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.