Mutation of a Conserved Glycine in the SH1-SH2 Helix Affects the Load-Dependent Kinetics of Myosin

Kad, Neil M.; Patlak, Joseph B.; Fagnant, Patricia M.; Trybus, Kathleen M.; Warshaw, David M.

doi:10.1529/biophysj.106.097618

Cited by 57 publications

(67 citation statements)

References 42 publications

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“…This finding has important implications for myosin force production and ATP utilization as sarcomere length changes throughout a contraction. Slower MgADP release at longer sarcomere length also provides supporting evidence for a strain-dependent MgADP release mechanism in slow-twitch skeletal fibers, consistent with single-molecule optical trap measurements describing this mechanism in multiple muscle myosin isoforms (31)(32)(33)(34).…”

Section: Discussionsupporting

confidence: 78%

“…Thus, it is likely that these structural alterations to the myofilaments also influence load-dependent mechanisms of MgADP release to slow cross-bridge detachment. Further studies have suggested that release of MgADP requires additional movement of the myosin head after the power stroke to induce a conformational change in the nucleotide binding pocket, freeing MgADP (31)(32)(33)(34)36,62). This ''double step'' was observed in both slow and fast skeletal myosins, and was associated with MgADP release from myosin, in single-molecule optical trap experiments (31).…”

Section: Discussionmentioning

confidence: 98%

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Myosin MgADP Release Rate Decreases as Sarcomere Length Increases in Skinned Rat Soleus Muscle Fibers

Fenwick

Leighton

Tanner

2016

Biophysical Journal

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Actin-myosin cross-bridges use chemical energy from MgATP hydrolysis to generate force and shortening in striated muscle. Previous studies show that increases in sarcomere length can reduce thick-to-thin filament spacing in skinned muscle fibers, thereby increasing force production at longer sarcomere lengths. However, it is unclear how changes in sarcomere length and lattice spacing affect cross-bridge kinetics at fundamental steps of the cross-bridge cycle, such as the MgADP release rate. We hypothesize that decreased lattice spacing, achieved through increased sarcomere length or osmotic compression of the fiber via dextran T-500, could slow MgADP release rate and increase cross-bridge attachment duration. To test this, we measured cross-bridge cycling and MgADP release rates in skinned soleus fibers using stochastic length-perturbation analysis at 2.5 and 2.0 mm sarcomere lengths as pCa and [MgATP] varied. In the absence of dextran, the force-pCa relationship showed greater Ca 2þ sensitivity for 2.5 vs. 2.0 mm sarcomere length fibers (pCa 50 ¼ 5.68 5 0.01 vs. 5.60 5 0.01). When fibers were compressed with 4% dextran, the length-dependent increase in Ca 2þ sensitivity of force was attenuated, though the Ca 2þ sensitivity of the force-pCa relationship at both sarcomere lengths was greater with osmotic compression via 4% dextran compared to no osmotic compression. Without dextran, the cross-bridge detachment rate slowed by~15% as sarcomere length increased, due to a slower MgADP release rate (11.2 5 0.5 vs. 13.5 5 0.7 s À1 ). In the presence of dextran, cross-bridge detachment was~20% slower at 2.5 vs. 2.0 mm sarcomere length due to a slower MgADP release rate (10.1 5 0.6 vs. 12.9 5 0.5 s À1 ). However, osmotic compression of fibers at either 2.5 or 2.0 mm sarcomere length produced only slight (and statistically insignificant) slowing in the rate of MgADP release. These data suggest that skeletal muscle exhibits sarcomere-length-dependent changes in cross-bridge kinetics and MgADP release that are separate from, or complementary to, changes in lattice spacing.

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Section: Discussionsupporting

confidence: 78%

Section: Discussionmentioning

confidence: 98%

Myosin MgADP Release Rate Decreases as Sarcomere Length Increases in Skinned Rat Soleus Muscle Fibers

Fenwick

Leighton

Tanner

2016

Biophysical Journal

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“…To reduce the stiffness of the laser traps, a partial force clamp was used (27). In brief, a 20-kHz closed loop feedback system was applied by linking the bead and laser trap positions via acoustic optical deflectors (28). The feedback frequency response is in excess of the corner frequency obtained from the Lorentzian form power spectrum of the trapped bead displacement in solution (Ͻ500 Hz (29)).…”

