Single-or dual-mode switching of semisynthetic ribonuclease S′ with an iminodiacetic acid moiety in response to the copper(II) concentration

Ribonuclease S′ bearing iminodiacetic acid as a metal-binding site was designed and semisynthesized by self-assembly of native S-protein with chemically modified S-peptide. Iminodiacetic acid-appended amino acid (Ida⁴) was synthesized and incorporated into the S-peptide sequence by solid-phase peptide synthesis based on Fmoc chemistry at a single site or double sites of the solvent-exposed side of the S-peptide. Circular dichroism (CD) spectroscopy of these S-peptides confirmed that the Cu^II ion induced an increase or decrease of a-helix conformation depending on the replacement position. S-Peptide/S-protein titration monitored by conventional enzymatic activity and UV or CD spectroscopy demonstrated that various S-peptides form a stable complex (Ida⁴-RNase S′) with S-protein, except when Met13 and Asp14 are replaced with Ida⁴. In Cu^II titration and thermal denaturation experiments with single-site replacement mutants, Cu^II binding occurred at 1:1 stoichiometry of Ida⁴/Cu^II with perturbation of the α-helix conformation. Both 2:1 and 2:2 stoichiometries were achieved by addition of Cu^II ions to double-site mutants, and were dependent on the Cu^II concentration. Most importantly, the A6/E9Ida⁴-RNase S′ mutant shows cooperative binding of Cu^II ion with two Ida⁴; holoenzyme stability is enhanced at 2:1 stoichiometry, but at 2:2 stoichiometry, two Ida⁴ sites independently bind two Cu^II ions, and the mutant is destabilized. Other double mutants showed simple destabilization of 3D structure upon Cu^II binding. The response of the enzymatic activity of these Ida⁴-RNase S′ to the concentration of Cu^II ion was evaluated by the hydrolysis of polyuridiric acid catalyzed by RNase S′ mutants. The Cu^II-induced activity change of single and double mutants agreed well with the structural response to Cu^II, that is, the activity of A6/E9Ida⁴-RNase S′ was enhanced upon cooperative Cu^II binding at 2:1 stoichiometry and then suppressed at the 2:2 ratio. The activity of all other mutants was simply suppressed by Cu^II ions. These results represent successful switching of A6/E9Ida⁴-RNase S′ activity in dual mode, that is, suppression (OFF) or enhancement (ON), depending on the environmental Cu^II concentration. Thus it has been established that rational design of a metal-binding site can confer the dual mode of response to a metal cation on the structure and activity of an enzyme.