Abstract
Transmembrane β-barrels of eukaryotic outer mitochondrial membranes (OMMs) are major channels of communication between the cytosol and mitochondria and are indispensable for cellular homeostasis. A structurally intriguing exception to all known transmembrane β-barrels is the unique odd-stranded, i.e. 19-stranded, structures found solely in the OMM. The molecular origins of this 19-stranded structure and its associated functional significance are unclear. In humans, the most abundant OMM transporter is the voltage-dependent anion channel. Here, using the human voltage-dependent anion channel as our template scaffold, we designed and engineered odd- and even-stranded structures of smaller (V216, V217, V218) and larger (V220, V221) barrel diameters. Determination of the structure, dynamics, and energetics of these engineered structures in bilayer membranes reveals that the 19-stranded barrel surprisingly holds modest to low stability in a lipid-dependent manner. However, we demonstrate that this structurally metastable protein possesses superior voltage-gated channel regulation, efficient mitochondrial targeting, and in vivo cell survival, with lipid-modulated stability, all of which supersede the occurrence of a metastable 19-stranded scaffold. We propose that the unique structural adaptation of these transmembrane transporters exclusively in mitochondria bears strong evolutionary basis and is functionally significant for homeostasis.
Highlights
Transmembrane b-barrels of eukaryotic outer mitochondrial membranes (OMMs) are major channels of communication between the cytosol and mitochondria and are indispensable for cellular homeostasis
All known bacterial outer membrane proteins are evenstranded b-barrels that reside in membranes rich in phosphatidylethanolamine (PE; ;70–80%) and phosphatidylglycerol (PG; ;20–30%)
In interesting contrast is the mitochondrial outer membrane, which is different from its prokaryotic ancestor in (i) lipidic composition, wherein mitochondrial membranes are enriched with phosphatidylcholine (PC; .50%) with ;30% PE, ;15% phosphatidylinositol, 1% cardiolipin (CL), and 0% PG [22, 23]; and (ii) possessing at least three abundant outer membrane protein b-barrels, namely voltage-dependent anion channel (VDAC), Tom40, and Mdm10 that adopt odd-stranded, i.e. 19-stranded b-barrel structures [9]
Summary
We engineered scaffolds with odd-stranded [17, 21] or even-stranded [16, 18, 20] barrels of varying pore diameters (Fig. 1 and Fig. S1). The addition of PE in PC membranes increases the stability of most the engineered V2 barrels, when compared with the DGeq of hV2WT in the same condition (Fig. 3C). Cells expressing V216–V218 show poor growth when compared with EV (Fig. 5), these variants form voltage-gated channels in vitro (Fig. 4), suggesting poor mitochondrial targeting of hV216–18 after removal of the 19th strand [30, 45] This observation signifies the importance of b19 in mitochondrial targeting of VDAC. V220 ’ V221 (Fig. 5), supporting our electrophysiology measurements that (i) the engineered 20- and 21-stranded barrels are functionally compromised compared with the 19-stranded scaffold, and (ii) cell survival under redox stress relies on efficient voltage-gated switch of the 19-stranded VDACs to the subconductance state. Our findings support the functional superiority of the 19stranded b-barrel in voltage gating and regulating mitochondrial bioenergetics
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