Octamer formation and coupling of cardiac sarcomeric mitochondrial creatine kinase are mediated by charged N-terminal residues.
Mitochondrial creatine kinases form octameric structures composed of four active and stable dimers. Octamer formation has been postulated to occur via interaction of the charged amino acids in the N-terminal peptide of the mature enzyme. We altered codons for charged amino acids in the N-terminal region of mature sarcomeric mitochondrial creatine kinase (sMtCK) to those encoding neutral amino acids. Transfection of normal sMtCK cDNA or those with the mutations R42G, E43G/H45G, and K46G into rat neonatal cardiomyocytes resulted in enzymatically active sMtCK expression in all. After hypoosmotic treatment of isolated mitochondria, mitochondrial inner membrane-associated and soluble sMtCK from the intermembranous space were measured. The R42G and E43G/H45G double mutation caused destabilization of the octameric structure of sMtCK and a profound reduction in binding of sMtCK to the inner mitochondrial membrane. The other mutant sMtCK proteins had modest reductions in binding. Creatine-stimulated respiration was markedly reduced in mitochondria isolated from cells transfected with the R42G mutant cDNA as compared with those transfected with normal sMtCK cDNA. We conclude that neutralization of charges in N-terminal peptide resulted in destabilization of octamer structure of sMtCK. Thus, charged amino acids at the N-terminal moiety of mature sMtCK are essential for octamer formation, binding of sMtCK with inner mitochondrial membrane, and coupling of sMtCK to oxidative phosphorylation.
Departments of Pediatrics and Molecular Biology and Pharmacology, Washington University School of Medicine and St. Louis Children's Hospital, St. Louis, Missouri 63110, USA.
Z A
ZA
Khuchua
W
W
Qin
J
J
Boero
J
J
Cheng
R M
RM
Payne
V A
VA
Saks
A W
AW
Strauss
D000599:Amino Acids, Diamino / Q000737:chemistry; D000818:Animals; D003402:Creatine Kinase / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D006639:Histidine / Q000737:chemistry; D007527:Isoenzymes; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008929:Mitochondria, Heart / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D010101:Oxygen Consumption; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012518:Sarcomeres / Q000201:enzymology*; D012995:Solubility
D000599:Amino Acids, Diamino; D007527:Isoenzymes; D008565:Membrane Proteins; D011994:Recombinant Proteins; D006639:Histidine; D003402:Creatine Kinase