Solution structures of apo and hole biotinyl domains from acetyl coenzyme A carboxylase of Escherichia coli determined by triple-resonance nuclear magnetic resonance spectroscopy

A subgene encoding the 87 C-terminal amino acids of the biotinyl carboxy ca rrier protein (BCCP) from the acetyl CoA carboxylase of Escherichia coli wa s overexpressed and the apoprotein biotinylated in vitro. The structures of both the apo and hole forms of the biotinyl domain were determined by mean s of multidimensional NMR spectroscopy. That of the hole domain was well-de fined, except for the 10 N-terminal residues, which form part of the flexib le linker between the biotinyl and subunit-binding domains of BCCP. In agre ement with X-ray crystallographic studies [Athappilly, F. K., and Hendricks on, W. A. (1995) Structure 3, 1407-1419], the structure comprises a flatten ed beta-barrel composed of two four-stranded beta-sheets with a 2-fold axis of quasi-symmetry and the biotinyl-lysine residue displayed in an exposed beta-turn on the side of the protein opposite from the N- and C-terminal re sidues. The biotin group is immobilized on the protein surface, with the ur eido ring held down by interactions with a protruding polypeptide "thumb" f ormed by residues 94-101. However,at the site of carboxylation, no evidence could be found in solution for the predicted hydrogen bond between the mai n chain O of Thr94 and the ureido H-N1'. The structure of the apo domain is essentially identical, although the packing of side chains is more favorab le in the hole domain, and this may be reflected in differences in the dyna mics of the two forms. The thumb region appears to be lacking in almost all other biotinyl domain sequences, and it may be that the immobilization of the biotinyl-lysine residue in the biotinyl domain of BCCP is an unusual re quirement, needed for the catalytic reaction of acetyl CoA carboxylase.