Protein-protein interaction revealed by NMR T-2 relaxation experiments: The lipoyl domain and E1 component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

T-2 relaxation experiments in combination with chemical shift and site-dire cted mutagenesis data were used to identify sites involved in weak but spec ific protein-protein interactions in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. The pyruvate decarboxylase compone nt, a heterotetramer E1(alpha(2)beta(2)), is responsible for the first comm itted and irreversible catalytic step. The accompanying reductive acetylati on of the lipoyl group attached to the dihydrolipoyl acetyltransferase (E2) component involves weak, transient but specific interactions between El an d the lipoyl domain of the E2 polypeptide chain. The interactions between t he free lipoyl domain (9 kDa) and free E1 alpha (41 kDa), E1 beta (35 kDa) and intact E1 alpha(2)beta(2) (152 kDa) components, all the products of gen es or sub-genes over-expressed in Escherichia coil, were investigated using heteronuclear 2D NMR spectroscopy. The experiments were conducted with uni formly N-15-labeled lipoyl domain and unlabeled El components. Major contac t points on the lipoyl domain were identified from changes in the backbone N-15 spin-spin relaxation time in the presence and absence of E1(alpha(2)be ta(2)) or its individual E1 alpha or E1 beta components. Although the Ela s ubunit houses the sequence motif associated with the essential cofactor, th iamin diphosphate, recognition of the lipoyl domain was distributed over si tes in both E1 alpha and E1 beta. A single point mutation (N40A) on the lip oyl domain significantly reduces its ability to be reductively acetylated b y the cognate El. None the less, the N40A mutant domain appears to interact with El similarly to the wild-type domain. This suggests that the lipoyl g roup of the N40A lipoyl domain is not being presented to El in the correct orientation, owing perhaps to slight perturbations in the lipoyl domain str ucture, especially in the lipoyllysine beta-turn region, as indicated by ch emical shift data. Interaction with El and subsequent reductive acetylation are not necessarily coupled. (C) 2000 Academic Press.