Department of Molecular Biology & Genetics, Cornell University, Ithaca, NY, 14853, USA.
Department of Biochemistry and Biophysics and the Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
J Biomol NMR. 2019 Sep;73(8-9):493-507. doi: 10.1007/s10858-019-00258-0. Epub 2019 Aug 12.
Peptidyl Prolyl Isomerases (PPIases) accelerate cis-trans isomerization of prolyl peptide bonds. In rice, the PPIase LRT2 is essential for lateral root initiation. LRT2 displays in vitro isomerization of a highly conserved W-P peptide bond (W-P) in the natural substrate OsIAA11. OsIAA11 is a transcription repressor that, in response to the plant hormone auxin, is targeted to ubiquitin-mediated proteasomal degradation via specific recognition of the cis isomer of its W-P peptide bond. OsIAA11 controls transcription of specific genes, including its own, that are required for lateral root development. This auxin-responsive negative feedback circuit governs patterning and development of lateral roots along the primary root. The ability to tune LRT2 activity via mutagenesis is crucial for understanding and modeling the role of this bimodal switch in the auxin circuit and lateral root development. We present characterization of the thermal stability and isomerization rates of several LRT2 mutants acting on the OsIAA11 substrate. The thermally stable mutants display activities lower than that of wild-type (WT) LRT2. These include binding diminished but catalytically active P125K, binding incompetent W128A, and binding capable but catalytically incompetent H133Q mutations. Additionally, LRT2 homologs hCypA from human, TaCypA from Triticum aestivum (wheat) and PPIB from E. coli were shown to have 110, 50 and 60% of WT LRT2 activity on the OsIAA11 substrate. These studies identify several thermally stable LRT2 mutants with altered activities that will be useful for establishing relationships between cis-trans isomerization, auxin circuit dynamics, and lateral root development in rice.
肽基脯氨酰顺反异构酶（PPIases）可加速脯氨酸肽键的顺反异构化。在水稻中，PPIase LRT2 对于侧根起始是必需的。LRT2 在体外对天然底物 OsIAA11 中的高度保守的 W-P 肽键（W-P）进行异构化。OsIAA11 是一种转录抑制剂，在植物激素生长素的作用下，通过其 W-P 肽键顺式异构体的特异性识别，被靶向到泛素介导的蛋白酶体降解。OsIAA11 控制特定基因的转录，包括其自身的基因，这些基因对于侧根发育是必需的。这个生长素反应性的负反馈回路控制侧根沿着主根的模式形成和发育。通过诱变来调节 LRT2 活性的能力对于理解和模拟这个双模态开关在生长素回路和侧根发育中的作用至关重要。我们介绍了几种作用于 OsIAA11 底物的 LRT2 突变体的热稳定性和异构化速率的特征。热稳定突变体的活性低于野生型（WT）LRT2。这些突变体包括结合能力降低但催化活性保留的 P125K、结合能力丧失的 W128A 和结合能力保留但催化能力丧失的 H133Q 突变体。此外，来自人类的 hCypA、来自小麦的 TaCypA 和来自大肠杆菌的 PPIB 的 LRT2 同源物在 OsIAA11 底物上显示出 WT LRT2 活性的 110%、50%和 60%。这些研究鉴定了几种热稳定的 LRT2 突变体，它们的活性发生了改变，这将有助于建立顺反异构化、生长素回路动力学和水稻侧根发育之间的关系。
