Nuclear-magnetic-resonance spectroscopy studies of the flexible linkages between lipoyl domains in the pyruvate dehydrogenase multienzyme complex from Escherichia coli

The pyruvate dehydrogenase (PDH) multienzyme complex of Escherichia coli ( M , 5 x 10') contains multiple copies of three different enzymes which function successively to catalyse the oxidative decarboxylation of pyruvate to acetylCoA (Reed, 1974; Perham, 1983). The structural core of the complex is composed of 24 copies of dihydrolipoamide acetyltransferase (E2p, EC 2.3.1.12) assembled with octahedral symmetry. An unusually sophisticated form of active site coupling in the complex is thought to be facilitated by the existence in the E2p subunits of particular regions of polypeptide chain which, to judge from ' H-n.m.r. spectroscopy, enjoy substantial conformational mobility (Perham et al., 1981; Packman et al., 1982). The primary structure of the E2p chain is distinctly segmental; in particular, the N-terminal half of the polypeptide chain comprises three highly homologous repeats of approx. 100 amino acids (Stephens et al., 1983). Each repeat contains a potential lipoyl-lysine residue and can be isolated as an intact functional lipoyl domain after limited proteolysis (Packman et al., 1984). Each lipoyl segment also contains a lengthy C-terminal region of 2&30 amino acids oddly rich in alanine and proline residues, interspersed with charged amino acids. I t is likely that these regions are associated with some of the major sharp resonances in the n.m.r. spectrum of the complex, and are therefore responsible for the conformational flexibility required of the lipoyl segments of the E2p chain in active site coupling (Graham el al.. 1986). To investigate this further, a peptide was synthesized with the sequence of the innermost of the three (alanine + proline)-rich regions. The I H-n.m.r. spectra of this peptide and the PDH complex show remarkable similarity (see Fig. I ) . Thus there is little doubt that the (alanine + proline)-rich regions of E2p are the source of many sharp signals seen in the spectrum of the complex. Moreover, the close similarity between the spectra suggests that the conformational flexibility of these regions in the complex may resemble that of the synthetic peptide. A detailed n.m.r. study of the structure of the synthetic peptide in aqueous solution was therefore undertaken. Non-exchangeable protons within amino acids were readily assigned to amino acid type from COSY spectra. The chemical shifts were close to those found in small peptides and the peak intensities correspond satisfactorily with the peptide composition. At first sight the structure of the peptide would appear to be that of a typical random coil. However, the spectra also revealed a significant disperson of the alanine Ca proton chemical shifts. The "Cn.m.r. chemical shifts of the alanine residues which precede proline, and of those which do not, were tentatively assigned by comparison with the I3C-n.m.r. spectra of the tripeptides Ala-Pro-Gly and Gly-Pro-Ala. For the Cp resonance of proline in particular, the signals due to the cis and trans conformers about the X-Pro peptide bond were well resolved. The Ala-Pro peptide bonds in the synthetic peptide were found to exist predominantly (> 97%) in the trans form. In contrast, the Ala-Pro peptide bond in the tripeptide AlaPro-Gly was approx. 75% trans. This was supported by reciprocal nuclear Overhauser enhancements (noes) seen