The Local Environment of Loop Switch 1 Modulates the Rate of ATP-Induced Dissociation of Human Cardiac Actomyosin.

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.

Electrostatic interaction of loop 1 and backbone of human cardiac myosin regulates the rate of ATP induced actomyosin dissociation.

Double mutation D208Q:K450L was introduced in the beta isoform of human cardiac myosin to remove the salt bridge D208:K450 connecting loop 1 and the seven-stranded beta sheet within the myosin head. Beta isoform-specific salt bridge D208:K450, restricting the flexibility of loop 1, was previously discovered in molecular dynamics simulations. Earlier it was proposed that loop 1 modulates nucleotide affinity to actomyosin and we hypothesized that the electrostatic interactions between loop 1 and myosin head backbone regulate ATP binding to and ADP dissociation from actomyosin, and therefore, the time of the strong actomyosin binding. To examine the hypothesis we expressed the wild type and mutant of the myosin head construct (1-843 amino acid residues) in differentiated C2C12 cells, and the kinetics of ATP-induced actomyosin dissociation and ADP release were characterized using stopped-flow spectrofluorometry. Both constructs exhibit a fast rate of ATP binding to actomyosin and a slow rate of ADP dissociation, showing that ADP release limits the time of the strongly bound state of actomyosin. We observed a faster rate of ATP-induced actomyosin dissociation with the mutant, compared to the wild type actomyosin. The rate of ADP release from actomyosin remains the same for the mutant and the wild type actomyosin. We conclude that the flexibility of loop 1 is a factor affecting the rate of ATP binding to actomyosin and actomyosin dissociation. The flexibility of loop 1 does not affect the rate of ADP release from human cardiac actomyosin.

Electrostatic interactions in the SH1-SH2 helix of human cardiac myosin modulate the time of strong actomyosin binding.

Two single mutations, R694N and E45Q, were introduced in the beta isoform of human cardiac myosin to remove permanent salt bridges E45:R694 and E98:R694 in the SH1-SH2 helix of the myosin head. Beta isoform-specific bridges E45:R694 and E98:R694 were discovered in the molecular dynamics simulations of the alpha and beta myosin isoforms. Alpha and beta isoforms exhibit different kinetics, ADP dissociates slower from actomyosin containing beta myosin isoform, therefore, beta myosin stays strongly bound to actin longer. We hypothesize that the electrostatic interactions in the SH1-SH2 helix modulate the affinity of ADP to actomyosin, and therefore, the time of the strong actomyosin binding. Wild type and the mutants of the myosin head construct (1-843 amino acid residues) were expressed in differentiated C2C12 cells, and the duration of the strongly bound state of actomyosin was characterized using transient kinetics spectrophotometry. All myosin constructs exhibited a fast rate of ATP binding to actomyosin and a slow rate of ADP dissociation, showing that ADP release limits the time of the strongly bound state of actomyosin. The mutant R694N showed a faster rate of ADP release from actomyosin, compared to the wild type and the E45Q mutant, thus indicating that electrostatic interactions within the SH1-SH2 helix region of human cardiac myosin modulate ADP release and thus, the duration of the strongly bound state of actomyosin.

CaATP prolongs strong actomyosin binding and promotes futile myosin stroke.

Calcium plays an essential role in muscle contraction, regulating actomyosin interaction by binding troponin of thin filaments. There are several buffers for calcium in muscle, and those buffers play a crucial role in the formation of the transient calcium wave in sarcomere upon muscle activation. One such calcium buffer in muscle is ATP. ATP is a fuel molecule, and the important role of MgATP in muscle is to bind myosin and supply energy for the power stroke. Myosin is not a specific ATPase, and CaATP also supports myosin ATPase activity. The concentration of CaATP in sarcomeres reaches 1% of all ATP available. Since 294 myosin molecules form a thick filament, naïve estimation gives three heads per filament with CaATP bound, instead of MgATP. We found that CaATP dissociates actomyosin slower than MgATP, thus increasing the time of the strong actomyosin binding. The rate of the basal CaATPase is faster than that of MgATPase, myosin readily produces futile stroke with CaATP. When calcium is upregulated, as in malignant hyperthermia, kinetics of myosin and actomyosin interaction with CaATP suggest that myosin CaATPase activity may contribute to observed muscle rigidity and enhanced muscle thermogenesis.

Electrostatic interactions in the force-generating region of the human cardiac myosin modulate ADP dissociation from actomyosin.

Human cardiac myosin has two isoforms, alpha and beta, sharing significant sequence similarity, but different in kinetics: ADP release from actomyosin is an order of magnitude faster in the alpha myosin isoform. Apparently, small differences in the sequence are responsible for distinct local inter-residue interactions within alpha and beta isoforms, leading to such a dramatic difference in the rate of ADP release. Our analysis of structural kinetics of alpha and beta isoforms using molecular dynamics simulations revealed distinct dynamics of SH1:SH2 helix within the force-generation region of myosin head. The simulations showed that the residue R694 of the helix forms two permanent salt bridges in the beta isoform, which are not present in the alpha isoform. We hypothesized that the isoform-specific electrostatic interactions play a role in the difference of kinetic properties of myosin isoforms. We prepared R694N mutant in the beta isoform background to destabilize electrostatic interactions in the force-generating region of the myosin head. Our experimental data confirm faster ADP release from R694N actomyosin mutant, but is not as dramatic as the difference of kinetics of ADP release in the alpha and beta isoforms.