W196 and the β-Hairpin Motif Modulate the Redox Switch of Conformation and the Biomolecular Interaction Network of the Apoptosis-Inducing Factor

The human apoptosis-inducing factor (hAIF) is a moonlight flavoprotein involved in mitochondrial respiratory complex assembly and caspase-independent programmed cell death. These functions might be modulated by its redox-linked structural transition that enables hAIF to act as a NAD(H/(+)) redox sensor. Upon reduction with NADH, hAIF undergoes a conformational reorganization in two specific insertions-the flexible regulatory C-loop and the 190-202 beta-harpin-promoting protein dimerization and the stabilization of a long-life charge transfer complex (CTC) that modulates its monomer-dimer equilibrium and its protein interaction network in healthy mitochondria. In this regard, here, we investigated the precise function of the beta-hairpin in the AIF conformation landscape related to its redox mechanism, by analyzing the role played by W196, a key residue in the interaction of this motif with the regulatory C-loop. Mutations at W196 decrease the compactness and stability of the oxidized hAIF, indicating that the beta-hairpin and C-loop coupling contribute to protein stability. Kinetic studies complemented with computational simulations reveal that W196 and the beta-hairpin conformation modulate the low efficiency of hAIF as NADH oxidoreductase, contributing to configure its active site in a noncompetent geometry for hydride transfer and to stabilize the CTC state by enhancing the affinity for NAD(+). Finally, the beta-hairpin motif contributes to define the conformation of AIF's interaction surfaces with its physiological partners. These findings improve our understanding on the molecular basis of hAIF's cellular activities, a crucial aspect for clarifying its associated pathological mechanisms and developing new molecular therapies.

Automated summary

The human apoptosis-inducing factor (hAIF) acts as a NAD(H/(+)) redox sensor, with its structural transition modulating its functions in mitochondrial respiratory complex assembly and programmed cell death. The interaction between W196 and the beta-hairpin motif plays a crucial role in stabilizing hAIF and configuring its active site for hydride transfer. Additionally, the beta-hairpin motif defines AIF's interaction surfaces with its physiological partners, enhancing our understanding of its cellular activities and associated pathological mechanisms.