Domain structure and ATP-induced conformational changes in Escherichia coli protease Lon revealed by limited proteolysis and autolysis.

Escherichia coli protease Lon (La) is an adenosine triphosphate (ATP)-regulated homo-oligomeric proteolytic complex responsible for the recognition and selective degradation of abnormal and unstable proteins. Each subunit of the protease Lon appears to consist of three functional domains: the C-terminal proteolytic containing a serine active site, the central displaying the ATPase activity, and the N-terminal with still obscure function. We have used limited proteolysis to probe the domain structure and nucleotide-induced conformational changes in the enzyme. Limited proteolysis of the native protease Lon generated a low number of stable fragments roughly corresponding to its functional domains. Conformational changes in the wild-type enzyme and its mutant forms in the presence or absence of adenine and guanine nucleotides were investigated by limited proteolysis. The nucleotide character was shown to play a key role for susceptibility of the protease Lon to limited proteolysis, in particular, for resistance of the ATPase functional domain. ATP and adenosine diphosphate displayed a protective effect of the ATPase domain of the enzyme. We suggest that these nucleotides induce conformational changes of the enzyme, transforming the ATPase domain from the most vulnerable part of the molecule into a spatially inaccessible one. Both limited proteolysis and autolysis demonstrate that the most stable part of the protease Lon molecule is its N-terminal region. Obvious resistance of the protease Lon C-terminus to proteolysis indicates that this region of the enzyme molecule including its substrate-binding and proteolytic domains has a well folded structure.