Section: Methodsmentioning

confidence: 99%

Load and Pi Control Flux through the Branched Kinetic Cycle of Myosin V

Kad

Trybus

Warshaw

2008

Journal of Biological Chemistry

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Myosin V is a processive actin-based motor protein that takes multiple 36-nm steps to deliver intracellular cargo to its destination. In the laser trap, applied load slows myosin V heavy meromyosin stepping and increases the probability of backsteps. In the presence of 40 mM phosphate (P i ), both forward and backward steps become less load-dependent. From these data, we infer that P i release commits myosin V to undergo a highly loaddependent transition from a state in which ADP is bound to both heads and its lead head trapped in a pre-powerstroke conformation. Increasing the residence time in this state by applying load increases the probability of backstepping or detachment. The kinetics of detachment indicate that myosin V can detach from actin at two distinct points in the cycle, one of which is turned off by the presence of P i . We propose a branched kinetic model to explain these data. Our model includes P i release prior to the most load-dependent step in the cycle, implying that P i release and load both act as checkpoints that control the flux through two parallel pathways.Myosin V is a cargo-carrying molecular motor that converts chemical energy from ATP hydrolysis into 36-nm hand-overhand strides, as it moves processively along actin tracks (1). A striking feature of the molecule is its long ␣-helical neck domain, which binds six calmodulins in series (2) and acts as a lever arm (3-6). Beyond the neck, the two heavy chains form a predominantly ␣-helical coiled-coil that ends in a globular cargo-binding domain, which is also involved in regulating the activity of the molecule (7,8).For myosin V to travel processively over long, 1-2-m distances, the ATPase activity and motion generation of the individual heads must be coordinated so that one head remains bound to actin as the other head steps forward (9). This coordination requires the heads to communicate, presumably through intramolecular strain that develops as the leading head attempts to swing its lever arm forward but is resisted by the strongly bound trailing head (5, 9 -11). This internal resistive load may slow the release of ATP hydrolysis products (i.e. ADP and/or P i ) from the active site of the leading head, whereas the positive strain experienced by the trailing head may accelerate their release (10 -14). The specific biochemical and mechanical states that each head transitions through during its processive run is far from certain, but we and others have proposed that myosin V proceeds through a branched kinetic scheme (15, 16), potentially offering the myosin V molecule alternate processive pathways as it negotiates the crowded cytoskeletal network of the cell (17) and the loads that this meshwork may present.To characterize the kinetic pathways of myosin V and the specific states that are sensitive to load, we have used the single molecule laser trap assay to examine the stepping kinetics of expressed double-headed myosin V heavy meromyosin in response to load. With increasing load, the attached lifetime following a forward step was s...

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“…Because ADP release is rate limiting for unloaded shortening velocity (17) and is thought to be a strain-dependent state in the actomyosin biochemical cycle (18)(19)(20)(21), the ADP-induced inhibition of velocity was measured in the presence and absence of load imposed by α-actinin (Fig. 3).…”

Section: Rlc Depletion Andmentioning

confidence: 99%

Cardiomyopathy-linked myosin regulatory light chain mutations disrupt myosin strain-dependent biochemistry

Greenberg

Kazmierczak

Szczesna‐Cordary

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Familial hypertrophic cardiomyopathy (FHC) is caused by mutations in sarcomeric proteins including the myosin regulatory light chain (RLC). Two such FHC mutations, R58Q and N47K, located near the cationic binding site of the RLC, have been identified from population studies. To examine the molecular basis for the observed phenotypes, we exchanged endogenous RLC from native porcine cardiac myosin with recombinant human ventricular wild type (WT) or FHC mutant RLC and examined the ability of the reconstituted myosin to propel actin filament sliding using the in vitro motility assay. We find that, whereas the mutant myosins are indistinguishable from the controls (WT or native myosin) under unloaded conditions, both R58Q-and N47K-exchanged myosins show reductions in force and power output compared with WT or native myosin. We also show that the changes in loaded kinetics are a result of mutation-induced loss of myosin strain sensitivity of ADP affinity. We propose that the R58Q and N47K mutations alter the mechanical properties of the myosin neck region, leading to altered loaddependent kinetics that may explain the observed mutant-induced FHC phenotypes.in vitro motility | load-dependent kinetics | familial hypertrophic cardiomyopathy | R58Q | N47K F amilial hypertrophic cardiomyopathy (FHC), the leading cause of sudden cardiac death in people under 30 (1), is characterized by several changes in cardiac structure including myofibrillar disarray and thickening of the left ventricle, papillary muscles, and/or septum. FHC is a disease of the sarcomere, resulting from mutations of cardiac proteins (reviewed in refs. 1 and 2) including the regulatory (RLC) and essential (ELC) light chains of myosin.Each myosin molecule is a hexamer composed of two myosin heavy chains (MHC), two RLCs, and two ELCs (3). The α-helical neck region of the MHC has been proposed to act as a lever arm, amplifying small conformational changes that originate at the myosin catalytic site into large movements needed to produce contractile force. The α-helical neck, supported by the two light chains, has been proposed to function as a compliant element (4-6), with the light chains imparting stiffness to the lever arm. Therefore, disruption of the light chain binding to the MHC could conceivably impair force generation as well as the transmission of external loads to the myosin active site, leading to a loss of myosin strain sensitivity.The RLC is a calmodulin homology protein that contains an EF-hand Ca 2+ -Mg 2+ binding site and also a highly conserved phosphorylatable serine, both sites located at the N terminus of the RLC. It has also been shown that both phosphorylation of the RLC (7, 8) and the presence of bound cation (9) play important roles in the structure and function of myosin, reinforcing the notion that there is a high degree of interdomain communication between these sites.Given the functional importance of the RLC-containing neck domain of myosin, it is not surprising that mutations of the RLC can cause FHC. Population studies have i...

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Mutation of a Conserved Glycine in the SH1-SH2 Helix Affects the Load-Dependent Kinetics of Myosin

Cited by 57 publications

References 42 publications

Myosin MgADP Release Rate Decreases as Sarcomere Length Increases in Skinned Rat Soleus Muscle Fibers

Myosin MgADP Release Rate Decreases as Sarcomere Length Increases in Skinned Rat Soleus Muscle Fibers

Load and Pi Control Flux through the Branched Kinetic Cycle of Myosin V

Cardiomyopathy-linked myosin regulatory light chain mutations disrupt myosin strain-dependent biochemistry

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