Function-Structure Studies on the isolated Domains of Lon Protease from Meiothermus taiwanensis WR-220

ATP-dependent Lon protease has been known as one of the most evolutionarily conserved proteins in living organisms, which is a homo-oligomeric multi-domain enzyme. Lon proteases are divided into two subfamilies, LonA and LonB. Each possesses both ATPase domain, belonging to the AAA+ superfamily and a proteolytic domain (P-domain) with a serine-lysine catalytic dyad. The difference between LonA and LonB is that LonA contains a large N-terminal domain, whereas LonB has a transmembrane domain that anchors the protein to the membrane. Lon proteases are well-known to regulate the metabolic processes and involve in protein quality control system. In this study, we analyzed the primary sequence of MtaLonA1 (793 a.a) and MtaLonA2 (815 a.a) from Meiothermus taiwanensis (WR-220) and constructed the truncated-domain MtaLonA1N (1-312 a.a), MtaLonA1A (313-585 a.a), MtaLonA1α (492-585 a.a), MtaLonA1C (586-793 a.a), MtaLonA1NA (1-585 a.a), MtaLonA1AC (313-793 a.a), MtaLonA2N (1-320 a.a), MtaLonA2A (321-601 a.a), MtaLonA2α (508-601 a.a), MtaLonA2C (602-815 a.a), MtaLonA2NA (1-601 a.a) and MtaLonA2AC (321-815 a.a). MtaLonA1, MtaLonA2 and their truncated proteins are overexpressed and purified to examine the functional and structural properties. The structural characteristic presented by circular dichroism revealed that all constructs were composed of α-helical as the major secondary structures and all possessed well-defined three-dimensional structures. For quaternary structure, MtaLonA1 reveals mainly as hexamer and MtaLonA2 might be as tetramer. In native-PAGE, MtaLonA1N-, A1NA- and MtaLonA2N-, A2NA-domain exhibited polymeric structures. Functional studies demonstrated that MtaLonA1 shows the protease, peptidase, ATPase and chaperone-like activities; whereas MtaLonA2 shows the protease, ATPase, DNA-binding activity and weaker chaperone-like activities than that of MtaLonA1. Among the truncated proteins of MtaLonA2, only MtaLonA2N (N-domain) shows no DNA-binding activity, and the MtaLonA2α-domain) reveals weaker DNA-binding capability compared to MtaLonA1α. Particularly, MtaLonA2AC has peptidase activity, protease activity, but only slight ATPase activity. MtaLonA1N-, MtaLonA1NA-, MtaLonA2N- and MtaLonA2NA domain have chaperone-like activities. These results suggested that N-terminal sequence is essential for oligomerization and chaperone-like activities. We found out the substrate, MtaHU β is specific to the MtaLonA2. Based on these results, we propose that MtaLonA1 and MtaLonA2 may have different functions and it is important to find out the specific natural substrates and crystallize structures of full-length enzymes.