Leucine 232 and hydrophobic residues at the ribosomal P stalk binding site are critical for biological activity of ricin

Yijun Zhou, Xiao Ping Li, Jennifer N. Kahn, John E. McLaughlin, Nilgun E. Tumer

Research output: Contribution to journalArticle

Abstract

Ricin interacts with the ribosomal P stalk to cleave a conserved adenine from the a-sarcin/ricin loop (SRL) of the rRNA. Ricin toxin A chain (RTA) uses Arg235 as the most critical arginine for binding to the P stalk through electrostatic interactions to facilitate depurination. Structural analysis showed that a P2 peptide binds to a hydrophobic pocket on RTA and the last two residues form hydrogen bonds with Arg235. The importance of hydrophobic residues relative to Arg235 in the interaction with the P stalk in vivo and on the toxicity of RTA is not known. Here, we mutated residues in the hydrophobic pocket to analyze their contribution to toxicity and depurination activity in yeast and in mammalian cells. We found that Leu232, Tyr183 and Phe240 contribute cumulatively to toxicity, with Leu232 being the most significant. A quadruplemutant, Y183A/L232A/R235A/F240A, which combined mutations in critical hydrophobic residues with R235A completely abolished the activity of RTA, indicating that Arg235 and hydrophobic residues are required for full biological activity. Y183A and F240A mutants had reduced activity on RNA, but higher activity on ribosomes compared with R235A in vitro, suggesting that they could partially regain activity upon interaction with ribosomes. These results expand the region of interaction between RTA and the P stalk critical for cellular activity to include the hydrophobic pocket and provide the first evidence that interaction of P stalk with the hydrophobic pocket promotes a conformational rearrangement of RTA to correctly position the active site residues for catalytic attack on the SRL.

Original languageEnglish (US)
Article numberBSR20192022
JournalBioscience Reports
Volume39
Issue number10
DOIs
StatePublished - Jan 1 2019

Fingerprint

Ricin
Bioactivity
Leucine
Binding Sites
Toxicity
Ribosomes
Regain
Adenine
Coulomb interactions
Static Electricity
Structural analysis
Yeast
Arginine
Hydrogen
Catalytic Domain
Hydrogen bonds
Yeasts
Cells
RNA
Mutation

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Biophysics
  • Biochemistry
  • Cell Biology

Cite this

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title = "Leucine 232 and hydrophobic residues at the ribosomal P stalk binding site are critical for biological activity of ricin",
abstract = "Ricin interacts with the ribosomal P stalk to cleave a conserved adenine from the a-sarcin/ricin loop (SRL) of the rRNA. Ricin toxin A chain (RTA) uses Arg235 as the most critical arginine for binding to the P stalk through electrostatic interactions to facilitate depurination. Structural analysis showed that a P2 peptide binds to a hydrophobic pocket on RTA and the last two residues form hydrogen bonds with Arg235. The importance of hydrophobic residues relative to Arg235 in the interaction with the P stalk in vivo and on the toxicity of RTA is not known. Here, we mutated residues in the hydrophobic pocket to analyze their contribution to toxicity and depurination activity in yeast and in mammalian cells. We found that Leu232, Tyr183 and Phe240 contribute cumulatively to toxicity, with Leu232 being the most significant. A quadruplemutant, Y183A/L232A/R235A/F240A, which combined mutations in critical hydrophobic residues with R235A completely abolished the activity of RTA, indicating that Arg235 and hydrophobic residues are required for full biological activity. Y183A and F240A mutants had reduced activity on RNA, but higher activity on ribosomes compared with R235A in vitro, suggesting that they could partially regain activity upon interaction with ribosomes. These results expand the region of interaction between RTA and the P stalk critical for cellular activity to include the hydrophobic pocket and provide the first evidence that interaction of P stalk with the hydrophobic pocket promotes a conformational rearrangement of RTA to correctly position the active site residues for catalytic attack on the SRL.",
author = "Yijun Zhou and Li, {Xiao Ping} and Kahn, {Jennifer N.} and McLaughlin, {John E.} and Tumer, {Nilgun E.}",
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Leucine 232 and hydrophobic residues at the ribosomal P stalk binding site are critical for biological activity of ricin. / Zhou, Yijun; Li, Xiao Ping; Kahn, Jennifer N.; McLaughlin, John E.; Tumer, Nilgun E.

In: Bioscience Reports, Vol. 39, No. 10, BSR20192022, 01.01.2019.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Leucine 232 and hydrophobic residues at the ribosomal P stalk binding site are critical for biological activity of ricin

AU - Zhou, Yijun

AU - Li, Xiao Ping

AU - Kahn, Jennifer N.

AU - McLaughlin, John E.

AU - Tumer, Nilgun E.

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AB - Ricin interacts with the ribosomal P stalk to cleave a conserved adenine from the a-sarcin/ricin loop (SRL) of the rRNA. Ricin toxin A chain (RTA) uses Arg235 as the most critical arginine for binding to the P stalk through electrostatic interactions to facilitate depurination. Structural analysis showed that a P2 peptide binds to a hydrophobic pocket on RTA and the last two residues form hydrogen bonds with Arg235. The importance of hydrophobic residues relative to Arg235 in the interaction with the P stalk in vivo and on the toxicity of RTA is not known. Here, we mutated residues in the hydrophobic pocket to analyze their contribution to toxicity and depurination activity in yeast and in mammalian cells. We found that Leu232, Tyr183 and Phe240 contribute cumulatively to toxicity, with Leu232 being the most significant. A quadruplemutant, Y183A/L232A/R235A/F240A, which combined mutations in critical hydrophobic residues with R235A completely abolished the activity of RTA, indicating that Arg235 and hydrophobic residues are required for full biological activity. Y183A and F240A mutants had reduced activity on RNA, but higher activity on ribosomes compared with R235A in vitro, suggesting that they could partially regain activity upon interaction with ribosomes. These results expand the region of interaction between RTA and the P stalk critical for cellular activity to include the hydrophobic pocket and provide the first evidence that interaction of P stalk with the hydrophobic pocket promotes a conformational rearrangement of RTA to correctly position the active site residues for catalytic attack on the SRL.

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