Structural evidence for co-evolution of the regulation of contraction and energy production in skeletal muscle |
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Authors: | Jeyasingham Marina D Artigues Antonio Nadeau Owen W Carlson Gerald M |
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Affiliation: | Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Mail Stop 3030, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA |
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Abstract: | Skeletal muscle phosphorylase kinase (PhK) is a Ca2+-dependent enzyme complex, (αβγδ)4, with the δ subunit being tightly bound endogenous calmodulin (CaM). The Ca2+-dependent activation of glycogen phosphorylase by PhK couples muscle contraction with glycogen breakdown in the “excitation-contraction-energy production triad.” Although the Ca2+-dependent protein-protein interactions among the relevant contractile components of muscle are well characterized, such interactions have not been previously examined in the intact PhK complex. Here we show that zero-length cross-linking of the PhK complex produces a covalent dimer of its catalytic γ and CaM subunits. Utilizing mass spectrometry, we determined the residues cross-linked to be in an EF hand of CaM and in a region of the γ subunit sharing high sequence similarity with the Ca2+-sensitive molecular switch of troponin I that is known to bind actin and troponin C, a homolog of CaM. Our findings represent an unusual binding of CaM to a target protein and supply an explanation for the low Ca2+ stoichiometry of PhK that has been reported. They also provide direct structural evidence supporting co-evolution of the coordinate regulation by Ca2+ of contraction and energy production in muscle through the sharing of a common structural motif in troponin I and the catalytic subunit of PhK for their respective interactions with the homologous Ca2+-binding proteins troponin C and CaM. |
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Keywords: | CaM, calmodulin CBD, calmodulin-binding domain EEDQ, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline MS, mass spectrometry PhK, phosphorylase kinase TnC, troponin C TnI, troponin I N-CBD, N-terminal CBD TOF, time of flight MALDI, matrix-assisted laser desorption/ionization |
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