Interaction of E1 and E3 components with the core proteins of the human pyruvate dehydrogenase complex

Abstract

The human (h) pyruvate dehydrogenase complex (hPDC) consists of multiple copies of several components: pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), dihydrolipoamide dehydrogenase (E3), E3-binding protein (BP), and specific kinases and phosphatases. Mammalian PDC has a well organized structure with an icosahedral symmetry of the central E2/BP core to which the other component proteins bind non-covalently. Both hE2 and hBP consist of three well defined domains, namely the lipoyl domain, the subunit-binding domain and the inner domain, connected with flexible linkers. hE1 (alpha(2)beta(2)) binds to the subunit-binding domain of hE2; whereas hE3 binds to the E3-binding domain of hBP. Among several residues of the C-terminal surface of the hE1beta E1betaD289 was found to interact with hE2K276. The C-terminal residue I329 of the hE1beta did not participate in binding to hE2. This latter finding shows specificity in the interaction between E1beta and E2 in hPDC. The selective binding between hE3 and the E3-binding domain of hBP was investigated using specific mutants. E3R460G and E3340K showed significant reductions in affinity for hBP as determined by surface plasmon resonance. Both residues are involved in the structural organization of the binding site on hE3. Substitution of I157, N137 and R155 of hBP resulted in variable increases in the K(D) for binding with wild-type hE3, suggesting that the binding results from several weak electrostatic bonds and hydrophobic interactions among residues of hBP with residues at the interface of dimeric hE3. These results provide insight in the mono-specificity of binding of E1 to E2 and E3 to BP in hPDC and showed the differences in the binding of peripheral components (E1 and E3) in human and bacterial PDCs.

Figures

Figure 1

Binding domains of E1 and E3 in different PDCs. L, L1, L2, L3 are lipoyl domains of bacterial E2 and human E2 and BP. Sb, S and S1 are the corresponding subunit-binding domains.

Figure 2

Activities of the wild-type and mutant hE1s. Activities were measured in the PDC assay (black bars) by the formation of NADH after reconstitution of hE1 with hE2-BP and hE3 in PDC and by the DCPIP assay (hatched bars) by the reduction of DCPIP. *, undetectable. Results are means ± SE (n = 4–6). Wild-type 100% activity for E1 in hPDC was 28 U/mg protein and in DCPIP-assay was 160 mU/mg protein.

Figure 3

Comparison of the fold increase in the KD determined by surface plasmon resonance for the wild-type and mutant hE1s interaction with the wild-type and mutant L2Ss. Binding parameters were determined by SPR. UD, undetectable.

Figure 4

Comparison of the binding of the wild-type and mutant hE3 with the wild-type L3S1 (upper panel) and binding of the wild-type hE3 with the wild-type and mutant L3S1s (lower panel). Binding of hE3 with the immobilized L3S1 was detected by SPR. E3 concentration was 50 nM (upper panel) and 39 nM (lower panel).

Figure 5

Structure of hE3 with the E3-binding domain of hBP. Residues of hBP are shown in dashed circles. The pdb file 1ZY8 was used. NOTE: Colored Figure 5 is for an on-line article and black/white Figure 5 is for a print copy.