protein kinase A (PKA) was first characterized in 1968 and is considered to be the prototype for the entire kinome. The inactive form of PKA is a heterotetramer of two catalytic subunits (PKAc) that are bound to each member of a regulatory subunit (PKAr) dimer. PKA activates when two cyclic-AMPs bind to each of the PKAr, which releases the PKAc to perform the phosphorylation reaction on a polypeptide substrate. PKAc has two lobes that flank the active site: a small N-terminal lobe that is primarily associated with binding and positioning ATP, and a large lobe that provides a docking surface for substrates or inhibitor proteins. PKAc has three major conformational states: open, intermediate and closed. Despite its early discovery, its activation and deactivation is still not fully understood. Although NMR spin relaxation experiments have probed local dynamics, the movements of the PKAc domains, that may be related to its biological activity, have not been directly examined. To gain new insight into the role that PKAc dynamics plays in the phosphotransfer reaction, we performed microsecond long molecular dynamics simulations of apo-PKAc, PKAc-ATP (intermediate) and PKAc-ATP-substrate peptide (closed, reactive) and PKAc-ATP-inhibitor peptide (closed, nonreactive) complexes. By applying a network-based analysis method, we found conformational communities for PKAc in the various states that made it possible to identify critical residues and structural transitions involved in the catalytic process.