scholarly journals MicroED structure of lipid-embedded mammalian mitochondrial voltage dependent anion channel

2020 ◽  
Author(s):  
Michael W. Martynowycz ◽  
Farha Khan ◽  
Johan Hattne ◽  
Jeff Abramson ◽  
Tamir Gonen
Keyword(s):  
Membrane Protein ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Anion Channel ◽  
Voltage Dependent Anion Channel ◽  
X Ray Crystallography ◽  
Voltage Dependent ◽  
Crystal Contacts ◽  
Viscous Media ◽  
Resolution Structure

AbstractA near-atomic resolution structure of the mouse voltage dependent anion channel (mVDAC) is determined by combining cryogenic focused ion-beam (FIB) milling and microcrystal electron diffraction (MicroED). The crystals were grown in a viscous modified bicelle suspension which limited their size and made them unsuitable for conventional X-ray crystallography. Individual thin, plate-like crystals were identified using scanning electron microscopy (SEM) and focused ion-beam (FIB) imaging at high magnification. Three crystals were milled into thin lamellae. MicroED data were collected from each lamellae and merged to increase completeness. Unmodelled densities were observed between protein monomers, suggesting the presence of lipids that likely mediate crystal contacts. This work demonstrates the utility of milling membrane protein microcrystals grown in viscous media using a focused ion-beam for subsequent structure determination by MicroED for samples that are not otherwise tractable by other crystallographic methods. To our knowledge, the structure presented here is the first of a membrane protein crystallized in a lipid matrix and solved by MicroED.

scholarly journals MicroED structure of lipid-embedded mammalian mitochondrial voltage-dependent anion channel

2020 ◽  
Vol 117 (51) ◽  
pp. 32380-32385
Author(s):  
Michael W. Martynowycz ◽  
Farha Khan ◽  
Johan Hattne ◽  
Jeff Abramson ◽  
Tamir Gonen
Keyword(s):  
Membrane Protein ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Anion Channel ◽  
Voltage Dependent Anion Channel ◽  
X Ray ◽  
X Ray Crystallography ◽  
Voltage Dependent ◽  
Viscous Media ◽  
Scanning Electron

A structure of the murine voltage-dependent anion channel (VDAC) was determined by microcrystal electron diffraction (MicroED). Microcrystals of an essential mutant of VDAC grew in a viscous bicelle suspension, making it unsuitable for conventional X-ray crystallography. Thin, plate-like crystals were identified using scanning-electron microscopy (SEM). Crystals were milled into thin lamellae using a focused-ion beam (FIB). MicroED data were collected from three crystal lamellae and merged for completeness. The refined structure revealed unmodeled densities between protein monomers, indicative of lipids that likely mediate contacts between the proteins in the crystal. This body of work demonstrates the effectiveness of milling membrane protein microcrystals grown in viscous media using a focused ion beam for subsequent structure determination by MicroED. This approach is well suited for samples that are intractable by X-ray crystallography. To our knowledge, the presented structure is a previously undescribed mutant of the membrane protein VDAC, crystallized in a lipid bicelle matrix and solved by MicroED.

scholarly journals Structure of the human voltage-dependent anion channel

2008 ◽  
Vol 105 (40) ◽  
pp. 15370-15375 ◽  
Author(s):  
Monika Bayrhuber ◽  
Thomas Meins ◽  
Michael Habeck ◽  
Stefan Becker ◽  
Karin Giller ◽  
...  
Keyword(s):  
Metabolic Flux ◽  
3D Structure ◽  
Anion Channel ◽  
Bioinformatic Analysis ◽  
Mitochondrial Outer Membrane ◽  
Voltage Dependent Anion Channel ◽  
X Ray Crystallography ◽  
Voltage Dependent ◽  
Α Helix ◽  
Induced Apoptosis

