MOLECULAR-PROPERTIES OF CLPAP PROTEASE OF ESCHERICHIA-COLI - ATP-DEPENDENT ASSOCIATION OF CLPA AND CLPP

The ClpAP protease from Escherichia coli consists of the ATP-binding r egulatory component, ClpA (subunit M-r 84 165), and the proteolytic co mponent, ClpP (subunit M-r 21 563). Our hydrodynamic studies demonstra te that the predominant forms of these proteins in solution correspond to those observed by electron microscopy. ClpP and proClpP(SA), which in electron micrographs appear to have subunits arranged in rings of seven subunits, were found by ultracentrifugation to have s(20,w) valu es of 12.2 and 13.2 S and molecular weights of 300 000 and 324 000 +/- 3000, respectively, indicating that the native form of each consists of two such rings. The two intact rings of ClpP were separated in the presence of greater than or equal to 0.1 M sulfate at low temperatures , suggesting that ring-ring contacts are polar in nature and more easi ly disrupted than subunit contacts within individual rings. Sedimentat ion equilibrium analysis indicated that ClpA purified without nucleoti de exists as an equilibrium mixture of monomers and dimers with K-a = (1.0 +/- 0.2) x 10(5) M-1 and that, upon addition of MgATP or adenosin e 5'-O-(3-thiotriphosphate), ClpA subunits associated to a form with M -r 505 000 +/- 5000, consistent with the hexameric structure seen by e lectron microscopy. Sedimentation velocity and gel-filtration analysis showed that the nucleotide-promoted hexamer of ClpA (s(20,w) = 17.2 S ) binds tightly to ClpP producing species with sao,, values of 21 and 27 S (f/f(0) = 1.5 and 1.8, respectively), consistent with electron mi crographs of ClpAP that show a single tetradecamer of ClpP associated with either one or two ClpA hexamers [Kessel et al. (1995) J. Mol. Bio l. 250, 587-594]. Under assay conditions in the presence of ATP and Mg 2+, the apparent dissociation constant of hexameric ClpA and tetradeca meric ClpP was similar to 4 +/- 2 nM. By the method of continuous vari ation, the optimal ratio of ClpA to ClpP in the active complex was 2:1 . The specific activities of limiting ClpA and ClpP determined in the presence of an excess of the other component indicated that the second molecule of ClpA provides very little additional activation of ClpP.