Photoaffinity labeling with cholesterol analogues precisely maps a cholesterol-binding site in voltage-dependent anion channel-1

Voltage-dependent anion channel-1 (VDAC1) is a highly regulated β-barrel membrane protein that mediates transport of ions and metabolites between the mitochondria and cytosol of the cell. VDAC1 co-purifies with cholesterol and is functionally regulated by cholesterol, among other endogenous lipids. Molecular modeling studies based on NMR observations have suggested five cholesterol-binding sites in VDAC1, but direct experimental evidence for these sites is lacking. Here, to determine the sites of cholesterol binding, we photolabeled purified mouse VDAC1 (mVDAC1) with photoactivatable cholesterol analogues and analyzed the photolabeled sites with both top-down mass spectrometry (MS), and bottom-up MS paired with a clickable, stable isotope-labeled tag, FLI-tag. Using cholesterol analogues with a diazirine in either the 7 position of the steroid ring (LKM38) or the aliphatic tail (KK174), we mapped a binding pocket in mVDAC1 localized to Thr83 and Glu73, respectively. When Glu73 was mutated to a glutamine, KK174 no longer photolabeled this residue, but instead labeled the nearby Tyr62 within this same binding pocket. The combination of analytical strategies employed in this work permits detailed molecular mapping of a cholesterol-binding site in a protein, including an orientation of the sterol within the site. Our work raises the interesting possibility that cholesterol-mediated regulation of VDAC1 may be facilitated through a specific binding site at the functionally important Glu73 residue

Protonation state of glutamate 73 regulates the formation of a specific dimeric association of mVDAC1.

The voltage-dependent anion channel (VDAC) is the most abundant protein in the outer mitochondrial membrane and constitutes the primary pathway for the exchange of ions and metabolites between the cytosol and the mitochondria. There is accumulating evidence supporting VDAC's role in mitochondrial metabolic regulation and apoptosis, where VDAC oligomerization has been implicated with these processes. Herein, we report a specific pH-dependent dimerization of murine VDAC1 (mVDAC1) identified by double electron-electron resonance and native mass spectrometry. Intermolecular distances on four singly spin-labeled mVDAC1 mutants were used to generate a model of the low-pH dimer, establishing the presence of residue E73 at the interface. This dimer arrangement is different from any oligomeric state previously described, and it forms as a steep function of pH with an apparent pKa of 7.4. Moreover, the monomer-dimer equilibrium affinity constant was determined using native MS, revealing a nearly eightfold enhancement in dimerization affinity at low pH. Mutation of E73 to either alanine or glutamine severely reduces oligomerization, demonstrating the role of protonated E73 in enhancing dimer formation. Based on these results, and the known importance of E73 in VDAC physiology, VDAC dimerization likely plays a significant role in mitochondrial metabolic regulation and apoptosis in response to cytosolic acidification during cellular stress

Purification and characterization of a respiratory supercomplex

Les membranes impliquées dans les processus bioénergétiques arborent une très grande densité de protéines, paramètre déterminant pour leur organisation supra-moléculaire. Dans ce travail, nous avons utilisé la bactérie thermophile Geobacillus stearothermophilus comme modèle pour étudier la formation de super-complexes de protéines membranaires, en vue d'une étude structurale. Nous avons purifié et caractérisé un super-complexe comprenant une menaquinol: cytochrome c oxydoréductase (b6c), un cytochrome c550 et une cytochrome c oxydase caa3. En combinant des titrations par spectroscopie optique et résonance paramagnétique électronique, nous avons pu déterminer les potentiels d'oxydo-réduction de la plupart des cofacteurs et combler ainsi une lacune dans l'étude des chaînes de transfert d'électrons utilisant des quinones à bas potentiel redox, les ménaquinones. Nous avons ainsi montré que les potentiels redox des cofacteurs du cytochrome b6c terminés par celui des quinones. Ce travail va à l'encontre de données partielles antérieures publiées, mais est en parfait accord avec les modèles du Q-cycle de Peter Mitchell. Les résultats obtenus ont des répercussions sur les rendements bioénergétiques des différents maillons de la chaîne de transfert.Bioenergetic membranes present a high protein density - a crucial factor for their organizationinto super-complexes. This project uses the thermophilic bacteria Geobacillus stearothermophilusas a model to study the formation of membrane protein super-complexes with the aim of structuralstudies. We purified and characterized a super-complex between a menaquinone : cytochromec oxidoreductase (b6c), a cytochrome c550, and a cytochrome c oxidase caa3. Using both opticaland EPR spectroscopy methods, we performed the redox titrations of most of the redox cofactorsof the super-complex. Thus, these results enable a new understanding of menaquinone-usingelectron transport chains, showing that quinones’ redox potential determines the redox potentialof the cytochrome b6c’s cofactors. The conclusions differ from previous partial data, althoughthey fit perfectly with Peter Mitchell’s model of the Q-cycle. These unexpected redox potentialsimpact bioenergetic yields at different levels of the electron transfer chain