The voltage-dependent anion channel (VDAC), also known as mitochondrial porin, is the most abundant protein in the mitochondrial outer membrane (MOM). VDAC is the channel known to guide the metabolic flux across the MOM and plays a key role in mitochondrially induced apoptosis. Here, we present the 3D structure of human VDAC1, which was solved conjointly by NMR spectroscopy and x-ray crystallography. Human VDAC1 (hVDAC1) adopts a β-barrel architecture composed of 19 β-strands with an α-helix located horizontally midway within the pore. Bioinformatic analysis indicates that this channel architecture is common to all VDAC proteins and is adopted by the general import pore TOM40 of mammals, which is also located in the MOM.

scholarly journals The Role of Voltage-Dependent Anion Channel in Mitochondrial Dysfunction and Human Disease

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1737
Author(s):  
Joyce T. Varughese ◽  
Susan K. Buchanan ◽  
Ashley S. Pitt
Keyword(s):  
Physiological Role ◽  
Anion Channel ◽  
Calcium Flux ◽  
Voltage Dependent Anion Channel ◽  
X Ray Crystallography ◽  
Mitochondrial Regulation ◽  
Interaction Partners ◽  
Voltage Dependent ◽  
Conductance States

The voltage-dependent anion channel (VDAC) is a β-barrel membrane protein located in the outer mitochondrial membrane (OMM). VDAC has two conductance states: an open anion selective state, and a closed and slightly cation-selective state. VDAC conductance states play major roles in regulating permeability of ATP/ADP, regulation of calcium homeostasis, calcium flux within ER-mitochondria contact sites, and apoptotic signaling events. Three reported structures of VDAC provide information on the VDAC open state via X-ray crystallography and nuclear magnetic resonance (NMR). Together, these structures provide insight on how VDAC aids metabolite transport. The interaction partners of VDAC, together with the permeability of the pore, affect the molecular pathology of diseases including Parkinson’s disease (PD), Friedreich’s ataxia (FA), lupus, and cancer. To fully address the molecular role of VDAC in disease pathology, major questions must be answered on the structural conformers of VDAC. For example, further information is needed on the structure of the closed state, how binding partners or membrane potential could lead to the open/closed states, the function and mobility of the N-terminal α-helical domain of VDAC, and the physiological role of VDAC oligomers. This review covers our current understanding of the various states of VDAC, VDAC interaction partners, and the roles they play in mitochondrial regulation pertaining to human diseases.

scholarly journals Porin 1 Modulates Autophagy in Yeast

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2416
Author(s):  
Filomena Broeskamp ◽  
Elizabeth S. M. Edrich ◽  
Oskar Knittelfelder ◽  
Lisa Neuhaus ◽  
Thorsten Meyer ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane Protein ◽  
De Novo ◽  
Anion Channel ◽  
Lipid Homeostasis ◽  
Mitochondrial Outer Membrane ◽  
Cell Organelle ◽  
Voltage Dependent Anion Channel ◽  
Whole Process ◽  
Voltage Dependent

Autophagy is a cellular recycling program which efficiently reduces the cellular burden of ageing. Autophagy is characterised by nucleation of isolation membranes, which grow in size and further expand to form autophagosomes, engulfing cellular material to be degraded by fusion with lysosomes (vacuole in yeast). Autophagosomal membranes do not bud from a single cell organelle, but are generated de novo. Several lipid sources for autophagosomal membranes have been identified, but the whole process of their generation is complex and not entirely understood. In this study, we investigated how the mitochondrial outer membrane protein porin 1 (Por1), the yeast orthologue of mammalian voltage-dependent anion channel (VDAC), affects autophagy in yeast. We show that POR1 deficiency reduces the autophagic capacity and leads to changes in vacuole and lipid homeostasis. We further investigated whether limited phosphatidylethanolamine (PE) availability in por1∆ was causative for reduced autophagy by overexpression of the PE-generating phosphatidylserine decarboxylase 1 (Psd1). Altogether, our results show that POR1 deficiency is associated with reduced autophagy, which can be circumvented by additional PSD1 overexpression. This suggests a role for Por1 in Psd1-mediated autophagy regulation.

