Slow skeletal muscle myosin-binding protein-C (MyBPC1) mediates recruitment of muscle-type creatine kinase (CK) to myosin.

Muscle contraction requires high energy fluxes, which are supplied by MM-CK (muscle-type creatine kinase) which couples to the myofibril. However, little is known about the detailed molecular mechanisms of how MM-CK participates in and is regulated during muscle contraction. In the present study, MM-CK is found to physically interact with the slow skeletal muscle-type MyBPC1 (myosin-binding protein C1). The interaction between MyBPC1 and MM-CK depended on the creatine concentration in a dose-dependent manner, but not on ATP, ADP or phosphocreatine. The MyBPC1-CK interaction favoured acidic conditions, and the two molecules dissociated at above pH 7.5. Domain-mapping experiments indicated that MM-CK binds to the C-terminal domains of MyBPC1, which is also the binding site of myosin. The functional coupling of myosin, MyBPC1 and MM-CK is further corroborated using an ATPase activity assay in which ATP expenditure accelerates upon the association of the three proteins, and the apparent K(m) value of myosin is therefore reduced. The results of the present study suggest that MyBPC1 acts as an adaptor to connect the ATP consumer (myosin) and the regenerator (MM-CK) for efficient energy metabolism and homoeostasis.

Sequential events in the irreversible thermal denaturation of human brain-type creatine kinase by spectroscopic methods.

The non-cooperative or sequential events which occur during protein thermal denaturation are closely correlated with protein folding, stability, and physiological functions. In this research, the sequential events of human brain-type creatine kinase (hBBCK) thermal denaturation were studied by differential scanning calorimetry (DSC), CD, and intrinsic fluorescence spectroscopy. DSC experiments revealed that the thermal denaturation of hBBCK was calorimetrically irreversible. The existence of several endothermic peaks suggested that the denaturation involved stepwise conformational changes, which were further verified by the discrepancy in the transition curves obtained from various spectroscopic probes. During heating, the disruption of the active site structure occurred prior to the secondary and tertiary structural changes. The thermal unfolding and aggregation of hBBCK was found to occur through sequential events. This is quite different from that of muscle-type CK (MMCK). The results herein suggest that BBCK and MMCK undergo quite dissimilar thermal unfolding pathways, although they are highly conserved in the primary and tertiary structures. A minor difference in structure might endow the isoenzymes dissimilar local stabilities in structure, which further contribute to isoenzyme-specific thermal stabilities.

A single residue substitution accounts for the significant difference in thermostability between two isoforms of human cytosolic creatine kinase.

Creatine kinase (CK) helps maintain homeostasis of intracellular ATP level by catalyzing the reversible phosphotransfer between ATP and phosphocreatine. In humans, there are two cytosolic CK isoforms, the muscle-type (M) and the brain-type (B), which frequently function as homodimers (hMMCK and hBBCK). Interestingly, these isoenzymes exhibit significantly different thermostabilities, despite high similarity in amino acid sequences and tertiary structures. In order to investigate the mechanism of this phenomenon, in this work, we first used domain swapping and site-directed mutagenesis to search for the key residues responsible for the isoenzyme-specific thermostability. Strikingly, the difference in thermostability was found to principally arise from one single residue substitution at position 36 (Pro in hBBCK vs. Leu in hMMCK). We then engaged the molecular dynamics simulations to study the molecular mechanism. The calculations imply that the P36L substitution introduces additional local interactions around residue 36 and thus further stabilizes the dimer interface through a complex interaction network, which rationalizes the observation that hMMCK is more resistant to thermal inactivation than hBBCK. We finally confirmed this molecular explanation through thermal inactivation assays on Asp36 mutants that were proposed to devastate the local interactions and thus the dimer associations in both isoenzymes.

Generation of the oxidized form protects human brain type creatine kinase against cystine-induced inactivation.

Cystine accumulation in cystinotic patients has been reported to inhibit brain type creatine kinase (BBCK), an important thiol-containing enzyme in energy homeostasis. In this research, we found that the oxidized form of BBCK (O-BBCK) was induced by cystine, and the intramolecular disulfide bond of O-BBCK was formed between Cys74 and Cys254. The wild type BBCK was found to be more resistant to the inactivation induced by cystine when compared to the single point mutant C74S or C254S. Meanwhile, the existence of GSH could protect the wild type BBCK more efficiently than the mutants. These observations suggested that the ability to generate the oxidized form could protect BBCK against the intracellular oxidative stress.

Dissimilarity in the folding of human cytosolic creatine kinase isoenzymes.

Creatine kinase (CK, EC 2.7.3.2) plays a key role in the energy homeostasis of excitable cells. The cytosolic human CK isoenzymes exist as homodimers (HMCK and HBCK) or a heterodimer (MBCK) formed by the muscle CK subunit (M) and/or brain CK subunit (B) with highly conserved three-dimensional structures composed of a small N-terminal domain (NTD) and a large C-terminal domain (CTD). The isoforms of CK provide a novel system to investigate the sequence/structural determinants of multimeric/multidomain protein folding. In this research, the role of NTD and CTD as well as the domain interactions in CK folding was investigated by comparing the equilibrium and kinetic folding parameters of HMCK, HBCK, MBCK and two domain-swapped chimeric forms (BnMc and MnBc). Spectroscopic results indicated that the five proteins had distinct structural features depending on the domain organizations. MBCK BnMc had the smallest CD signals and the lowest stability against guanidine chloride-induced denaturation. During the biphasic kinetic refolding, three proteins (HMCK, BnMc and MnBc), which contained either the NTD or CTD of the M subunit and similar microenvironments of the Trp fluorophores, refolded about 10-fold faster than HBCK for both the fast and slow phase. The fast folding of these three proteins led to an accumulation of the aggregation-prone intermediate and slowed down the reactivation rate thereby during the kinetic refolding. Our results suggested that the intra- and inter-subunit domain interactions modified the behavior of kinetic refolding. The alternation of domain interactions based on isoenzymes also provides a valuable strategy to improve the properties of multidomain enzymes in biotechnology.

Dissecting the key residues crucial for the species-specific thermostability of muscle-type creatine kinase.

Species-specific protein thermal stability is closely correlated to the living conditions of the organism, especially to its body temperature. In this research, human and zebrafish muscle-type creatine kinases (MMCKs) were taken as model proteins to investigate the molecular adaptation of proteins in poikilothermal and homoiothermal animals. Both the optimal temperature for catalysis and the thermal stability of human MMCK was much higher than those of zebrafish MMCK. Sequence alignment identified 9 amino acid variations conserved in either the teleost MMCKs or the mammal and electric ray MMCKs. Bidirectional mutations were performed to find the residues with beneficial mutations. The results showed that two residues close to the dimer interface of MMCK, the 46th and 146th residue, were crucial for species-specific thermal stability.

Isoenzyme-specific thermostability of human cytosolic creatine kinase.

Creatine kinase (CK) is a key enzyme involved in intracellular energy homeostasis. The distinct tissue distribution of muscle CK (MMCK) and brain CK (BBCK) implies that they function under conditions facing dissimilar environmental stresses. We found that MMCK and BBCK were significantly different in their stability and reversibility against heat stress. MMCK was more stable than BBCK, and BBCK was only marginally stable and began to inactivate at temperatures just above normal body temperature. The thermal inactivation of MMCK was fully irreversible, whereas that of BBCK was highly reversible at temperatures below 55 degrees C. These differences in stability were proposed to be closely correlated to the isoenzymes' adaptation to the distinct tissue environments.