Origami in outer membrane mimetics: correlating the first detailed images of refolded VDAC with over 20 years of biochemical data

Abstract

Mitochondrial porin forms an aqueous pore in the outer membrane, through which selective passage of small metabolites and ions occurs, thereby regulating both mitochondrial function and cellular respiration. Investigations of the structure and function of porin have been performed with whole mitochondria, membrane vesicles, artificial membranes, and in detergent solutions, resulting in numerous models of porin structure. The mechanisms by which this protein functions are undoubtedly linked to its structure, which remained elusive until 2008, with reports of 3 high-resolution structures of this voltage-dependent, anion-selective channel (VDAC). The barrel structure is relatively simple yet unique: it is arranged as 19 anti-parallel beta-strands, with beta-strands 1 and 19 aligned parallel to each other to close the barrel. The N-terminal helical component is located within the lumen of the channel, although its precise structure and location in the lumen varies. With the basic barrel structure in hand, the data obtained in attempts to model the structure and understand porin over the past 20 years can be re-evaluated. Herein, using the mammalian VDAC structures as templates, the amassed electrophysiological and biochemical information has been reassessed with respect to the functional mechanisms of VDAC activity, with a focus on voltage-dependent gating.

The N-terminus of VDAC: Structure, mutational analysis, and a potential role in regulating barrel shape

“…Taken together, these observations are suggestive of transient disruptions of the hydrogen bonding network in that segment of the protein. Other experiments using biotinylated [9] or FLAG-tagged [57] residues revealed sites in this region of the protein that were accessible to streptavidin or anti-FLAG antibody, respectively from both sides of VDAC embedded in artificial membranes, again consistent with a non-rigid barrel (see [49] for discussion). Finally, NMR signals assigned to V143 (ß9) were undetectable, rather than shifted, in hVDAC lacking the first 20 residues [58].…”

“…The roles of particular residues in the function of the N-terminus have been investigated ( [47], [48], [34], reviewed in [35], [49]). Yeast VDAC variants D15K and K19E (equivalent to D16K and K20E for mammalian VDAC1, Fig.…”

“…Based on the observation of barrels composed solely of ß-strands, the gating process has been envisioned as movement of the N-terminus or internal loop(s) either within [49] or in and out [54] of the barrel. In alignment with mechanisms proposed for gating of bacterial porins, these models invoke a fairly rigid barrel (see [55] and discussion in [21]).…”

The mitochondrial voltage-dependent anion channel 1 in tumor cells

“…A breaking of the helix at glycine 11 could increase mobility in this region. Another important VDAC1 structural element is the stretch of multiple glycine residues ( 21 GlyTyrGlyPheGly 25 ) present in the sequence [1,5] that connects the N-terminal domain to β-strand 1 of the barrel and that is highly conserved among mammals [56]. It was proposed that this glycine-rich sequence provides the flexibility required for N-terminal region translocation out of the internal pore of the channel [49].…”

Evidence Supporting the 19 β-Strand Model for Tom40 from Cysteine Scanning and Protease Site Accessibility Studies

“…There is general acceptance of the 19-strand porin structure (hereafter known as the "three-dimensional model") in the literature (13,29,30). However, it has been pointed out that the three-dimensional model conflicts with conclusions based on many previous biochemical and functional studies of porin (31)(32)(33)(34)(35)(36)(37)(38)(39).…”