Determination of the Conformation of the Human VDAC1 N‐Terminal Peptide, a Protein Moiety Essential for the Functional Properties of the Pore

Abstract

Mitochondrial porin or VDAC (voltage-dependent anion-selective channel) is the most abundant protein in the mitochondrial outer membrane. The structure of VDAC has been predicted to be a transmembrane beta-barrel with an alpha-helix at the N terminus. It is a matter of debate as to whether this putative alpha-helix plays a structural role as a component of the pore walls or a function in the pore activity. We have synthesised the human VDAC1 (HVDAC1) N-terminal peptide Ac-AVPPTYADLGKSARDVFTK-NH2 (Prn2-20) and determined its structure by CD and NMR spectroscopy. CD studies show that the Prn2-20 peptide exists in aqueous solvent as an unstructured peptide with no stable secondary structure. In membrane-mimetic SDS micelles or water/trifluoroethanol, however, it assumes an amphipathic alpha-helix conformation between Tyr5 and Val16, as deduced from NMR. No ordered structure was observed in dodecyl beta-maltoside. Differential scanning calorimetric measurements were carried out in order to examine the membrane affinity of the peptide. Upon interaction with the negatively charged 1,2 dipalmitoyl-sn-glycero-3-phosphoserine membrane, Prn2-20 exhibited distinctive behaviour, suggesting that electrostatics play an important role. Interaction between the peptide and artificial bilayers indicates that the peptide lies on the membrane surface. Recombinant HVDAC1 deletion mutants, devoid of seven or 19 N-terminal amino acids, were used for transfection of eukaryotic cells. Over-expression of HVDAC1 increases the number of Cos cells with depolarised mitochondria, and this effect is progressively reduced in cells transfected with HVDAC1 lacking those seven or 19 amino acids. The mitochondrial targeting of the deletion mutants is unaffected. The overall picture emerging from our experiments is that the VDAC N-terminal peptide plays a role in the proper function of this protein during apoptotic events.

Swapping of the N‐terminus of VDAC1 with VDAC3 restores full activity of the channel and confers anti‐aging features to the cell

“…The Nterminal sequence is located inside the channel, forming a partial obstruction of the wide pore, possibly exerting a structural reinforcement to the channel walls. In agreement with this finding, CD and NMR spectroscopy of the isolated N-terminal peptide showed that there is the need of a special environment to force the peptide in an ordered a-helix secondary structure [20]. In water, for example, it is usually disordered.…”

“…This structure represents an addition to the b-barrel pore structure and was proposed to have functionally relevant properties [20][21][22][23][24][25][26]35]. It has been recently proposed that the final N-terminal sequence (i.e.…”

The VDAC1 N-terminus is essential both for apoptosis and the protective effect of anti-apoptotic proteins

“…As such, this helix is ideally positioned to regulate the conductance of ions and metabolites passing through the VDAC pore. These structural and other studies (De Pinto et al, 2007) further indicate that the helical region of the VDAC N-terminal domain forms a distorted α-helix that does not span the whole sequence but only part of it. Although these recently determined VDAC1 structures suggest that the hydrophilic N-terminus of the protein is nestled within the pore (Bayrhuber et al, 2008;Hiller et al, 2008;Ujwal et al, 2008), other approaches point to the N-terminal α-helix as being exposed to the cytoplasm (De Pinto et al, 2003), to cross the membrane (Colombini, 2004) or to lie on the membrane surface (Reymann et al, 1995).…”

“…The N-terminal domain of VDAC1 has been the object of considerable interest and was proposed to possess functionally relevant properties (Colombini et al, 1987;De Pinto et al, 2007;Peng et al, 1992;Stanley et al, 1995;Ujwal et al, 2008;Yehezkel et al, 2007). VDAC provides the major pathway for nucleotides and other metabolites moving across the OMM.…”

Probing the orientation of yeast VDAC1 in vivo

“…The FLAG1, which is 11-20 residues from the N-terminus, will most likely have a trans exposure (see Fig. 1); especially if the Nterminus is as dynamic as proposed [8,13,[22][23][24][25]. Residues 1-10 continue through the lumen towards exposure of the extreme Nterminal residue to the cis side.…”