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 mol-ecule, 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 oli-gomerization, 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). ClpY∆L1 (ClpY deleted of loop 1) oligomers revealed an excessive SulA-binding activity. With ClpQ, it showed increased proteolytic activity for SulA degradation. Yet, ClpY∆L2 formed fewer oligomers that retained less proteolytic activity, but still had increased SulA-binding activity. In contrast, ClpY∆pore I had a lower SulA-binding activity. ClpY∆ pore I ∆L2 showed the lowest SulA-binding activity. In addi-tion, 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, ac-cordingly, ClpY permits the formation of SulA-ClpY_(6x), with ATP(s), and this complex then docks through ClpQ_(6x) for ultimate proteolytic degrada-tion.