Skeletal muscle phosphorylase kinase (PhK) is a Ca
2+-dependent enzyme complex, (
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βγδ)
4, with the δ 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 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 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.