Structural and functional characterization of Solanum tuberosum VDAC36

Abstract

As it forms water-filled channel in the mitochondria outer membrane and diffuses essential metabolites such as NADH and ATP, the VDAC (voltage-dependent anion channel) protein family plays a central role in all eukaryotic cells. In comparison with their mammalian homologues, little is known about the structural and functional properties of plant VDACs. In the present contribution, one of the two VDACs isoforms of Solanum tuberosum, stVDAC36, has been successfully overexpressed and refolded by an in-house method, as demonstrated by the information on its secondary and tertiary structure gathered from circular dichroism and intrinsic fluorescence. Cross-linking and molecular modelling studies have evidenced the presence of dimers and tetramers, and they suggest the formation of an intermolecular disulphide bond between two stVDAC36 monomers. The pore-forming activity was also assessed by liposome swelling assays, indicating a typical pore diameter between 2.0 and 2.7 nm. Finally, insights about the ATP binding inside the pore are given by docking studies and electrostatic calculations.

Historical Perspective of Pore-Forming Activity Studies of Voltage-Dependent Anion Channel (Eukaryotic or Mitochondrial Porin) Since Its Discovery in the 70th of the Last Century

“…The voltage dependence of eukaryotic porins from a variety of eukaryotic organisms was investigated in detail in many studies: Paramecium ( Schein et al, 1976 ; Doring and Colombini, 1984 ; Ludwig et al, 1989 ); Mammals: rat ( Roos et al, 1982 ; Colombini, 1983 ; Ludwig et al, 1986 ), rabbit ( De Pinto et al, 1987a ), bovine ( De Pinto et al, 1987a ), pig ( De Pinto et al, 1987a ), and human brain ( Bureau et al, 1992 ); Fish: Anguilla anguilla ( De Pinto et al, 1991c ); Plants: potato ( Heins et al, 1994 ; Lopes-Rodrigues et al, 2020 ), pea ( Fischer et al, 1994 ), corn ( Smack and Colombini, 1985 ; Aljamal et al, 1993 ; Fischer et al, 1994 ), wheat ( Blumenthal et al, 1993 ), and pea root plastid porin ( Fischer et al, 1994 ; Popp et al, 1997 ); Other organisms: Neurospora crassa ( Freitag et al, 1982c ), yeast ( Ludwig et al, 1988 ), and Dictyostelium ( Troll et al, 1992 ); and Flies: Protophormia ( Wiesner et al, 1996 ) and Drosophila ( De Pinto et al, 1989a ; Aiello et al, 2004 ; Komarov et al, 2004 ). Common to all of these studies is that the eukaryotic porins of all these eukaryotes formed high-conducting channels in reconstituted systems.…”

“…Sterols were also necessary when the properties of mutated N. crassa porin were studied in lipid bilayer membranes. Following the renaturation process of different eukaryotic porins, it seems that sterols seem to be necessary, although they may modulate the properties of the pores, in particular of plant porins ( Popp et al, 1995 ; Carbonara et al, 1996 ; Mlayeh et al, 2010 ; Lopes-Rodrigues et al, 2020 ; Saidani et al, 2021 ). However, in mass production and functional renaturation of two human isoforms of human porin (hVDAC1 and hVDAC2) and of potato VDAC36, no indication for the need of cholesterol/sterol for porin structure and function was observed ( Engelhardt et al, 2007 ; Lopes-Rodrigues et al, 2020 ; Najbauer et al, 2021 ).…”

“…Following the renaturation process of different eukaryotic porins, it seems that sterols seem to be necessary, although they may modulate the properties of the pores, in particular of plant porins ( Popp et al, 1995 ; Carbonara et al, 1996 ; Mlayeh et al, 2010 ; Lopes-Rodrigues et al, 2020 ; Saidani et al, 2021 ). However, in mass production and functional renaturation of two human isoforms of human porin (hVDAC1 and hVDAC2) and of potato VDAC36, no indication for the need of cholesterol/sterol for porin structure and function was observed ( Engelhardt et al, 2007 ; Lopes-Rodrigues et al, 2020 ; Najbauer et al, 2021 ). On the other hand, ergosterol interacts with eukaryotic porin of N. crassa ( Bay and Court, 2009 ) and stigmasterol seems to be important for proper function of bean seed VDAC ( Saidani et al, 2021 ) and sterols were found to be important for renaturation of VDAC from pea root plastids ( Popp et al, 1995 ).…”

Self-standing, conducting and capacitive biomimetic hybrid nanomembranes for selective molecular ion separation

“…28 As a result of its low tendency to associate into large hierarchical structures (i.e. cross-linking experiments showed the formation of dimers and tetramers only 25 ), VDAC36 protein cannot be distinguished at the surface. However, visualization of the intrinsic protein fluorescence by confocal microscopy demonstrated the successful incorporation of VDAC36.…”

“…2.0 vs 1.2 nm), 21 the former is more suitable for the diffusion of molecular ions than the latter. 25 In addition, VDACs are able to switch from an open state (high conductance) to a closed state (low conductance) depending on the environmental conditions, being possible to modulate its diffusion activity. Consequently, we have examined the response of the developed films as a function of the charge of the molecular ion, the concentration of the molecular ion and the state of VDAC.…”