Hsieh F.-CChang L.-KTsai C.-HKuan J.-EWu K.-FWu CWHEI-FEN WU2021-07-262021-07-262020221260https://www.scopus.com/inward/record.uri?eid=2-s2.0-85102240396&doi=10.2323%2fjgam.2019.12.003&partnerID=40&md5=f9c3c0d40d045a6a9e505ef0d1f46af9https://scholars.lib.ntu.edu.tw/handle/123456789/572734An 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. ? 2020 Applied Microbiology, Molecular and Cellular Biosciences Research Foundation.journal article10.2323/jgam.2019.12.003324350022-s2.0-85102240396