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Identification of functional differences between recombinant human α and β cardiac myosin motors.

Deacon JC, Bloemink MJ, Rezavandi H, Geeves MA, Leinwand LA - Cell. Mol. Life Sci. (2012)

Bottom Line: For these parameters, α-subfragment 1 (S1) is far more similar to adult fast skeletal muscle myosin isoforms than to the slow β isoform despite 91% sequence identity between the motor domains of α- and β-myosin.Among the features that differentiate α- from β-S1: the ATP hydrolysis step of α-S1 is ~ten-fold faster than β-S1, α-S1 exhibits ~five-fold weaker actin affinity than β-S1, and actin·α-S1 exhibits rapid ADP release, which is >ten-fold faster than ADP release for β-S1.Overall, the cycle times are ten-fold faster for α-S1 but the portion of time each myosin spends tightly bound to actin (the duty ratio) is similar.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology and Biofrontiers Institute, University of Colorado, MCDB, Boulder, CO 80309, USA.

ABSTRACT
The myosin isoform composition of the heart is dynamic in health and disease and has been shown to affect contractile velocity and force generation. While different mammalian species express different proportions of α and β myosin heavy chain, healthy human heart ventricles express these isoforms in a ratio of about 1:9 (α:β) while failing human ventricles express no detectable α-myosin. We report here fast-kinetic analysis of recombinant human α and β myosin heavy chain motor domains. This represents the first such analysis of any human muscle myosin motor and the first of α-myosin from any species. Our findings reveal substantial isoform differences in individual kinetic parameters, overall contractile character, and predicted cycle times. For these parameters, α-subfragment 1 (S1) is far more similar to adult fast skeletal muscle myosin isoforms than to the slow β isoform despite 91% sequence identity between the motor domains of α- and β-myosin. Among the features that differentiate α- from β-S1: the ATP hydrolysis step of α-S1 is ~ten-fold faster than β-S1, α-S1 exhibits ~five-fold weaker actin affinity than β-S1, and actin·α-S1 exhibits rapid ADP release, which is >ten-fold faster than ADP release for β-S1. Overall, the cycle times are ten-fold faster for α-S1 but the portion of time each myosin spends tightly bound to actin (the duty ratio) is similar. Sequence analysis points to regions that might underlie the basis for this finding.

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Myosin contractile cycle. Myosin motors shown graphically interacting with actin filaments and nucleotides as is modeled to occur in the contractile cycle. ATP, ADP, and phosphate are represented by T, D, and Pi, respectively. Strong actin–myosin binding is indicated by black motor domains and low actin-affinity states by white motor domains. Steps occurring while bound to actin are indicated as step 1′–5′, and those while detached from actin as step 1–5. The highlighted path is the main active contractile cycle. Steps 1 and 1′ are dependent upon the equilibrium constants of ATP binding K1 and K′1, respectively. Steps 2 and 2′ are dependent upon the rate constants of a conformational change in the motor domain associated with loss of actin affinity k+2 and k′+2 respectively. Step 3 is dependent upon the rate constant of ATP hydrolysis k+3 + k−3. Steps 4 and 4′ are dependent upon the rate constants of Pi release k+4 and  respectively. Step 5 and 5′ are dependent upon the rate constants of ADP release k−ADP and , respectively. Dissociation of myosin from actin in the absence of nucleotide is governed by the dissociation constant KA. Dissociation of myosin from actin in the presence of ADP is governed by the dissociation constant KDA. Dissociation of myosin from actin after Step 2′ is essentially diffusion-limited
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Fig1: Myosin contractile cycle. Myosin motors shown graphically interacting with actin filaments and nucleotides as is modeled to occur in the contractile cycle. ATP, ADP, and phosphate are represented by T, D, and Pi, respectively. Strong actin–myosin binding is indicated by black motor domains and low actin-affinity states by white motor domains. Steps occurring while bound to actin are indicated as step 1′–5′, and those while detached from actin as step 1–5. The highlighted path is the main active contractile cycle. Steps 1 and 1′ are dependent upon the equilibrium constants of ATP binding K1 and K′1, respectively. Steps 2 and 2′ are dependent upon the rate constants of a conformational change in the motor domain associated with loss of actin affinity k+2 and k′+2 respectively. Step 3 is dependent upon the rate constant of ATP hydrolysis k+3 + k−3. Steps 4 and 4′ are dependent upon the rate constants of Pi release k+4 and respectively. Step 5 and 5′ are dependent upon the rate constants of ADP release k−ADP and , respectively. Dissociation of myosin from actin in the absence of nucleotide is governed by the dissociation constant KA. Dissociation of myosin from actin in the presence of ADP is governed by the dissociation constant KDA. Dissociation of myosin from actin after Step 2′ is essentially diffusion-limited

Mentions: Myosins are the molecular motors responsible for muscle contraction via the ATP-driven cross-bridge cycle, outlined in Fig. 1. Current interest in the myosin family of motors is focused on how this ATP-driven cross-bridge cycle is adapted for a wide range of different mechanochemical functions. Conventional myosins are the best-known family of motors, consisting of two heavy chains (MyHC) and two pairs of light chains: regulatory light chains (RLC) and essential light chains (ELC). The C-termini of the MyHCs dimerize and form a coiled-coil tail and the N-termini form the two myosin ‘heads’ or ‘motor-domains’. A lever arm, stabilized by binding of the ELC and RLC, transfers the conformational changes occurring in the motor domain into directional movement along the actin filament [1]. The single globular motor domain (often referred to as S1) is responsible for the motor function of myosin and contains the sites for both actin and nucleotide binding [2, 3].Fig. 1


Identification of functional differences between recombinant human α and β cardiac myosin motors.