Efficient, high-throughput ligand incorporation into protein microcrystals by on-grid soaking

2020 ◽  
Author(s):  
Michael W. Martynowycz ◽  
Tamir Gonen
Keyword(s):  
High Resolution ◽  
Electron Diffraction ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Proteinase K ◽  
X Ray ◽  
X Ray Crystallography ◽  
High Resolution Structure ◽  
Efficient Uptake ◽  
Resolution Structure

AbstractA method for soaking ligands into protein microcrystals on TEM grids is presented. Every crystal on the grid is soaked simultaneously using only standard cryoEM vitrification equipment. The method is demonstrated using proteinase K microcrystals soaked with the 5-amino-2,4,6-triodoisophthalic acid (I3C) magic triangle. A soaked microcrystal is milled to a thickness of 200nm using a focused ion-beam, and microcrystal electron diffraction (MicroED) data are collected. A high-resolution structure of the protein with four ligands at high occupancy is determined. Compared to much larger crystals investigated by X-ray crystallography, both the number of ligands bound and their occupancy was higher in MicroED. These results indicate that soaking ligands into microcrystals in this way results in a more efficient uptake than in larger crystals that are typically used in drug discovery pipelines by X-ray crystallography.

scholarly journals VDAC1 in the diseased myocardium and the effect of VDAC1-interacting compound on atrial fibrosis induced by hyperaldosteronism

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Hadar Klapper-Goldstein ◽  
Ankit Verma ◽  
Sigal Elyagon ◽  
Roni Gillis ◽  
Michael Murninkas ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Apoptotic Cell ◽  
Anion Channel ◽  
Immunohistochemical Analysis ◽  
Voltage Dependent Anion Channel ◽  
Cardiac Tissues ◽  
Therapeutic Implications ◽  
Voltage Dependent ◽  
Models Of Lupus

AbstractThe voltage-dependent anion channel 1 (VDAC1) is a key player in mitochondrial function. VDAC1 serves as a gatekeeper mediating the fluxes of ions, nucleotides, and other metabolites across the outer mitochondrial membrane, as well as the release of apoptogenic proteins initiating apoptotic cell death. VBIT-4, a VDAC1 oligomerization inhibitor, was recently shown to prevent mitochondrial dysfunction and apoptosis, as validated in mouse models of lupus and type-2 diabetes. In the present study, we explored the expression of VDAC1 in the diseased myocardium of humans and rats. In addition, we evaluated the effect of VBIT-4 treatment on the atrial structural and electrical remodeling of rats exposed to excessive aldosterone levels. Immunohistochemical analysis of commercially available human cardiac tissues revealed marked overexpression of VDAC1 in post-myocardial infarction patients, as well as in patients with chronic ventricular dilatation\dysfunction. In agreement, rats exposed to myocardial infarction or to excessive aldosterone had a marked increase of VDAC1 in both ventricular and atrial tissues. Immunofluorescence staining indicated a punctuated appearance typical for mitochondrial-localized VDAC1. Finally, VBIT-4 treatment attenuated the atrial fibrotic load of rats exposed to excessive aldosterone without a notable effect on the susceptibility to atrial fibrillation episodes induced by burst pacing. Our results indicate that VDAC1 overexpression is associated with myocardial abnormalities in common pathological settings. Our data also indicate that inhibition of the VDAC1 can reduce excessive fibrosis in the atrial myocardium, a finding which may have important therapeutic implications. The exact mechanism\s of this beneficial effect need further studies.

scholarly journals Identification of two voltage-dependent anion channel-like protein sequences conserved in Kinetoplastida

2012 ◽  
Vol 8 (3) ◽  
pp. 446-449 ◽  
Author(s):  
Nadine Flinner ◽  
Enrico Schleiff ◽  
Oliver Mirus
Keyword(s):  
Outer Membrane ◽  
Trypanosoma Brucei ◽  
Protein Sequences ◽  
Anion Channel ◽  
Mitochondrial Outer Membrane ◽  
Voltage Dependent Anion Channel ◽  
Voltage Dependent

The eukaryotic porin superfamily consists of two families, voltage-dependent anion channel (VDAC) and Tom40, which are both located in the mitochondrial outer membrane. In Trypanosoma brucei , only a single member of the VDAC family has been described. We report the detection of two additional eukaryotic porin-like sequences in T. brucei . By bioinformatic means, we classify both as putative VDAC isoforms.

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