1H, 13C and 15N NMR assignments of cyclophilin LRT2 (OsCYP2) from rice

Acevedo, Lucila A.; Nicholson, Linda

doi:10.1007/s12104-018-9803-x

Cited by 2 publications

(3 citation statements)

References 14 publications

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“…Plasmids, Proteins, and Peptides. LRT2 was purified as previously described (42), except that lysogeny broth media was used for expression of unlabeled protein, and M9 minimal media enriched only with 15 The OsIAA11 72-125 gene was purchased from Genscript with a human rhinovirus-3C protease (3CPro) cleavage site at the N-terminal end. BamHI and HindIII cut sites were introduced via PCR.…”

Section: Methodsmentioning

confidence: 99%

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

Acevedo

Kwon

Nicholson

2019

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Section: Methodsmentioning

confidence: 99%

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

Acevedo

Kwon

Nicholson

2019

Proc. Natl. Acad. Sci. U.S.A.

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“…The plasmids for expression of WT LRT2, hCypA (a gift from C. Kalodimos, Rutgers University, Piscataway, NJ) and OsIAA11 72−125 in E. coli have been described previously 16,32,33 . Mutations in the LRT2 coding sequence were achieved either by site directed mutagenesis Polymerase Chain Reaction (PCR) or by overlap extension PCR 34 .…”

Section: Plasmids Protein Expression and Purificationmentioning

confidence: 99%

“…All LRT2 mutants, hCypA, PPIB, and TaCyp were expressed and purified as previously described for WT LRT2 32 , with the exception that for hCypA (which lacks a cleavage site) the His-tag was not cleaved. Each protein was concentrated with a 10K or 3K MWC centrifugal device (Pall Corporation, Ann Arbor, MI, or Millipore Corporation, Bedford, MA) to either 5μM in assay buffer (50mM KCl, 20mM KPO 4 , 1mM TCEP, pH=6.67) for thermostability testing or to higher concentrations in NMR buffer (50mM KCl, 20mM KPO 4 , 3mM TCEP, 5mM NaN 3 , 0.1% P.I.…”

Section: Plasmids Protein Expression and Purificationmentioning

confidence: 99%

Tuning a timing device that regulates lateral root development in rice

et al. 2019

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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 ( 104 W-P 105 ) in the natural substrate OsIAA11. OsIAA11 is a transcription repressor that, in response to the plant hormone auxin, is targeted to ubiquitinmediated proteasomal degradation via specific recognition of the cis isomer of its 104 W-P 105 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.

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1H, 13C and 15N NMR assignments of cyclophilin LRT2 (OsCYP2) from rice

Cited by 2 publications

References 14 publications

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

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

Tuning a timing device that regulates lateral root development in rice

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