Helix–strand interaction regulates stability and aggregation of the human mitochondrial membrane protein channel VDAC3

Abstract

Voltage-dependent anion channels (VDACs) are β-sheet–rich transmembrane β-barrels that are vital for metabolite transport across the mitochondrial membrane. Under cellular stress, human VDACs hetero-oligomerize and coaggregate with proteins that can form amyloidogenic and neurodegenerative deposits, implicating a role for VDACs in proteotoxicity. However, whether VDACs possess intrinsic interaction sites that can lead to protein aggregation is not known. Here, we couple a systematic thiol replacement strategy with far-UV circular dichroism spectropolarimetry and UV scattering spectroscopy to map aggregation-prone regions of human VDACs, using isoform 3 as our model VDAC. We show that the region comprising strands β7–β9 is highly aggregation prone. Further, we find that an α1–β7–β9 interaction (involving the hVDAC3 N-terminal α1 helix) can lower protein aggregation, whereas perturbations of this interaction promote VDAC aggregation. We also show that hVDAC3 aggregation proceeds via a partially unfolded structure. Our findings allow us to propose a plausible mechanism for the role of human VDACs in forming proteotoxic aggregates in the cell. The key target sites on VDACs—strands β7–β9—may be useful for developing VDAC aggregation inhibitors.

Single-residue physicochemical characteristics kinetically partition membrane protein self-assembly and aggregation

“…1C). Similar observations have been made for the human mitochondrial voltage-dependent anion channel (VDAC) protein barrel (29).…”

“…Put simply, Ail aggregates when it is unfolded by heating. Other ␤-barrel OMPs such as OmpA, OmpLA, OmpT, OmpW, OmpX, and human mitochondrial porins (12,29) show similar behavior, which arises from the unusually high kinetic stability of these protein scaffolds. Therefore, kinetically stabilized OMPs are aggregation-prone.…”

“…separating the transition from folded to the unfolded state (1,2,29). For example, unfolding of Ail, which is folded in micelles, requires temperatures Ͼ55°C (Fig.…”

“…Next, to deduce how interface aromatics regulate the stability and aggregation of Ail, we monitored the activation energy barrier (E act ) separating the native and aggregated protein states. Most OMPs are kinetically trapped in high-energy states (1,2,29). This allows the ␤-barrel to resist unfolding under adverse cellular conditions.…”

“…Protein unfolding largely precedes aggregation (18) (Fig. 5A, Mechanism 1), but proteins that self-associate in their partially folded states and lead to aggregation have also been documented (29) (Fig. 5A, Mechanism 2).…”