Structural evidence for co-evolution of the regulation of contraction and energy production in skeletal muscle.
Skeletal muscle phosphorylase kinase (PhK) is a Ca(2+)-dependent enzyme complex, (alpha beta gamma delta)(4), with the delta subunit being tightly bound endogenous calmodulin (CaM). The Ca(2+)-dependent activation of glycogen phosphorylase by PhK couples muscle contraction with glycogen breakdown in the "excitation-contraction-energy production triad." Although the Ca(2+)-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 gamma 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 gamma subunit sharing high sequence similarity with the Ca(2+)-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 Ca(2+) stoichiometry of PhK that has been reported. They also provide direct structural evidence supporting co-evolution of the coordinate regulation by Ca(2+) 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 Ca(2+)-binding proteins troponin C and CaM.
Jeyasingham
Marina D
MD
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Mail Stop 3030, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA.
Artigues
Antonio
A
Nadeau
Owen W
OW
Carlson
Gerald M
GM
D000818:Animals; D002118:Calcium / Q000502:physiology; D002147:Calmodulin / Q000737:chemistry / Q000502:physiology; D020134:Catalytic Domain; D002853:Chromatography, Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry; D019281:Dimerization; D004734:Energy Metabolism; D006801:Humans; D009119:Muscle Contraction; D018482:Muscle, Skeletal / Q000502:physiology*; D010764:Phosphorylase Kinase / Q000737:chemistry* / Q000502:physiology; D021241:Spectrometry, Mass, Electrospray Ionization; D019209:Troponin C / Q000502:physiology; D019210:Troponin I / Q000502:physiology
D002147:Calmodulin; D003432:Cross-Linking Reagents; D019209:Troponin C; D019210:Troponin I; D010764:Phosphorylase Kinase; D002118:Calcium