Electrostatic contributions to site specific DNA cleavage by EcoRV endonuclease

Nancy C Horton, Christopher Otey, Shelley Lusetti, My D. Sam, Jonathan Kohn, Amy M. Martin, Vidya Ananthnarayan, John J. Perona

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Mutational analysis of amino acids at the periphery of the EcoRV endonuclease active site suggests that moderate-range electrostatic effects play a significant role in modulating the efficiency of phosphoryl transfer. Asp36 and Lys38 located on minor-groove binding surface loops approach within 7-9 Å of the scissile phosphates of the DNA. While the rates of single-site mutations removing the carboxylate or amine moieties at these positions are decreased 103-105-fold compared to that of wild-type EcoRV, we find that double mutants which rebalance the charge improve catalysis by up to 500-fold. Mutational analysis also suggests that catalytic efficiency is influenced by Lys173, which is buried at the base of a deep depression penetrating from a distal surface of the enzyme. The Lys173 amine group lies just 6 Å from the amine group of the conserved essential Lys92 side chain in the active site. Kinetic and crystallographic analyses of the EcoRV E45A mutant enzyme further show that the Glu45 carboxylate group facilitates an extensive set of conformational transitions which occur upon DNA binding. The crystal structure of E45A bound to DNA and Mn2+ ions reveals significant conformational alterations in a small α-helical portion of the dimer interface located adjacent to the DNA minor groove. This leads to a tertiary reorientation of the two monomers as well as shifting of the key major-groove binding recognition loops. Because the Glu45 side chain does not appear to play a direct structural role in maintaining the active site, these rearrangements may instead originate in an altered electrostatic potential caused by removal of the negative charge. A Mn2+ binding site on the scissile phosphate is also disrupted in the E45A structure such that inner-sphere metal interactions made by the scissile DNA phosphate and conserved Asp90 carboxylate are each replaced with water molecules in the mutant. These findings argue against a proposed role for Asp36 as the general base in EcoRV catalysis, and reveal that the induced-fit conformational changes necessary for active site assembly and metal binding are significantly modulated by the electrostatic potential in this region.

Original languageEnglish (US)
Pages (from-to)10754-10763
Number of pages10
JournalBiochemistry
Volume41
Issue number35
DOIs
StatePublished - Sep 3 2002

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DNA Cleavage
Static Electricity
Electrostatics
Catalytic Domain
DNA
Amines
Phosphates
Catalysis
Metals
Enzymes
Dimers
Monomers
Crystal structure
Binding Sites
GATATC-specific type II deoxyribonucleases
Ions
Amino Acids
Mutation
Molecules
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Horton, N. C., Otey, C., Lusetti, S., Sam, M. D., Kohn, J., Martin, A. M., ... Perona, J. J. (2002). Electrostatic contributions to site specific DNA cleavage by EcoRV endonuclease. Biochemistry, 41(35), 10754-10763. https://doi.org/10.1021/bi020305l

Electrostatic contributions to site specific DNA cleavage by EcoRV endonuclease. / Horton, Nancy C; Otey, Christopher; Lusetti, Shelley; Sam, My D.; Kohn, Jonathan; Martin, Amy M.; Ananthnarayan, Vidya; Perona, John J.

In: Biochemistry, Vol. 41, No. 35, 03.09.2002, p. 10754-10763.

Research output: Contribution to journalArticle

Horton, NC, Otey, C, Lusetti, S, Sam, MD, Kohn, J, Martin, AM, Ananthnarayan, V & Perona, JJ 2002, 'Electrostatic contributions to site specific DNA cleavage by EcoRV endonuclease', Biochemistry, vol. 41, no. 35, pp. 10754-10763. https://doi.org/10.1021/bi020305l
Horton NC, Otey C, Lusetti S, Sam MD, Kohn J, Martin AM et al. Electrostatic contributions to site specific DNA cleavage by EcoRV endonuclease. Biochemistry. 2002 Sep 3;41(35):10754-10763. https://doi.org/10.1021/bi020305l
Horton, Nancy C ; Otey, Christopher ; Lusetti, Shelley ; Sam, My D. ; Kohn, Jonathan ; Martin, Amy M. ; Ananthnarayan, Vidya ; Perona, John J. / Electrostatic contributions to site specific DNA cleavage by EcoRV endonuclease. In: Biochemistry. 2002 ; Vol. 41, No. 35. pp. 10754-10763.
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abstract = "Mutational analysis of amino acids at the periphery of the EcoRV endonuclease active site suggests that moderate-range electrostatic effects play a significant role in modulating the efficiency of phosphoryl transfer. Asp36 and Lys38 located on minor-groove binding surface loops approach within 7-9 {\AA} of the scissile phosphates of the DNA. While the rates of single-site mutations removing the carboxylate or amine moieties at these positions are decreased 103-105-fold compared to that of wild-type EcoRV, we find that double mutants which rebalance the charge improve catalysis by up to 500-fold. Mutational analysis also suggests that catalytic efficiency is influenced by Lys173, which is buried at the base of a deep depression penetrating from a distal surface of the enzyme. The Lys173 amine group lies just 6 {\AA} from the amine group of the conserved essential Lys92 side chain in the active site. Kinetic and crystallographic analyses of the EcoRV E45A mutant enzyme further show that the Glu45 carboxylate group facilitates an extensive set of conformational transitions which occur upon DNA binding. The crystal structure of E45A bound to DNA and Mn2+ ions reveals significant conformational alterations in a small α-helical portion of the dimer interface located adjacent to the DNA minor groove. This leads to a tertiary reorientation of the two monomers as well as shifting of the key major-groove binding recognition loops. Because the Glu45 side chain does not appear to play a direct structural role in maintaining the active site, these rearrangements may instead originate in an altered electrostatic potential caused by removal of the negative charge. A Mn2+ binding site on the scissile phosphate is also disrupted in the E45A structure such that inner-sphere metal interactions made by the scissile DNA phosphate and conserved Asp90 carboxylate are each replaced with water molecules in the mutant. These findings argue against a proposed role for Asp36 as the general base in EcoRV catalysis, and reveal that the induced-fit conformational changes necessary for active site assembly and metal binding are significantly modulated by the electrostatic potential in this region.",
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