Site-directed Mutagenesis and Characterization of Uracil-DNA Glycosylase Inhibitor Protein: ROLE OF SPECIFIC CARBOXYLIC AMINO ACIDS IN COMPLEX FORMATION WITH ESCHERICHIA COLI URACIL-DNA GLYCOSYLASE

Amy J Lundquist,Richard D Beger,Samuel E Bennett,Philip H Bolton,Dale W Mosbaugh

doi:10.1074/jbc.272.34.21408

Amy J Lundquist, Richard D Beger + Show 3 more

Open Access

https://doi.org/10.1074/jbc.272.34.21408

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Abstract

Bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) protein inactivates uracil-DNA glycosylase (Ung) by acting as a DNA mimic to bind Ung in an irreversible complex. Seven mutant Ugi proteins (E20I, E27A, E28L, E30L, E31L, D61G, and E78V) were created to assess the role of various negatively charged residues in the binding mechanism. Each mutant Ugi protein was purified and characterized with respect to inhibitor activity and Ung binding properties relative to the wild type Ugi. Analysis of the Ugi protein solution structures by nuclear magnetic resonance indicated that the mutant Ugi proteins were folded into the same general conformation as wild type Ugi. All seven of the Ugi proteins were capable of forming a Ung.Ugi complex but varied considerably in their individual ability to inhibit Ung activity. Like the wild type Ugi, five of the mutants formed an irreversible complex with Ung; however, the binding of Ugi E20I and E28L to Ung was shown to be reversible. The tertiary structure of [13C,15N]Ugi in complex with Ung was determined by solution state multi-dimensional nuclear magnetic resonance and compared with the unbound Ugi structure. Structural and functional analysis of these proteins have elucidated the two-step mechanism involved in Ung.Ugi association and irreversible complex formation.

Highlights

Bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) protein inactivates uracil-DNA glycosylase (Ung) by acting as a DNA mimic to bind Ung in an irreversible complex
Site-directed Mutagenesis of the Uracil-DNA Glycosylase Inhibitor Gene—To investigate the role of specific negatively charged amino acid residues in the Ung/Ugi interaction, sitedirected mutagenesis producing single amino acid substitutions was performed on the ugi gene
The results suggested that Ugi E20I competes very poorly with wild type Ugi since the maximum amount of [35S]Ugi based on the molar amount of Ung was found in complex for all ratios utilizing Ugi E20I

Summary

EXPERIMENTAL PROCEDURES

Materials—Restriction endonucleases (EcoRI, EcoRV, HindIII, PstI, SacI, and XmnI), T4 polynucleotide kinase, T4 DNA polymerase, and T4 DNA ligase were purchased from New England Biolabs. The third step of the procedure involved subcloning the ugi genes containing site-directed mutations from pSugi to the pKK223-3 derived overexpression vector pZWtac, replacing the wild type ugi gene Both pSugi and pZWtac DNA (1 ␮g) were separately digested with excess HindIII and EcoRI, and the products were resolved by 0.8% agarose (low melting point) gel electrophoresis. Purification of [3H]Ung1⁄7Ugi Complexes—Wild type Ugi or various site-directed mutants of Ugi protein were mixed with [3H]Ung in buffer A (30 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol, 5% (w/v) glycerol) containing 50 mM NaCl and incubated at 25 °C for 10 min and at 4 °C for 20 min. Interaction energies were calculated by combining the van der Waals, electrostatic, and hydrogen bond energies of the enzyme-inhibitor complex and unbound Ugi. Changes in interaction energies, ⌬Eint, are defined as the difference in the interaction energies of the uracil-DNA glycosylase1⁄7Ugi wild type and mutant complex. The differences in the change in the interactive energies ⌬⌬Eint are defined by subtracting the difference of the ⌬Eint of the wild type and mutant Ugi from the ⌬Eint of the mutant Ugi-containing complex

RESULTS

Loop regions

DISCUSSION