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Abstract
Two isoforms of human cardiac myosin, alpha and beta, share significant sequence similarities but show different kinetics. The alpha isoform is a faster motor; it spends less time being strongly bound to actin during the actomyosin cycle. With alpha isoform, actomyosin dissociates faster upon ATP binding, and the affinity of ADP to actomyosin is weaker. One can suggest that the isoform-specific actomyosin kinetics is regulated at the nucleotide binding site of human cardiac myosin. Myosin is a P-loop ATPase; the nucleotide-binding site consists of P-loop and loops switch 1 and 2. All three loops position MgATP for successful hydrolysis. Loops sequence is conserved in both myosin isoforms, and we hypothesize that the isoform-specific structural element near the active site regulates the rate of nucleotide binding and release. Previously we ran molecular dynamics simulations and found that loop S291-E317 near loop switch 1 is more compact and exhibits larger fluctuations of the position of amino acid residues in beta isoform than in alpha. In alpha isoform, the loop forms a salt bridge with loop switch 1, the bridge is not present in beta isoform. Two isoleucines I303 and I313 of loop S291-E317 are replaced with valines in alpha isoform. We introduced a double mutation I303V:I313V in beta isoform background and studied how the mutation affects the rate of ATP binding and ADP dissociation from actomyosin. We found that ATP-induced actomyosin dissociation occurs faster in the mutant, but the rate of ADP release remains the same as in the wild-type beta isoform. Due to the proximity of loop S291-E317 and loop switch 1, a faster rate of ATP-induced actomyosin dissociation indicates that loop S291-E317 affects structural dynamics of loop switch 1, and that loop switch 1 controls ATP binding to the active site. A similar rate of ADP dissociation from actomyosin in the mutant and wild-type myosin constructs indicates that loop switch 1 does not control ADP release from actomyosin.
Highlights
Myosins perform various motor functions in living organisms, regardless of high conservation of the core sequence and structure of the motor domain in all myosin classes [1,2].One can speculate that the fold of the myosin head determines the general function of the molecule, and the kinetic properties of different myosins are determined by the sequencespecific residue–residue interactions
ATP-induced actomyosin dissociation indicates that loop S291-E317 affects structural dynamics of loop switch 1, and that loop switch 1 controls ATP binding to the active site
We showed that electrostatic interactions in the SH1:SH2 region of the myosin head [4] and the salt bridge between loop 1 and myosin backbone [5] modulate the kinetics of nucleotide binding and release from human cardiac actomyosin
Summary
Myosins perform various motor functions in living organisms, regardless of high conservation of the core sequence and structure of the motor domain in all myosin classes [1,2].One can speculate that the fold of the myosin head determines the general function of the molecule, and the kinetic properties of different myosins are determined by the sequencespecific residue–residue interactions. We compared the fluctuation of the position of amino acid residues within structural elements of the myosin head and assessed permanent, isoform-specific salt bridges [3]. We showed that electrostatic interactions in the SH1:SH2 region of the myosin head [4] and the salt bridge between loop 1 and myosin backbone [5] modulate the kinetics of nucleotide binding and release from human cardiac actomyosin. Both loop 1 and the SH1:SH2 region are located relatively far from the active site of the myosin head, but they are connected to the loops of the active site. Changed electrostatic interactions within the SH1:SH2 region increase the rate of ADP release and do not affect the rate of ATP binding to actomyosin
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