Octamer Formation and Coupling of Cardiac Sarcomeric Mitochondrial Creatine Kinase Are Mediated by Charged N-terminal Residues

Abstract

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. Sarcomeric mitochondrial creatine kinase (sMtCK)1 is a member of a gene family of four homologous CK genes (1-4). The M-CK and B-CK genes encode soluble, cytoplasmic homodimeric enzymes necessary for ATP production from creatine phosphate at sites of high energy utilization, such as the contractile apparatus. These cytosolic CKs share about 85% similarity to each other in a particular species, and each has 77-91% homology among vertebrate species. Two additional genes encode different mitochondrial CKs, which are localized on the outer surface of the inner mitochondrial membrane (IMM) (2, 3). sMtCK expression is restricted to heart and skeletal muscle, but ubiquitous MtCK is present in many tissues, especially brain and smooth muscle. Mammalian MtCKs are only 60 -65% similar to cytosolic CKs and about 80% homologous to each other, but sMtCKs share 95% identity across species. This suggests that, in addition to enzymatic functions, ubiquitous MtCK and sMtCK may have slightly different structural roles requiring sequence conservation across species.We previously isolated and characterized cDNA clones encoding human (2, 3) and rat (7) MtCKs. The rat sMtCK cDNA coding region consists of 1260 nucleotides, and the first 117 bp encode the transit peptide, responsible for transport of de novo synthesized precursor sMtCK from cytosol into mitochondria. During this translocation of pre-sMtCK to the intermembranous space, the transit peptide is proteolytically removed, allowing formation of the active, mature subunit (2, 4).Both MtCK proteins can f...

C-terminal Lysines Determine Phospholipid Interaction of Sarcomeric Mitochondrial Creatine Kinase

“…topology could accommodate interaction with four ANT dimers, giving rise to a molar dimer ratio of about 1:1 in such a proteolipid complex as already speculated earlier (56). The CL interactions of MtCK, together with its octameric structure, are important prerequisites for the formation of the functional microcompartments with ANT (3,30). These microcompartments control metabolite channeling between CK and ANT (Fig.…”

“…It may allow for close co-localization of MtCK and ANT in proteolipid complexes, possibly through CL membrane patches. In support of this, mutations that reduce MtCK membrane binding through destabilization of its octameric structure also reduce creatinestimulated respiration in cardiomyocyte mitochondria (30). In fact, it is well known that CL-protein interactions are important not only for activity and structural integrity of mitochondrial inner membrane proteins but also for subunit assembly and supercomplex formation (31)(32)(33)(34).…”

Creatine kinase levels in patients with bipolar disorder: depressive, manic, and euthymic phases

“…12,14,15 Furthermore, there is strong evidence that metabolic impairment and mitochondrial dysfunction are involved in the pathophysiology of BD. [16][17][18][19][20] The phosphocreatine/ CK energy circuit, which is important for maintaining normal energy homeostasis, 21,22 has a number of integrated functions, such as temporary energy buffering and energy transfer, as well as regulating metabolic capacity. 23 In view of these data, we can suggest that the increased CK activity seen in manic BD patients is a compensatory mechanism related to the mitochondrial damage that occurs in BD.…”

Acute toxicity of doxorubicin on isolated perfused heart: response of kinases regulating energy supply

“…In contrast to dimeric cytosolic CK isoenzymes, mitochondrial isoenzymes occur as two interconvertible oligomeric species, dimers and octamers (38). The latter is predominant in vivo and is considered as the physiologically active species that binds to mitochondrial membranes and functions in cellular energy transduction (23). In both control and DXR-treated samples, sMtCK dimers were hardly detectable in CPAE performed under standard conditions (Figs.…”