Probing Conformational Changes within LC2 Domains of Cardiac Myosin

Eldin, Patrick; Cathiard, Anne-Marie; Anoal, Monique; Leger, Jocelyne; Mornet, Dominique; Léger, Jean J.; Cornillon, Bernard

doi:10.1021/bi00208a005

Cited by 2 publications

(2 citation statements)

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“…One could speculate that both of these processes may operate as adaptive and/or protective mechanisms to either attenuate the effect of the FHC mutations and/or improve performance of the working muscle. Alterations introduced by the FHC mutations most likely interfere with the interaction of the RLC with the heavy chain of myosin (40) and affect the function of myosin cross-bridges during force generation (41). The region of the myosin heavy chain that contains the RLC has been postulated to undergo conformational changes that are important for working muscle (42,43).…”

Section: Discussionmentioning

confidence: 99%

Familial Hypertrophic Cardiomyopathy Mutations in the Regulatory Light Chains of Myosin Affect Their Structure, Ca2+Binding, and Phosphorylation

Szczêsna

Ghosh

et al. 2001

Journal of Biological Chemistry

105

154

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The effect of the familial hypertrophic cardiomyopathy mutations, A13T, F18L, E22K, R58Q, and P95A, found in the regulatory light chains of human cardiac myosin has been investigated. The results demonstrate that E22K and R58Q, located in the immediate extension of the helices flanking the regulatory light chain Ca 2؉ binding site, had dramatically altered Ca 2؉ binding properties. The K Ca value for E22K was decreased by ϳ17-fold compared with the wild-type light chain, and the R58Q mutant did not bind Ca 2؉ . Interestingly, Ca 2؉binding to the R58Q mutant was restored upon phosphorylation, whereas the E22K mutant could not be phosphorylated. In addition, the ␣-helical content of phosphorylated R58Q greatly increased with Ca 2؉ binding. The A13T mutation, located near the phosphorylation site (Ser-15) of the human cardiac regulatory light chain, had 3-fold lower K Ca than wild-type light chain, whereas phosphorylation of this mutant increased the Ca 2؉ affinity 6-fold. Whereas phosphorylation of wildtype light chain decreased its Ca 2؉ affinity, the opposite was true for A13T. The ␣-helical content of the A13T mutant returned to the level of wild-type light chain upon phosphorylation. The phosphorylation and Ca 2؉ binding properties of the regulatory light chain of human cardiac myosin are important for physiological function, and alteration any of these could contribute to the development of hypertrophic cardiomyopathy.There is substantial evidence that myosin regulatory light chains (RLC) 1 play a primary regulatory role in scallop and smooth muscle contraction, but their functional role in mammalian striated (skeletal and cardiac) muscle contraction is unclear. RLC, together with the essential light chain, stabilizes the 8.5-nm-long ␣-helical neck of the myosin head, with the N terminus of RLC wrapped around the heavy chain (1). Smooth muscle contraction is initiated by RLC phosphorylation with a Ca 2ϩ -calmodulin-activated myosin light chain kinase (MLCK) (2, 3). However, in skeletal and cardiac muscle, RLC phosphorylation does not activate contraction but appears to play a modulatory role (4). It was shown that RLC phosphorylation increased the Ca 2ϩ sensitivity of force in skinned skeletal (5-7) and cardiac (8) muscle fibers. In the human heart, several RLC isoforms are expressed (9, 10) preferentially in the atrium and in the ventricle. Recent studies have revealed that the ventricular RLC is one of the sarcomeric proteins associated with familial hypertrophic cardiomyopathy (FHC) (11,12). FHC is an autosomal dominant disease, characterized by left ventricular hypertrophy, myofibrillar disarray, and sudden death. It is caused by missense mutations in various genes that encode for ␤-myosin heavy chain (13), myosin-binding protein C (14), ventricular RLC and essential light chain (11,12,15), troponin T (16), troponin I (17), ␣-tropomyosin (18), actin (19), and titin (20). Depending on the affected gene, and the site of the mutation, FHC has variable presentation with regard to its degree and severity and t...

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Section: Discussionmentioning

confidence: 99%

Familial Hypertrophic Cardiomyopathy Mutations in the Regulatory Light Chains of Myosin Affect Their Structure, Ca2+Binding, and Phosphorylation

Szczêsna

Ghosh

et al. 2001

Journal of Biological Chemistry

105

154

View full text Add to dashboard Cite

show abstract

“…For immunostaining, whole EBs or dissected beating areas (that had been replated on gelatin‐ and laminin‐coated glass coverslips for 2–3 days) were rinsed with PBS and fixed with 3% paraformaldehyde for 15 min at room temperature. Cells were then permeabilized with 0.5% Triton X‐100, rinsed with PBS, blocked with PBS+1% BSA, and stained with the following primary antibodies for 1 hr at 37°C: mouse monoclonal anti‐Oct4 (sc‐5279, Santa Cruz Biotechnology Inc., Santa Cruz, CA); rabbit anti‐α‐cardiac actin (Clement et al, 2003); goat anti‐CD31 (sc‐1506, Santa Cruz); mouse monoclonal anti‐CD34 (clone BIRMA‐K3, Dako, Glostrup, Denmark); mouse monoclonal anti‐β‐tubulin type III (T8860, Sigma‐Aldrich GmbH); mouse monoclonal anti‐myosin antibody (ALX‐BC‐1150‐S, Alexis Biochemicals, Lausen, Switzerland), which specifically recognizes myosin light chain 2 ventricular (mlc2v) (Eldin et al, 1994); mouse monoclonal anti‐myosin heavy chain (MHC) (clone MF20, Developmental Studies Hybridoma Bank, University of Iowa, Iowa, IA). EBs were then rinsed with PBS+Tween 0.1%, blocked with 10% goat serum (Sigma‐Aldrich GmbH), and incubated with appropriate secondary antibodies (from Molecular Probes, Eugene, OR; Southern Biotechnology, Birmingham, AL; or Sigma‐Aldrich GmbH).…”

Section: Methodsmentioning

confidence: 99%