Cooperative Interactions in the Hammerhead Ribozyme Drive pKa Shifting of G12 and Its Stacked Base C17

Erica A. Frankel, Christopher A. Strulson, Christine Dolan Keating, Philip C. Bevilacqua

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

General acid-base catalysis is a key mechanistic strategy in protein and RNA enzymes. Ribozymes use hydrated metal ions, nucleobases, and organic cofactors to carry this out. In most small ribozymes, a guanosine is positioned to participate in proton transfer with the nucleophilic 2′-OH. The unshifted pKa values for nucleobases and solvated metal ions are far from neutrality, however, and thus nonideal for general acid-base catalysis. Herein, evidence is provided for cooperative interaction in the hammerhead ribozyme among the guanine that interacts with the nucleophilic 2′-OH, G12, the -1 nucleobase C17, and Mg2+ ions. We introduce global fitting for analyzing ribozyme rate-pH data parametric in Mg2+ concentration and benchmark this method on data from the hepatitis delta virus ribozyme. We then apply global fitting to new rate-pH data for the hammerhead ribozyme using a minimal three-dimensional, four-channel cooperative model. The value for the pKa of G12 that we obtain is channel-dependent and varies from 8.1 to 9.9, shifting closest toward neutrality in the presence of two cationic species: C17H+ and a Mg2+ ion. The value for the pKa of the -1 nucleotide, C17, is increased a remarkable 3.5-5 pKa units toward neutrality. Shifting of the pKa of C17 appears to be driven by an electrostatic sandwich of C17 between carbonyl groups of the 5′-neighboring U and of G12 and involves cation-π interactions. Rate-pH profiles reveal that the major reactive channel under biological Mg2+ and pH involves a cationic C17 rather than a second metal ion. Substitution of a cationic base for a metal underscores the versatility of RNA.

Original languageEnglish (US)
Pages (from-to)2537-2548
Number of pages12
JournalBiochemistry
Volume56
Issue number20
DOIs
StatePublished - May 23 2017

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Catalytic RNA
Ions
Metal ions
Metals
Catalysis
RNA
Acids
Proton transfer
Guanosine
Guanine
Hepatitis Delta Virus
Benchmarking
Viruses
Cations
Electrostatics
Static Electricity
Substitution reactions
Nucleotides
Protons
hammerhead ribozyme

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

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title = "Cooperative Interactions in the Hammerhead Ribozyme Drive pKa Shifting of G12 and Its Stacked Base C17",
abstract = "General acid-base catalysis is a key mechanistic strategy in protein and RNA enzymes. Ribozymes use hydrated metal ions, nucleobases, and organic cofactors to carry this out. In most small ribozymes, a guanosine is positioned to participate in proton transfer with the nucleophilic 2′-OH. The unshifted pKa values for nucleobases and solvated metal ions are far from neutrality, however, and thus nonideal for general acid-base catalysis. Herein, evidence is provided for cooperative interaction in the hammerhead ribozyme among the guanine that interacts with the nucleophilic 2′-OH, G12, the -1 nucleobase C17, and Mg2+ ions. We introduce global fitting for analyzing ribozyme rate-pH data parametric in Mg2+ concentration and benchmark this method on data from the hepatitis delta virus ribozyme. We then apply global fitting to new rate-pH data for the hammerhead ribozyme using a minimal three-dimensional, four-channel cooperative model. The value for the pKa of G12 that we obtain is channel-dependent and varies from 8.1 to 9.9, shifting closest toward neutrality in the presence of two cationic species: C17H+ and a Mg2+ ion. The value for the pKa of the -1 nucleotide, C17, is increased a remarkable 3.5-5 pKa units toward neutrality. Shifting of the pKa of C17 appears to be driven by an electrostatic sandwich of C17 between carbonyl groups of the 5′-neighboring U and of G12 and involves cation-π interactions. Rate-pH profiles reveal that the major reactive channel under biological Mg2+ and pH involves a cationic C17 rather than a second metal ion. Substitution of a cationic base for a metal underscores the versatility of RNA.",
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Cooperative Interactions in the Hammerhead Ribozyme Drive pKa Shifting of G12 and Its Stacked Base C17. / Frankel, Erica A.; Strulson, Christopher A.; Keating, Christine Dolan; Bevilacqua, Philip C.

