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Quantification of reaction cycle parameters for an essential molecular switch in an auxin-responsive transcription circuit in rice

Significance The two-state nature (cis and trans) of the peptide bond between any amino acid residue and proline provides a bimodal switch with an intrinsically slow flipping rate. The cis−trans exchange rate is accelerated by isomerase enzymes, bringing this protein switch into relevant timescales for biological signaling. Turning on and off the expression of specific genes in response to hormone signals is a critical aspect of organism development. In rice, the initiation of lateral root development requires a prolyl cis−trans switch and its associated isomerase. Knowledge of the parameters that define this catalyzed reaction provides important predictive power for understanding the impact of this molecular switch as a timing device in a developmental process, and has broad implications across conserved systems. Protein-based molecular switches play critical roles in biological processes. The importance of the prolyl cis−trans switch is underscored by the ubiquitous presence of peptidyl prolyl isomerases such as cyclophilins that accelerate the intrinsically slow isomerization rate. In rice, a tryptophan−proline (W-P) cis−trans switch in transcription repressor protein OsIAA11 along with its associated cyclophilin LRT2 are essential components in a negative feedback gene regulation circuit that controls lateral root initiation in response to the plant hormone auxin. Importantly, no quantitative characterizations of the individual (microscopic) thermodynamic and kinetic parameters for any cyclophilin-catalyzed W-P isomerization have been reported. Here we present NMR studies that determine and independently validate these parameters for LRT2 catalysis of the W-P motif in OsIAA11, providing predictive power for understanding the role of this switch in the auxin-responsive circuit and the resulting lateral rootless phenotype in rice. We show that the observed isomerization rate is linearly dependent on LRT2 concentration but is independent of OsIAA11 concentration over a wide range, and LRT2 is optimally tuned to maintain OsIAA11 at its cis−trans equilibrium to supply the slower downstream cis-specific proteasomal degradation with maximal OsIAA11 substrate. This indicates that accelerating the LRT2-catalyzed isomerization would not accelerate OsIAA degradation, whereas decreasing this rate via targeted mutation could reveal relationships between circuit dynamics and lateral root development. Moreover, we show that sequences flanking the highly conserved Aux/IAA W-P motif do not impact LRT2 catalysis, suggesting that the parameters determined here are broadly applicable across highly conserved cyclophilins and their Aux/IAA targets.