doi:10.1155/2021/6673661engRomero-Tamayo, S.Laplaza, R.Velazquez-Campoy, A.Villanueva, R.Medina, M.Ferreira, P.W196 and the ß -Hairpin Motif Modulate the Redox Switch of Conformation and the Biomolecular Interaction Network of the Apoptosis-Inducing FactorART-2021-123304The 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 ß-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 ß-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 ß-hairpin and C-loop coupling contribute to protein stability. Kinetic studies complemented with computational simulations reveal that W196 and the ß-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 ß-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.2021http://zaguan.unizar.es/record/9976510.1155/2021/6673661http://zaguan.unizar.es/record/99765oai:zaguan.unizar.es:99765info:eu-repo/grantAgreement/ES/DGA/ARAIDinfo:eu-repo/grantAgreement/ES/DGA-FEDER/E35-20Rinfo:eu-repo/grantAgreement/ES/MICINN-AEI/PID2019-103901GB-I00info:eu-repo/grantAgreement/ES/MINECO-AEI-FEDER/BIO2016-75183-POxidative Medicine and Cellular Longevity 2021 (2021), 6673661 [19 pp]byhttp://creativecommons.org/licenses/by/3.0/es/info:eu-repo/semantics/openAccess