Chemically induced partial unfolding of the multifunctional Apurinic/apyrimidinic endonuclease 1

Apurinic/apyrimidinic endonuclease I (APE1) acts as both an endonuclease and a redox factor to ensure cell survival. The two activities require different conformations of APE1. As an endonuclease, APE1 is fully folded. As a redox factor, APE1 must be partially unfolded to expose the buried residue Cys65, which reduces transcription factors including AP-1, NF-{kappa}B, and HIF-1 and thereby enables them to bind DNA. To determine a molecular basis for partial unfolding associated with APE1s redox activity, we characterized specific interactions of a known redox inhibitor APX3330 with APE1 through waterLOGSY and 1H-15N HSQC NMR approaches using ethanol and acetonitrile as co-solvents. We find that APX3330 binds to the endonuclease active site in both co-solvents and to a distant small pocket in acetonitrile. Prolonged exposure of APE1 with APX3330 in acetonitrile resulted in a time-dependent loss of 1H-15N HSQC chemical shifts ([~]35%), consistent with partial unfolding. Regions that are partially unfolded include adjacent N- and C-terminal beta strands within one of the two sheets comprising the core, which converge within the small binding pocket defined by the CSPs. Removal of APX3330 via dialysis resulted in a slow reappearance of the 1H-15N HSQC chemical shifts suggesting that the effect of APX3330 is reversible. APX3330 significantly decreases the melting temperature of APE1 but has no effect on endonuclease activity using a standard assay in either co-solvent. Our results provide insights on reversible partial unfolding of APE1 relevant for its redox function as well as the mechanism of redox inhibition by APX3330. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/547112v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@13f9fe2org.highwire.dtl.DTLVardef@19cbcborg.highwire.dtl.DTLVardef@1927c7aorg.highwire.dtl.DTLVardef@2a3cc0_HPS_FORMAT_FIGEXP M_FIG C_FIG