Protein O-fucosylation is an essential post-translational modification, involved in the folding of target proteins and in the role of these target proteins during embryonic development and adult tissue homeostasis, among other things. Two different enzymes are responsible for this modification, Protein O-fucosyltransferase 1 and 2 (POFUT1 and POFUT2, respectively). Both proteins have been characterised biologically and enzymatically but nothing is known at the molecular or structural level. Here we describe the first crystal structure of a catalytically functional POFUT1 in an apo-form and in complex with GDP-fucose and GDP. The enzyme belongs to the GT-B family and is not dependent on manganese for activity. GDP-fucose/GDP is localised in a conserved cavity connected to a large solvent exposed pocket, which we show is the binding site of epidermal growth factor (EGF) repeats in the extracellular domain of the Notch Receptor. Through both mutational and kinetic studies we have identified which residues are involved in binding and catalysis and have determined that the Arg240 residue is a key catalytic residue. We also propose a novel SN1-like catalytic mechanism with formation of an intimate ion pair, in which the glycosidic bond is cleaved before the nucleophilic attack; and theoretical calculations at a DFT (B3LYP/6-31+G(d,p) support this mechanism. Thus, the crystal structure together with our mutagenesis studies explain the molecular mechanism of POFUT1 and provide a new starting point for the design of functional inhibitors to this critical enzyme in the future.

The apoptosis-inducing factor (AIF) is a mitochondrial-flavoprotein that, after cell death induction, is distributed to the nucleus to mediate chromatinolysis. In mitochondria, AIF is present in a monomer–dimer equilibrium that after reduction by NADH gets displaced toward the dimer. The crystal structure of the human AIF (hAIF):NAD(H)-bound dimer revealed one FAD and, unexpectedly, two NAD(H) molecules per protomer. A 1:2 hAIF:NAD(H) binding stoichiometry was additionally confirmed in solution by using surface plasmon resonance. The here newly discovered NAD(H)-binding site includes residues mutated in human disorders, and accommodation of the coenzyme in it requires restructuring of a hAIF portion within the 509–560 apoptogenic segment. Disruption of interactions at the dimerization surface by production of the hAIF E413A/R422A/R430A mutant resulted in a nondimerizable variant considerably less efficiently stabilizing charge-transfer complexes upon coenzyme reduction than WT hAIF. These data reveal that the coenzyme-mediated monomer–dimer transition of hAIF modulates the conformation of its C-terminal proapoptotic domain, as well as its mechanism as reductase. These observations suggest that both the mitochondrial and apoptotic functions of hAIF are interconnected and coenzyme controlled: a key information in the understanding of the physiological role of AIF in the cellular life and death cycle.

The human Apoptosis Inducing Factor (hAIF) is a bifunctional NAD(P)H-dependent flavoreductase involved in both mitochondrial energy metabolism and caspase-independent cell death. Even though several studies indicate that both functions are redox controlled by NADH binding, the exact role of hAIF as a reductase in healthy mitochondria remains unknown. Upon reduction by NADH, hAIF dimerizes and produces very stable flavin/nicotinamide charge transfer complexes (CTC), by stacking of the oxidized nicotinamide moiety of the NAD(+) coenzyme against the re-face of the reduced flavin ring of its FAD cofactor. Such complexes are critical to restrict the hAIF efficiency as a reductase. The molecular basis of the hAIF reductase activity is here investigated by analyzing the role played by residues contributing to the interaction of the FAD isoalloxazine ring and of the nicotinamide moiety of NADH at the active site. Mutations at K177 and E314 produced drastic effects on the hAIF ability to retain the FAD cofactor, indicating that these residues are important to set up the holo-enzyme active site conformation. Characterization of P173G hAIF indicates that the stacking of P173 against the isoalloxazine ring is relevant to determine the flavin environment and to modulate the enzyme affinity for NADH. Finally, the properties of the F310G and H454S hAIF mutants indicate that these two positions contribute to form a compact active site essential for NADH binding, CTC stabilization, and NAD(+) affinity for the reduced state of hAIF. These features are key determinants of the particular behavior of hAIF as a NADH-dependent oxidoreductase.

Aims: The human apoptosis-inducing factor (hAIF) supports OXPHOS biogenesis and programmed cell death, with missense mutations producing neurodegenerative phenotypes. hAIF senses the redox environment of cellular compartments, stabilizing a charge transfer complex (CTC) dimer that modulates the protein interaction network. In this context, we aimed to evaluate the subcellular pH, CTC formation, and pathogenic mutations effects on hAIF stability, and a thermal denaturation high-throughput screening (HTS) assay to discover AIF binders. Results: Apoptotic hAIF D1-101 is not stable at intermembrane mitochondrial space (IMS) pH, but the 77-101 residues confer stability to the mitochondrial isoform. hAIF and its CTC populate different conformational ensembles with redox switch to the CTC producing a less stable and compact protein. The pathogenic G308E, DR201, and E493V mutations modulate hAIF stability; particularly, DR201 causes a population shift to a less stable conformation that remodels active site structure and dynamics. We have identified new molecules that modulate the hAIF reduced nicotinamide adenine dinucleotide (NADH)/oxidized nicotinamide adenine dinucleotide (NAD +) association/dissociation equilibrium and regulate its catalytic efficiency. Innovation: Biophysical methods allow evaluating the regulation of hAIF functional ensembles and to develop an HTS assay to discover small molecules that might modulate hAIF stability and activities. Conclusions: The mitochondrial soluble 54-77 portion stabilizes hAIF at the IMS pH. NADH-redox-linked conformation changes course with strong NAD + binding and protein dimerization, but they produce a negative impact in overall hAIF stability. Loss of functionality in the R201 deletion is due to distortion of the active site architecture. We report molecules that may serve as leads in the development of hAIF bioactive compounds.

The apoptosis inducing factor (AIF) was first discovered as a caspase-independent apoptosis effector that promoted cell death upon release from the mitochondria (triggered by pro-apoptotic stimuli) and relocalization into the nucleus, where it promotes chromatin condensation and DNA fragmentation. AIF is a mammalian mitochondrial FAD-dependent flavoenzyme, ubiquitous in vertebrate cells,and with orthologs in all eukaryotes. Beyond its role in apoptosis AIF has additional functions in mitochondria, mainly related with the redox function of its flavin adenine dinucleotide cofactor (FAD), which despite being poorly understood are vital. Thus, defects in AIF trigger major dysfunctions in oxidative phosphorylation, and cause severe illnesses related with neurodegeneration as a consequence of mitochondriopathies. AIF folds in three modules: a FAD-binding, a nicotine adenine dinucleotide (NADH)-binding and a C-terminal modules. Upon reduction of the flavin cofactor by NADH, conformational changes leading to AIF dimerization are proposed as a key early event in the mitochondrial sensing/signaling functions of AIF. The recent interest in the design of new therapies to modulate caspase-independent apoptosis pathways also makes AIF a potential pharmacological target to treat pathological disorders related with AIF dependent mitochondriopathies. Therefore, the first step in this direction must be to understand the molecular basis of the AIF redox reactions and their relationship with the apoptotic function. Here, we examine recent research towards the molecular mechanisms linked to the AIF oxido-reduction properties.