Purification et caractérisation d'un super-complexe respiratoire

Bioenergetic membranes present a high protein density - a crucial factor for their organizationinto super-complexes. This project uses the thermophilic bacteria Geobacillus stearothermophilusas a model to study the formation of membrane protein super-complexes with the aim of structuralstudies. We purified and characterized a super-complex between a menaquinone : cytochromec oxidoreductase (b6c), a cytochrome c550, and a cytochrome c oxidase caa3. Using both opticaland EPR spectroscopy methods, we performed the redox titrations of most of the redox cofactorsof the super-complex. Thus, these results enable a new understanding of menaquinone-usingelectron transport chains, showing that quinones’ redox potential determines the redox potentialof the cytochrome b6c’s cofactors. The conclusions differ from previous partial data, althoughthey fit perfectly with Peter Mitchell’s model of the Q-cycle. These unexpected redox potentialsimpact bioenergetic yields at different levels of the electron transfer chain.Les membranes impliquées dans les processus bioénergétiques arborent une très grande densité de protéines, paramètre déterminant pour leur organisation supra-moléculaire. Dans ce travail, nous avons utilisé la bactérie thermophile Geobacillus stearothermophilus comme modèle pour étudier la formation de super-complexes de protéines membranaires, en vue d'une étude structurale. Nous avons purifié et caractérisé un super-complexe comprenant une menaquinol: cytochrome c oxydoréductase (b6c), un cytochrome c550 et une cytochrome c oxydase caa3. En combinant des titrations par spectroscopie optique et résonance paramagnétique électronique, nous avons pu déterminer les potentiels d'oxydo-réduction de la plupart des cofacteurs et combler ainsi une lacune dans l'étude des chaînes de transfert d'électrons utilisant des quinones à bas potentiel redox, les ménaquinones. Nous avons ainsi montré que les potentiels redox des cofacteurs du cytochrome b6c terminés par celui des quinones. Ce travail va à l'encontre de données partielles antérieures publiées, mais est en parfait accord avec les modèles du Q-cycle de Peter Mitchell. Les résultats obtenus ont des répercussions sur les rendements bioénergétiques des différents maillons de la chaîne de transfert

An assessment of how VDAC structures have impacted our understanding of their function

International audienc

VDAC interactions with lipids and their effect on isoform specificity

International audienceThe Voltage-Dependent Anion Channel (VDAC) is a β-barrel, the most abundant protein of the mitochondrial outer membrane, allowing passage of ions and metabolites (such as ATP, ADP, NADH⋯) in and out of the mitochondria. Besides its transport function, VDAC is also implicated in mitochondrial regulation with roles in apoptosis, calcium homeostasis or neurodegeneration. However, its role in those different pathways remains to be understood at the molecular level. VDAC possesses 3 isoforms in mammals (VDAC1, 2 and 3), sharing a high sequence identity, the same architecture and an overlapping tissue distribution. They display very similar transport properties such as conductance, selectivity and voltage-induced closure while their isoform specificity originates from their different interaction with partner proteins. This may determine their unique role in mitochondrial regulation, where each isoform seems to have different functions and a different set of partners: VDAC1 is implicated in neurodegeneration (Alzheimer and Parkinson disease), VDAC2 in apoptosis and calcium homeostasis, and VDAC3 probably in spermatogenesis and oxidative stress. Using a combination of biochemistry, molecular dynamics and electrophysiology, we investigated the lipid organization around each VDAC isoform and their impact on VDAC function. Our data suggest that the three isoforms have different affinities for lipids, and that could contribute to the selection of interacting partners