DNA binding is required for the apoptogenic action of apoptosis inducing factor

Abstract

The execution of apoptosis or programmed cell death comprises both caspase-dependent and caspase-independent processes. Apoptosis inducing factor (AIF) was identified as a major player in caspase-independent cell death. It induces chromatin condensation and initial DNA cleavage via an unknown molecular mechanism. Here we report the crystal structure of human AIF at 1.8 A resolution. The structure reveals the presence of a strong positive electrostatic potential at the AIF surface, although the calculated isoelectric point for the entire protein is neutral. We show that recombinant AIF interacts with DNA in a sequence-independent manner. In addition, in cells treated with an apoptotic stimulus, endogenous AIF becomes co-localized with DNA at an early stage of nuclear morphological changes. Structure-based mutagenesis shows that DNA-binding defective mutants of AIF fail to induce cell death while retaining nuclear translocation. The potential DNA-binding site identified from mutagenesis also coincides with computational docking of a DNA duplex. These observations suggest that AIF-induced nuclear apoptosis requires a direct interaction with DNA.

Physical interaction of apoptosis-inducing factor with DNA and RNA

“…This effect was attenuated for AIF mutants in which positively charged amino acids were mutated to neutral ones (mutants AIF510: K510A, K518A and AIF255: K255A, R265A), with a higher effect of the AIF50 mutation as compared to the AIF255 mutation (Figure 2a). The attenuated AIF-DNA binging detected in this system is compatible with the notion that the binding of AIF to DNA is dictated by electrostatic interactions (Ye et al, 2002) (Figure 2a). Recombinant AIF from S. cerevisiae also bound to isolated DNA in vitro (Figure 2b).…”

“…In the presence of Mg 2 þ , AIF also caused the formation of networks with intermolecular contact, when added to double-stranded, linearized DNA ( Figure 6A), suggesting protein-protein interactions between different AIF molecules. Such AIF-AIF protein interactions have been previously described, based on the crystallographic analysis of the protein (Mate et al, 2002;Ye et al, 2002). The preponderant AIF effects (binding and condensation) were absent when AIF was added to relaxed, circular DNA (not shown) and attenuated when AIF was probed on negatively supercoiled DNA ( Figure 6C).…”

“…However, the predominant localization of extra-mitochondrial AIF was the nucleus. This has been extensively documented for human and mouse AIF (Loeffler et al, 2001;Ye et al, 2002;Cande et al, 2002Cande et al, , 2004a, and also applies to the AIF ortholog from yeast (S. cerevisiae). Yeast AIF localized at distinct foci within the nucleus upon induction of apoptosis by chronological aging.…”

“…R265, K446 and R450 are the basic residues on the AIF surface which are most frequently targeted for electro- AIF interactions with nucleic acids N Vahsen et al static interactions. Interestingly, these residues were shown to play a key role in the AIF-DNA binding (Ye et al, 2002), suggesting that the DNA and RNAbinding sites on AIF overlap. The docking results do not seem to be affected by the nature of the nucleotide bases.…”

“…Independent experiments performed in Saccharomyces cerevisiae, which contains AIF and cyclophilin orthologs, confirmed the prodeath activity of the AIF-cyclophilin A interaction . Current evidence suggests that AIF binds to DNA through electrostatic interactions (Ye et al, 2002) and that it serves as a sort of scaffold to recruit further proteins belonging to the so-called degradosome to the chromatin of dying cells (Parrish and Xue, 2003).…”