Restricted motion of the lipoyl-lysine swinging ann in the pyruvate dehydrogenase complex of Escherichia coli

The three lipoyl (E2plip) domains of the dihydrolipoyl acetyltransferase co mponent of the pyruvate dehydrogenase (PDH) complex of Escherichia coli hou se the lipoyl-lysine side chain essential for active-site coupling and subs trate channelling within the complex. The structure of the unlipoylated for m of the innermost domain (E2plip(apo)) was determined by multidimensional NMR spectroscopy and found to resemble closely that of a nonfunctional hybr id domain determined previously [Green et al. (1995) J. Mol. Biol. 248, 328 -343]. The domain comprises two four-stranded beta-sheets, with the target lysine residue residing at the tip of a type-I beta-turn in one of the shee ts; the N- and C-termini lie close together at the opposite end of the mole cule in the other beta-sheet. Measurement of N-15 NMR relaxation parameters and backbone hydrogen/deuterium (H/D) exchange rates reveals that the resi dues in and surrounding the lipoyl-lysine beta-turn in the E2plip(apo) form of the domain become less flexible after lipoylation of the lysine residue . This implies that the lipoyl-lysine side chain may not sample the full ra nge of conformational space once thought. Moreover, reductive acetylation o f the lipoylated domain (E2plip(holo) -> E2plip(redac)) was accompanied by large changes in chemical shift between the two forms, and multiple resonan ces were observed for several residues. This implies a change in conformati on and the existence of multiple conformations of the domain on reductive a cetylation, which may be important in stabilizing this catalytic intermedia te.