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

Protein-based molecular switches play critical roles in biological processes. The importance of the prolylcistransswitch 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)cistransswitch 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 itscistransequilibrium to supply the slower downstreamcis-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.

 
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NSF-PAR ID:
10084367
Author(s) / Creator(s):
; ;
Publisher / Repository:
Proceedings of the National Academy of Sciences
Date Published:
Journal Name:
Proceedings of the National Academy of Sciences
Volume:
116
Issue:
7
ISSN:
0027-8424
Page Range / eLocation ID:
p. 2589-2594
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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