Deacon JC, Bloemink MJ, Rezavandi H, Geeves MA, Leinwand LA - Cell. Mol. Life Sci. (2012)

Myosin contractile cycle. Myosin motors shown graphically interacting with actin filaments and nucleotides as is modeled to occur in the contractile cycle. ATP, ADP, and phosphate are represented by T, D, and Pi, respectively. Strong actin–myosin binding is indicated by black motor domains and low actin-affinity states by white motor domains. Steps occurring while bound to actin are indicated as step 1′–5′, and those while detached from actin as step 1–5. The highlighted path is the main active contractile cycle. Steps 1 and 1′ are dependent upon the equilibrium constants of ATP binding K1 and K′1, respectively. Steps 2 and 2′ are dependent upon the rate constants of a conformational change in the motor domain associated with loss of actin affinity k+2 and k′+2 respectively. Step 3 is dependent upon the rate constant of ATP hydrolysis k+3 + k−3. Steps 4 and 4′ are dependent upon the rate constants of Pi release k+4 and  respectively. Step 5 and 5′ are dependent upon the rate constants of ADP release k−ADP and , respectively. Dissociation of myosin from actin in the absence of nucleotide is governed by the dissociation constant KA. Dissociation of myosin from actin in the presence of ADP is governed by the dissociation constant KDA. Dissociation of myosin from actin after Step 2′ is essentially diffusion-limited
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375423&req=5

Fig1: Myosin contractile cycle. Myosin motors shown graphically interacting with actin filaments and nucleotides as is modeled to occur in the contractile cycle. ATP, ADP, and phosphate are represented by T, D, and Pi, respectively. Strong actin–myosin binding is indicated by black motor domains and low actin-affinity states by white motor domains. Steps occurring while bound to actin are indicated as step 1′–5′, and those while detached from actin as step 1–5. The highlighted path is the main active contractile cycle. Steps 1 and 1′ are dependent upon the equilibrium constants of ATP binding K1 and K′1, respectively. Steps 2 and 2′ are dependent upon the rate constants of a conformational change in the motor domain associated with loss of actin affinity k+2 and k′+2 respectively. Step 3 is dependent upon the rate constant of ATP hydrolysis k+3 + k−3. Steps 4 and 4′ are dependent upon the rate constants of Pi release k+4 and respectively. Step 5 and 5′ are dependent upon the rate constants of ADP release k−ADP and , respectively. Dissociation of myosin from actin in the absence of nucleotide is governed by the dissociation constant KA. Dissociation of myosin from actin in the presence of ADP is governed by the dissociation constant KDA. Dissociation of myosin from actin after Step 2′ is essentially diffusion-limited
Mentions: Myosins are the molecular motors responsible for muscle contraction via the ATP-driven cross-bridge cycle, outlined in Fig. 1. Current interest in the myosin family of motors is focused on how this ATP-driven cross-bridge cycle is adapted for a wide range of different mechanochemical functions. Conventional myosins are the best-known family of motors, consisting of two heavy chains (MyHC) and two pairs of light chains: regulatory light chains (RLC) and essential light chains (ELC). The C-termini of the MyHCs dimerize and form a coiled-coil tail and the N-termini form the two myosin ‘heads’ or ‘motor-domains’. A lever arm, stabilized by binding of the ELC and RLC, transfers the conformational changes occurring in the motor domain into directional movement along the actin filament [1]. The single globular motor domain (often referred to as S1) is responsible for the motor function of myosin and contains the sites for both actin and nucleotide binding [2, 3].Fig. 1

Bottom Line: For these parameters, α-subfragment 1 (S1) is far more similar to adult fast skeletal muscle myosin isoforms than to the slow β isoform despite 91% sequence identity between the motor domains of α- and β-myosin.Among the features that differentiate α- from β-S1: the ATP hydrolysis step of α-S1 is ~ten-fold faster than β-S1, α-S1 exhibits ~five-fold weaker actin affinity than β-S1, and actin·α-S1 exhibits rapid ADP release, which is >ten-fold faster than ADP release for β-S1.Overall, the cycle times are ten-fold faster for α-S1 but the portion of time each myosin spends tightly bound to actin (the duty ratio) is similar.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology and Biofrontiers Institute, University of Colorado, MCDB, Boulder, CO 80309, USA.

ABSTRACT
The myosin isoform composition of the heart is dynamic in health and disease and has been shown to affect contractile velocity and force generation. While different mammalian species express different proportions of α and β myosin heavy chain, healthy human heart ventricles express these isoforms in a ratio of about 1:9 (α:β) while failing human ventricles express no detectable α-myosin. We report here fast-kinetic analysis of recombinant human α and β myosin heavy chain motor domains. This represents the first such analysis of any human muscle myosin motor and the first of α-myosin from any species. Our findings reveal substantial isoform differences in individual kinetic parameters, overall contractile character, and predicted cycle times. For these parameters, α-subfragment 1 (S1) is far more similar to adult fast skeletal muscle myosin isoforms than to the slow β isoform despite 91% sequence identity between the motor domains of α- and β-myosin. Among the features that differentiate α- from β-S1: the ATP hydrolysis step of α-S1 is ~ten-fold faster than β-S1, α-S1 exhibits ~five-fold weaker actin affinity than β-S1, and actin·α-S1 exhibits rapid ADP release, which is >ten-fold faster than ADP release for β-S1. Overall, the cycle times are ten-fold faster for α-S1 but the portion of time each myosin spends tightly bound to actin (the duty ratio) is similar. Sequence analysis points to regions that might underlie the basis for this finding.

Show MeSH
Related in: MedlinePlus