Roles of double-loop (130~159 aa and 175~209 aa) in ClpY(HslU)-I domain for SulA substrate degradation by ClpYQ(HslUV) protease in Escherichia coli.

An Escherichia coli ATP-dependent two-component protease, ClpYQ(HslUV), targets the SulA molecule, an SOS induced protein. ClpY recognizes, unfolds and translocates the substrates into the proteolytic site of ClpQ for degradation. ClpY is divided into three domains N, I and C. The N domain is an ATPase; the C domain allows for oligomerization, while the I domain coordinates substrate binding. In the ClpYQ complex, two layer pore sites, pore I and II, are in the center of its hexameric rings. However, the actual roles of two outer-loop (130~159 aa, L1 and 175~209 aa, L2) of the ClpY-I domain for the degradation of SulA are unclear. In this study, with ATP, the MBP-SulA molecule was bound to ClpY oligomer(s). ClpYΔL1 (ClpY deleted of loop 1) oligomers revealed an excessive SulA-binding activity. With ClpQ, it showed increased proteolytic activity for SulA degradation. Yet, ClpYΔL2 formed fewer oligomers that retained less proteolytic activity, but still had increased SulA-binding activity. In contrast, ClpYΔpore I had a lower SulA-binding activity. ClpYΔ pore I ΔL2 showed the lowest SulA-binding activity. In addition, ClpY (Q198L, Q200L), with a double point mutation in loop 2, formed stable oligomers. It also had a subtle increase in SulA-binding activity, but displayed less proteolytic activity. As a result, loop 2 has an effect on ClpY oligomerization, substrate binding and delivery. Loop 1 has a role as a gate, to prevent excessive substrate binding. Thus, accordingly, ClpY permits the formation of SulA-ClpY(6x), with ATP(s), and this complex then docks through ClpQ(6x) for ultimate proteolytic degradation.

Stepwise activity of ClpY (HslU) mutants in the processive degradation of Escherichia coli ClpYQ (HslUV) protease substrates.

In Escherichia coli, ClpYQ (HslUV) is a two-component ATP-dependent protease composed of ClpY (HslU), an ATPase with unfolding activity, and ClpQ (HslV), a peptidase. In the ClpYQ proteolytic complex, the hexameric rings of ClpY (HslU) are responsible for protein recognition, unfolding, and translocation into the proteolytic inner chamber of the dodecameric ClpQ (HslV). Each of the three domains, N, I, and C, in ClpY has its own distinct activity. The double loops (amino acids [aa] 137 to 150 and 175 to 209) in domain I of ClpY are necessary for initial recognition/tethering of natural substrates such as SulA, a cell division inhibitor protein. The highly conserved sequence GYVG (aa 90 to 93) pore I site, along with the GESSG pore II site (aa 265 to 269), contribute to the central pore of ClpY in domain N. These two central loops of ClpY are in the center of its hexameric ring in which the energy of ATP hydrolysis allows substrate translocation and then degradation by ClpQ. However, no data have been obtained to determine the effect of the central loops on substrate binding or as part of the processivity of the ClpYQ complex. Thus, we probed the features of ClpY important for substrate engagement and protease processivity via random PCR or site-specific mutagenesis. In yeast two-hybrid analysis and pulldown assays, using isolated ClpY mutants and the pore I or pore II site of ClpY, each was examined for its influence on the adjoining structural regions of the substrates. The pore I site is essential for the translocation of the engaged substrates. Our in vivo study of the ClpY mutants also revealed that an ATP-binding site in domain N, separate from its role in polypeptide (ClpY) oligomerization, is required for complex formation with ClpQ. Additionally, we found that the tyrosine residue at position 408 in ClpY is critical for stabilization of hexamer formation between subunits. Therefore, our studies suggest that stepwise activities of the ClpYQ protease are necessary to facilitate the processive degradation of its natural substrates.