Converter domain mutations in myosin alter structural kinetics and motor function

Abstract

Edited by Karen G. Fleming Myosins are molecular motors that use a conserved ATPase cycle to generate force. We investigated two mutations in the converter domain of myosin V (R712G and F750L) to examine how altering specific structural transitions in the motor ATPase cycle can impair myosin mechanochemistry. The corresponding mutations in the human ␤-cardiac myosin gene are associated with hypertrophic and dilated cardiomyopathy, respectively. Despite similar steady-state actin-activated ATPase and unloaded in vitro motility-sliding velocities, both R712G and F750L were less able to overcome frictional loads measured in the loaded motility assay. Transient kinetic analysis and stopped-flow FRET demonstrated that the R712G mutation slowed the maximum ATP hydrolysis and recovery-stroke rate constants, whereas the F750L mutation enhanced these steps. In both mutants, the fast and slow power-stroke as well as actin-activated phosphate release rate constants were not significantly different from WT. Time-resolved FRET experiments revealed that R712G and F750L populate the pre-and post-power-stroke states with similar FRET distance and distance distribution profiles. The R712G mutant increased the mole fraction in the post-power-stroke conformation in the strong actin-binding states, whereas the F750L decreased this population in the actomyosin ADP state. We conclude that mutations in key allosteric pathways can shift the equilibrium and/or alter the activation energy associated with key structural transitions without altering the overall conformation of the pre-and post-powerstroke states. Thus, therapies designed to alter the transition between structural states may be able to rescue the impaired motor function induced by disease mutations. . 4 The abbreviations used are: HCM, hypertrophic cardiomyopathy; M2␤, human ␤-cardiac myosin; MV, chicken myosin Va; DCM, dilated cardiomyopathy; FlAsH, fluorescein bis-arsenical hairpin binding dye; QSY, QSY TM 9 C5-maleimide; CaM, calmodulin; mantADP, 2Ј-deoxy-ADP labeled with N-methylanthraniloy at the 3Ј-ribose position; mant, N-methylanthraniloy; PBP-MDCC, phosphate-binding protein labeled with 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin; (TR) 2 FRET, transient time-resolved FRET; ELC, essential light chain; PDB, Protein Data Bank. cro ARTICLE 1554 Figure 4. Actin-activated product release. Sequential mix single-turnover experiments were performed by mixing MV with ATP, aging the reaction to form the M.ADP.P i state (0.45 M), and then mixing with different concentrations of actin in the presence of MDCC-PBP (5 M ). A, the fluorescence transients were fit to a single-exponential function (phosphate burst) followed by a linear or slow exponential rise. B, the phosphate release rate constants were plotted as a function of actin concentration and fit to a hyperbolic function to estimate the maximum rate of phosphate release. C, acto-MV (0.25 M) in presence mantADP (5 M) was mixed with saturating ATP (1 mM) at 25°C to determine the ADP release rate constan...

Dilated cardiomyopathy mutation in the converter domain of human cardiac myosin alters motor activity and response to omecamtiv mecarbil

“…One major factor that may account for the differences in ATP hydrolysis is the light chain content. Interactions between the converter domain and essential light chain have been demonstrated (27,28), and myosins that vary only by the light chain content were found to have different ATP hydrolysis rate constants (29,30). Interestingly, the light chain content of the C2C12 cell expressed/purified myosin could vary depending on how long the cells are differentiated before harvest (e.g.…”

“…Palmer et al (41) observed that cardiac muscle from heterozygous F764L knockout mice exhibited significantly reduced cross-bridge stiffness, and therefore proposed that the F764L leads to a more compliant converter domain and myosin cross-bridge. Our recent work investigated the force-generating properties of the corresponding mutation (F750L) in myosin V, and found that this mutant is less able to overcome frictional loads compared with WT in the loaded in vitro motility assay (30). The F750L mutation also alters the recovery stroke rate constant and shifts the equilibrium between conformational states of the lever arm in several nucleotide states.…”

Actin-binding compounds that affect the kinetics of the interaction of cardiac myosin with actin

“…Although most of the compounds in the present study have been used to treat non-contractile dysfunction in humans (as anti-cancer, anti-psychotic, and anti-malarial agents), it is possible that some of them, or derivatives of them, have therapeutic potential for cardiomyopathy where attenuation of contractility is beneficial, as in a hypercontractile state caused by HCM mutations in myosin or actin (38,39). Regulation of striated muscle contractility is not only thin-filament dependent, but also thick-filament dependent, whereby the myosin binding affinity to actin is attenuated via phosphorylation of the light chains (40) and myosin-binding protein C (41,42), changes in flexibility and conformation (LC domain (43), converter region (44), actin binding region (3,45), and small molecules (Mava and OM) (39) that affect the myosin ATPase cycle. In the present study, we have shown that the compounds increase ATP affinity and decrease the rate constant for the subsequent isomerization steps in ternary complex formation (and hence the release of S1 from actin), which is likely to produce actin-bound non-force producing heads, resulting in decreased ATPase activity.…”

Structural and Computational Insights into a Blebbistatin-Bound Myosin•ADP Complex with Characteristics of an ADP-Release Conformation along the Two-Step Myosin Power Stoke

“…Pivotal to this allosteric communication is the central seven-stranded β-sheet (the transducer) (twisted in the rigor state, but untwisted in the pre-power stroke state) in the myosin motor domain, which couples the large actin-binding cleft (closed while strongly attached to F-actin, and open in actin-detached states) with the active site phosphate-sensing elements—the P-loop, switch-1 and switch-2—(closed in the hydrolysis-competent pre-power stroke state, but partially or fully opened in all other states) as well as through the relay helix (bent in the pre-power stroke state, while straight in the rigor state) and SH1/SH2-region with the mechanical converter and lever arm (down position in the rigor state, and up position in the pre-power stroke state). We and others have shown that mutations in these key regions disturb the motor function by interfering with chemomechanical coupling pathways [ 4 , 5 , 6 , 7 , 8 , 9 ].…”