HOFFMAN, G.G.; SONA, S.*; ELLINGTON, W.R.; Florida St. Univ., Tallahassee: Evolution of the Octameric State in Mitochondrial Creatine Kinases

Mitochondrial creatine kinase (MtCK) in protostomes, deuterostome invertebrates, protochordates and vertebrates exists primarily in the octameric state under physiological conditions. The octamer is thought to �bridge� contact site formation in the intermembrane space thereby creating a dynamic microcompartment facilitating phosphorylation of creatine by intramitochondrial ATP and vectorial transport of phosphocreatine into the cytoplasm. An absolutely conserved tryptophan residue (W264 in chicken sarcomeric MtCK) located at the dimer-dimer interface and present in ALL octameric MtCKs, appears to play a critical role in octamer formation and/or stability. MtCKs from sponges and the cnidarian Hydractinia do not have this Trp residue but instead have Tyr, His or Asn residues. Recombinant MtCK from the sponge Tethya (Sona et al., Biochem Biophys Res Comm 317, 1207-1214 [2004]) is clearly dimeric. To assess the role of W264 equivalent residue in octamer formation and stability we have mutated an expression construct for the octameric MtCK from the polychaete Chaetopterus and used dynamic light scattering to assess quaternary structure of the expressed protein. The non-conservative mutation of Trp to Tyr as well as the conservative mutations Trp to Cys, Trp to Leu and Trp to Phe all destabilized the octameric Chaetopterus MtCK yielding only dimers with specific activities comparable to the wild type. Mutation of three different constructs of the dimeric Tethya MtCK from Tyr to Trp failed to produce octamers. Clearly, the W264 equivalent residue is critical for octamer stability but represents one of many dimer-dimer interactions required for octamerization. (Supported by Am Hrt Assoc Predoctoral Fellowship grant 1306-544-45 to GGH and NSF grant IOB-0130024 to WRE)