A Transient π-π or Cation-π Interaction between Degron and Degrader Dual Residues: A Key Step for the Substrate Recognition and Discrimination in the Processive Degradation of SulA by ClpYQ (HslUV) Protease in Escherichia coli

The Escherichia coli ATP-dependent ClpYQ protease constitutes ClpY ATPase/unfoldase and ClpQ peptidase. The Tyr(91st) residue within the central pore-I site of ClpY-hexamer is important for unfolding and translocating substrates into the catalytic site of ClpQ. We have identified the degron site (GFIMRP(147th)) of SulA, a cell-division inhibitor recognized by ClpYQ and that the Phe(143rd) residue in degron site is necessary for SulA native folded structure. However, the functional association of this degron site with the ClpYQ degrader is unknown. Here, we investigated the molecular insights into substrate recognition and discrimination by the ClpYQ protease. We found that the point mutants ClpY(Y91F)Q, ClpY(Y91H)Q, and ClpY(Y91W)Q, carrying a ring structure at the 91st residue of ClpY, efficiently degraded their natural substrates, evidenced by the suppressed bacterial methyl-methane-sulfonate (MMS) sensitivity, the reduced beta-galactosidase activity of cpsB::lacZ, and the lowest amounts of MBP-SulA in both in vivo and in vitro degradation analyses. Alternatively, mimicking the wild-type SulA, SulA(F143H), SulA(F143K) and SulA(F143W), harboring a ring structure or a cation side-group in 143(rd) residue of SulA, were efficiently degraded by ClpYQ in the bacterial cells, also revealing shorter half-lives at 41 degrees C and higher binding affinities towards ClpY in pull-down assays. Finally, ClpY(Y91F)Q and ClpY(Y91H)Q, were capable of effectively degrading SulA(F143H) and SulA(F143K), highlighting a correspondingly functional interaction between the SulA 143rd and ClpY 91st residues. According to the interchangeable substituted amino acids, our results uniquely indicate that a transient pi-pi or cation-pi interaction between the SulA 143rd and ClpY 91st residues could be aptly gripped between the degron site of substrates and the pore site of proteases (degraders) for substrate recognition and discrimination of the processive degradation.