Redox- and Ligand Binding-Dependent Conformational Ensembles in the Human Apoptosis-Inducing Factor Regulate Its Pro-Life and Cell Death Functions.

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 hAIFΔ1-101 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, ΔR201, and E493V mutations modulate hAIF stability; particularly, ΔR201 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 closed conformation of the LDL receptor is destabilized by the low Ca(++) concentration but favored by the high Mg(++) concentration in the endosome.

The LDL receptor (LDLR) internalizes LDL and VLDL particles. In the endosomes, it adopts a closed conformation important for recycling, by interaction of two modules of the ligand binding domain (LR4-5) and a ß-propeller motif. Here, we investigate by SPR the interactions between those two modules and the ß-propeller. Our results indicate that the two modules cooperate to bind the ß-propeller. The binding is favored by low pH and by high [Ca(++)]. Our data show that Mg(++), at high concentration in the endosome, favors the formation of the closed conformation by replacing the structuring effect of Ca(++) in LR5. We propose a sequential model of LDL release where formation of the close conformation follows LDL release.

Intradomain Confinement of Disulfides in the Folding of Two Consecutive Modules of the LDL Receptor.

The LDL receptor internalizes circulating LDL and VLDL particles for degradation. Its extracellular binding domain contains ten (seven LA and three EGF) cysteine-rich modules, each bearing three disulfide bonds. Despite the enormous number of disulfide combinations possible, LDLR oxidative folding leads to a single native species with 30 unique intradomain disulfides. Previous folding studies of the LDLR have shown that non native disulfides are initially formed that lead to compact species. Accordingly, the folding of the LDLR has been described as a "coordinated nonvectorial" reaction, and it has been proposed that early compaction funnels the reaction toward the native structure. Here we analyze the oxidative folding of LA4 and LA5, the modules critical for ApoE binding, isolated and in the LA45 tandem. Compared to LA5, LA4 folding is slow and inefficient, resembling that of LA5 disease-linked mutants. Without Ca++, it leads to a mixture of many two-disulfide scrambled species and, with Ca++, to the native form plus two three-disulfide intermediates. The folding of the LA45 tandem seems to recapitulate that of the individual repeats. Importantly, although the folding of the LA45 tandem takes place through formation of scrambled isomers, no interdomain disulfides are detected, i.e. the two adjacent modules fold independently without the assistance of interdomain covalent interactions. Reduction of incredibly large disulfide combinatorial spaces, such as that in the LDLR, by intradomain confinement of disulfide bond formation might be also essential for the efficient folding of other homologous disulfide-rich receptors.

Low-density lipoprotein receptor is a calcium/magnesium sensor - role of LR4 and LR5 ion interaction kinetics in low-density lipoprotein release in the endosome.

The low-density lipoprotein receptor (LDLR) captures circulating lipoproteins and delivers them in the endosome for degradation. Its function is essential for cholesterol homeostasis, and mutations in the LDLR are the major cause of familiar hypercholesterolemia. The release of LDL is usually attributed to endosome acidification. As the pH drops, the affinity of the LDLR/LDL complex is reduced, whereas the strength of a self-complex formed between two domains of the receptor (i.e. the LDL binding domain and the ß-propeller domain) increases. However, an alternative model states that, as a consequence of a drop in both pH and Ca(2+) concentration, the LDLR binding domain is destabilized in the endosome, which weakens the LDLR/LDL complex, thus liberating the LDL particles. In the present study, we test a key underlying assumption of the second model, namely that the lipoprotein binding repeats of the receptor (specifically repeats 4 and 5, LR4 and LR5) rapidly sense endosomal changes in Ca(2+) concentration. Our kinetic and thermodynamic analysis of Ca(2+) and Mg(2+) binding to LR4 and LR5, as well as to the tandem of the two (LR4-5), shows that both repeats spontaneously release Ca(2+) in a time scale much shorter than endosomal delivery of LDL, thus acting as Ca(2+) sensors that become unfolded under endosomal conditions. Our analysis additionally explains the lower Ca(2+) affinity of repeat LR4, compared to LR5, as arising from a very slow Ca(2+) binding reaction in the former, most likely related to the lower conformational stability of apolipoprotein LR4, compared to apolipoprotein LR5, as determined from thermal unfolding experiments and molecular dynamics simulations.

LDL receptor/lipoprotein recognition: endosomal weakening of ApoB and ApoE binding to the convex face of the LR5 repeat.

The molecular mechanism of lipoprotein binding by the low-density lipoprotein (LDL) receptor (LDLR) is poorly understood, one reason being that structures of lipoprotein-receptor complexes are not available. LDLR uses calcium-binding repeats (LRs) to interact with apolipoprotein B and apolipoprotein E (ApoB and ApoE). We have used NMR and SPR to characterize the complexes formed by LR5 and three peptides encompassing the putative binding regions of ApoB (site A and site B) and ApoE. The three peptides bind at the hydrophilic convex face of LR5, forming complexes that are weakened at low [Ca(2+) ] and low pH. Thus, endosomal conditions favour dissociation of LDLR/lipoprotein complexes regardless of whether active displacement of bound lipoproteins by the ß-propeller in LDLR takes place. The multiple ApoE copies in ß very low density lipoproteins (ß-VLDLs), and the presence of two competent binding sites (A and B) in LDLs, suggest that LDLR chelates lipoproteins and enhances complex affinity by using more than one LR.