In: Biochemistry, Vol. 56, No. 20, 23.05.2017, p. 2537-2548.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Cooperative Interactions in the Hammerhead Ribozyme Drive pKa Shifting of G12 and Its Stacked Base C17

AU - Frankel, Erica A.

AU - Strulson, Christopher A.

AU - Keating, Christine Dolan

AU - Bevilacqua, Philip C.

PY - 2017/5/23

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N2 - General acid-base catalysis is a key mechanistic strategy in protein and RNA enzymes. Ribozymes use hydrated metal ions, nucleobases, and organic cofactors to carry this out. In most small ribozymes, a guanosine is positioned to participate in proton transfer with the nucleophilic 2′-OH. The unshifted pKa values for nucleobases and solvated metal ions are far from neutrality, however, and thus nonideal for general acid-base catalysis. Herein, evidence is provided for cooperative interaction in the hammerhead ribozyme among the guanine that interacts with the nucleophilic 2′-OH, G12, the -1 nucleobase C17, and Mg2+ ions. We introduce global fitting for analyzing ribozyme rate-pH data parametric in Mg2+ concentration and benchmark this method on data from the hepatitis delta virus ribozyme. We then apply global fitting to new rate-pH data for the hammerhead ribozyme using a minimal three-dimensional, four-channel cooperative model. The value for the pKa of G12 that we obtain is channel-dependent and varies from 8.1 to 9.9, shifting closest toward neutrality in the presence of two cationic species: C17H+ and a Mg2+ ion. The value for the pKa of the -1 nucleotide, C17, is increased a remarkable 3.5-5 pKa units toward neutrality. Shifting of the pKa of C17 appears to be driven by an electrostatic sandwich of C17 between carbonyl groups of the 5′-neighboring U and of G12 and involves cation-π interactions. Rate-pH profiles reveal that the major reactive channel under biological Mg2+ and pH involves a cationic C17 rather than a second metal ion. Substitution of a cationic base for a metal underscores the versatility of RNA.

AB - General acid-base catalysis is a key mechanistic strategy in protein and RNA enzymes. Ribozymes use hydrated metal ions, nucleobases, and organic cofactors to carry this out. In most small ribozymes, a guanosine is positioned to participate in proton transfer with the nucleophilic 2′-OH. The unshifted pKa values for nucleobases and solvated metal ions are far from neutrality, however, and thus nonideal for general acid-base catalysis. Herein, evidence is provided for cooperative interaction in the hammerhead ribozyme among the guanine that interacts with the nucleophilic 2′-OH, G12, the -1 nucleobase C17, and Mg2+ ions. We introduce global fitting for analyzing ribozyme rate-pH data parametric in Mg2+ concentration and benchmark this method on data from the hepatitis delta virus ribozyme. We then apply global fitting to new rate-pH data for the hammerhead ribozyme using a minimal three-dimensional, four-channel cooperative model. The value for the pKa of G12 that we obtain is channel-dependent and varies from 8.1 to 9.9, shifting closest toward neutrality in the presence of two cationic species: C17H+ and a Mg2+ ion. The value for the pKa of the -1 nucleotide, C17, is increased a remarkable 3.5-5 pKa units toward neutrality. Shifting of the pKa of C17 appears to be driven by an electrostatic sandwich of C17 between carbonyl groups of the 5′-neighboring U and of G12 and involves cation-π interactions. Rate-pH profiles reveal that the major reactive channel under biological Mg2+ and pH involves a cationic C17 rather than a second metal ion. Substitution of a cationic base for a metal underscores the versatility of RNA.

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