Formation of a complex of the catalytic subunit of pyruvate dehydrogenase phosphatase isoform 1 (PDP1c) and the L2 domain forms a Ca2+ binding site and captures PDP1c as a monomer.

Pyruvate dehydrogenase phosphatase isoform 1 (PDP1) is a heterodimer with a catalytic subunit (PDP1c) and a regulatory subunit (PDP1r). The activities of PDP1 or just PDP1c are greatly increased by Ca(2+)-dependent binding to the L2 (inner lipoyl) domain of the dihydrolipoyl acetyltransferase (E2) core. Using EGTA-Ca buffers, the dependence of PDP1 or PDP1c on the level of free Ca(2+) was evaluated in activity and L2 binding studies. An increase in the Mg(2+) concentration decreased the Ca(2+) concentration required for half-maximal activation of PDP1 from 3 to 1 microM, but this parameter was unchanged at 3 microM with PDP1c. Near 1 microM Ca(2+), tight binding of PDP1 but not PDP1c to gel-anchored L2 required Mg(2+). With just Ca(2+) included, some PDP1c separated from PDP1r and remained more tightly bound to L2 than intact PDP1. Thus, formation of the PDP1c.Ca(2+).L2 complex is supported by micromolar Ca(2+) concentrations and becomes sensitive to the Mg(2+) level when PDP1c is bound to PDP1r. Sedimentation velocity and equilibrium studies revealed that PDP1c exists as a reversible monomer/dimer mixture with an equilibrium dissociation constant of 8.0 +/- 2.5 microM. L2 binds tightly and preferentially to the PDP1c monomer. Approximately 45 PDP1c monomers bind to the E2 60mer with a K(d) of approximately 0.3 microM. Isothermal titration calorimetry and (45)Ca(2+) binding studies failed to detect binding of Ca(2+) (<100 microM) to L2 or PDP1c, alone, but readily detected binding to L2 and PDP1c. Therefore, both proteins are required for formation of a complex with tightly held Ca(2+), and complex formation hinders the tendency of PDP1c to form a dimer.