Structural Basis for Inactivation of the Human Pyruvate Dehydrogenase Complex by Phosphorylation: Role of Disordered Phosphorylation Loops

Abstract

SUMMARY We report the crystal structures of the phosporylated pyruvate dehydrogenase (E1p) component of the human pyruvate dehydrogenase complex (PDC). The complete phosphorylation at Ser264-α (site 1) of a variant E1p protein was achieved using robust pyruvate dehydrogenase kinase 4 free of the PDC core. We show that unlike its unmodified counterpart, the presence of a phosphoryl group at Ser264-α prevents the cofactor thiamine diphosphate-induced ordering of the two loops carrying the three phosphorylation sites. The disordering of these phosphorylation loops is caused by a previously unrecognized steric clash between the phosphoryl group at site 1 and a nearby Ser266-α, which nullifies a hydrogen-bonding network essential for maintaining the loop conformations. The disordered phosphorylation loops impede the binding of lipoyl domains of the PDC core to E1p negating the reductive acetylation step. This results in the disruption of the substrate channeling in the PDC, leading to the inactivation of this catalytic machine.

The Pyruvate Dehydrogenase Complexes: Structure-based Function and Regulation

“…There are three phosphorylation sites in E1h-␣ that could be phosphorylated independently, leading to PDC inactivation: site 1 at Ser-264-␣, site 2 at Ser-271-␣, and site 3 at Ser-203-␣ (65-68). In the E1h structure, site 1 and site 2 are located on a highly conserved phosphorylation loop A, whereas site 3 is on phosphorylation loop B (31,33,69). These loops are ordered on ThDP binding and disordered in its absence.…”

“…On phosphorylation of site 1 (both Ser-264-␣ residues in the ␣ 2 ␤ 2 heterotetramer were phosphorylated), the phosphorylation loops were disordered even in the presence of ThDP, leading to loss of PDC activity (67,69). Apparently, upon Ser-264-␣ phosphorylation, the bulky phosphoryl group produces a steric clash that disrupts the H-bond network involving residues from phosphorylation loop A and Tyr-33-␤Ј from the E1p-␤ subunit, resulting in a disordered conformation of both phosphorylation loops (69). In E1h, the presence of both loops in the ordered conformation is a requirement for lipoyl domain recognition and substrate channeling to E2h (69).…”

“…Apparently, upon Ser-264-␣ phosphorylation, the bulky phosphoryl group produces a steric clash that disrupts the H-bond network involving residues from phosphorylation loop A and Tyr-33-␤Ј from the E1p-␤ subunit, resulting in a disordered conformation of both phosphorylation loops (69). In E1h, the presence of both loops in the ordered conformation is a requirement for lipoyl domain recognition and substrate channeling to E2h (69). An alternative explanation based on the structure of E1h S264E (site 1 pseudo-phosphorylated) suggested that both steric and electrostatic factors affect substrate channeling between E1h and E2h (33).…”

Structure-guided Development of Specific Pyruvate Dehydrogenase Kinase Inhibitors Targeting the ATP-binding Pocket

“…Dihydrolipoamide mimetics, including AZD7545 (23) and secondary amides of SDZ048-619 (24), have also been developed. This family of compounds inhibits PDK2 activity by impeding PDK binding to the E2/E3BP core of PDC (25). Paradoxically, these dihydrolipoamide mimetics strongly stimulates PDC core-free PDK4 activity in vitro, which precludes these compounds as bona fide PDK inhibitors (26).…”

Pivotal Role of the C-terminal DW-motif in Mediating Inhibition of Pyruvate Dehydrogenase Kinase 2 by Dichloroacetate

“…Each PDK isoform exhibits different site specificity; all four isoforms phosphorylate sites 1 and 2 at different rates, but only PDK1 modifies site 3 (10,11). Phosphorylation at site 1 prevents cofactor thiamine diphosphate-induced ordering of the loop conformations in the E1p active site, which interrupts lipoic acid-bearing domain binding, resulting in the inactivation of PDC (12).…”