Interactions of apurinic/apyrimidinic endonuclease with a redox inhibitor: evidence for an alternate conformation of the enzyme.
Apurinic/apyrimidinic endonuclease (APE1) is an essential base excision repair protein that also functions as a reduction and oxidation (redox) factor in mammals. Through a thiol-based mechanism, APE1 reduces a number of important transcription factors, including AP-1, p53, NF-κB, and HIF-1α. What is known about the mechanism to date is that the buried residues Cys 65 and Cys 93 are critical for APE1's redox activity. To further detail the redox mechanism, we developed a chemical footprinting-mass spectrometric assay using N-ethylmaleimide (NEM), an irreversible Cys modifier, to characterize the interaction of the redox inhibitor, E3330, with APE1. When APE1 was incubated with E3330, two NEM-modified products were observed, one with two and a second with seven added NEMs; this latter product corresponds to a fully modified APE1. In a similar control reaction without E3330, only the +2NEM product was observed in which the two solvent-accessible Cys residues, C99 and C138, were modified by NEM. Through hydrogen-deuterium amide exchange with analysis by mass spectrometry, we found that the +7NEM-modified species incorporates approximately 40 more deuterium atoms than the native protein, which exchanges nearly identically as the +2NEM product, suggesting that APE1 can be trapped in a partially unfolded state. E3330 was also found to increase the extent of disulfide bond formation involving redox critical Cys residues in APE1 as assessed by liquid chromatography and tandem mass spectrometry, suggesting a basis for its inhibitory effects on APE1's redox activity. Collectively, our results suggest that APE1 adopts a partially unfolded state, which we propose is the redox active form of the enzyme.
Department of Chemistry, Washington University in St. Louis, St. Louis, Missouri 63130, United States.
Dian
D
Su
Sarah
S
Delaplane
Meihua
M
Luo
Don L
DL
Rempel
Bich
B
Vu
Mark R
MR
Kelley
Michael L
ML
Gross
Millie M
MM
Georgiadis
D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology*; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D005033:Ethylmaleimide / Q000378:metabolism*; D006801:Humans; D010084:Oxidation-Reduction / Q000187:drug effects*; D011422:Propionates / Q000494:pharmacology*; D011487:Protein Conformation / Q000187:drug effects; D021241:Spectrometry, Mass, Electrospray Ionization; D013439:Sulfhydryl Reagents / Q000378:metabolism*; D013696:Temperature
D016227:Benzoquinones; D011422:Propionates; D013439:Sulfhydryl Reagents; C075569:E 3330; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D003545:Cysteine; D005033:Ethylmaleimide