TY - JOUR

T1 - Residue coevolution reveals functionally important intramolecular interactions in formamidopyrimidine-DNA glycosylase

AU - Endutkin, Anton V.

AU - Koptelov, Simeon S.

AU - Popov, Alexander V.

AU - Torgasheva, Natalya A.

AU - Lomzov, Alexander A.

AU - Tsygankova, Alphiya R.

AU - Skiba, Tatiana V.

AU - Afonnikov, Dmitry A.

AU - Zharkov, Dmitry O.

N1 - Publisher Copyright: © 2018 Elsevier B.V.

PY - 2018/9/1

Y1 - 2018/9/1

N2 - In protein evolution, functionally important intramolecular interactions, such as polar bridges or hydrophobic interfaces, tend to be conserved. We have analyzed coevolution of physicochemical properties in pairs of amino acid residues in the formamidopyrimidine–DNA glycosylase (Fpg) protein family, identified three conserved polar bridges (Arg54–Glu131, Gln234–Arg244, and Tyr170–Ser208 in the E. coli protein) located in known functional regions of the protein, and analyzed their roles by site-directed mutagenesis. The structure and molecular dynamic modeling showed that the coevolving pairs do not form isolated bridges but rather participate in tight local clusters of hydrogen bonds. The Arg54–Glu131 bridge, connecting the N- and C-terminal domains, was important for DNA binding, as its abolishment or even ion pair reversal inactivated Fpg and greatly decreased the enzyme's affinity for DNA. Mutations of the Gln234–Arg244 bridge, located at the base of the single Fpg β-hairpin zinc finger, did not affect the activity but sharply decreased the melting temperature of the protein, with the bridge reversal partially restoring the thermal stability. Finally, Tyr170 mutation to Phe decreased Fpg binding but did not fully inactivate the protein, whereas Ser208 replacement with Ala had no effect; molecular dynamics showed that in both wild-type and S208 A Fpg, Tyr170 quickly re-orients to form an alternative set of hydrogen bonds. Thus, the coevolution analysis approach, combined with biochemical and computational studies, provides a powerful tool for understanding intramolecular interactions important for the function of DNA repair enzymes.

AB - In protein evolution, functionally important intramolecular interactions, such as polar bridges or hydrophobic interfaces, tend to be conserved. We have analyzed coevolution of physicochemical properties in pairs of amino acid residues in the formamidopyrimidine–DNA glycosylase (Fpg) protein family, identified three conserved polar bridges (Arg54–Glu131, Gln234–Arg244, and Tyr170–Ser208 in the E. coli protein) located in known functional regions of the protein, and analyzed their roles by site-directed mutagenesis. The structure and molecular dynamic modeling showed that the coevolving pairs do not form isolated bridges but rather participate in tight local clusters of hydrogen bonds. The Arg54–Glu131 bridge, connecting the N- and C-terminal domains, was important for DNA binding, as its abolishment or even ion pair reversal inactivated Fpg and greatly decreased the enzyme's affinity for DNA. Mutations of the Gln234–Arg244 bridge, located at the base of the single Fpg β-hairpin zinc finger, did not affect the activity but sharply decreased the melting temperature of the protein, with the bridge reversal partially restoring the thermal stability. Finally, Tyr170 mutation to Phe decreased Fpg binding but did not fully inactivate the protein, whereas Ser208 replacement with Ala had no effect; molecular dynamics showed that in both wild-type and S208 A Fpg, Tyr170 quickly re-orients to form an alternative set of hydrogen bonds. Thus, the coevolution analysis approach, combined with biochemical and computational studies, provides a powerful tool for understanding intramolecular interactions important for the function of DNA repair enzymes.

KW - Coevolution

KW - DNA glycosylases

KW - DNA repair

KW - Formamidopyrimidine–DNA glycosylase

KW - Structure

KW - Formamidopyrimidine-DNA glycosylase

KW - OXIDATIVELY DAMAGED DNA

KW - STEADY-STATE KINETICS

KW - CRYSTAL-STRUCTURE

KW - ESCHERICHIA-COLI

KW - GRAPHICAL USER-INTERFACE

KW - BASE EXCISION-REPAIR

KW - SUBSTRATE DISCRIMINATION

KW - MOLECULAR-DYNAMICS SIMULATIONS

KW - LESION RECOGNITION

KW - FPG PROTEIN

KW - DNA/metabolism

KW - Sequence Analysis, Protein

KW - DNA-Formamidopyrimidine Glycosylase/chemistry

KW - Escherichia coli/enzymology

KW - Mutagenesis, Site-Directed

KW - Molecular Dynamics Simulation

KW - DNA Repair

KW - Escherichia coli Proteins/chemistry

KW - Protein Conformation

KW - Kinetics

KW - Evolution, Molecular

UR - http://www.scopus.com/inward/record.url?scp=85050133009&partnerID=8YFLogxK

U2 - 10.1016/j.dnarep.2018.07.004

DO - 10.1016/j.dnarep.2018.07.004

M3 - Article

C2 - 30032016

AN - SCOPUS:85050133009

VL - 69

SP - 24

EP - 33

JO - DNA Repair

JF - DNA Repair

SN - 1568-7864

ER -