W196 and the <i>β</i>‐Hairpin Motif Modulate the Redox Switch of Conformation and the Biomolecular Interaction Network of the Apoptosis‐Inducing Factor
Silvia Romero-Tamayo, Rubén Laplaza, Adrián Velázquez‐Campoy, Raquel Villanueva, Milagros Medina, Patrícia Ferreira
Abstract
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 β ‐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 β ‐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 β ‐hairpin and C‐loop coupling contribute to protein stability. Kinetic studies complemented with computational simulations reveal that W196 and the β ‐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 β ‐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.