Restructuring an interdomain linker in the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex of Escherichia coli.

The lipoyl, subunit-binding and catalytic domains of the dihydrolipoamide acetyltransferase subunits (E2p) of the Escherichia coli pyruvate dehydrogenase complex are connected by linker sequences which are characteristically rich in alanine and proline residues. By facilitating domain movement these linkers are thought to promote interactions between the three types of active site that participate in the catalytic cycle of the complex. To investigate functional constraints associated with linker composition and sequence, the natural linker of an E2p subunit containing one lipoyl domain was replaced by shorter sequences containing: mixtures of alanine plus proline residues; mainly alanine; mainly proline; and mainly charged residues. Each artificial linker possessed a central histidine residue for assessing linker flexibility by 1H-NMR spectroscopy. The resultant complexes exhibited 181% (proline), 74-79% (alanine plus proline), 63% (alanine) and 7% (charged residues) of parental activity compared with a value of 75% expected for a complex with a comparably shortened linker. The 1H-NMR spectra showed that the alanine plus proline linkers are flexible but the alanine linker and the proline linker are relatively inflexible. Substantial variations in linker sequence and composition were tolerated without loss of function, and the enhanced activity conferred by the proline linker was attributed to the combined effects of length and relative inflexibility.