Crystal packing analysis of murine VDAC1 crystals in a lipidic environment reveals novel insights on oligomerization and orientation

Packing analysis of mVDAC1 crystals

The oligomerization of VDAC is widely viewed as an essential component for a number of fundamental mitochondrial functions - ranging from scaffolding for the binding of modulator proteins from both sides of the OMM to facilitating the release of cytochrome c during apoptosis.^16-18 Biochemical studies have shown VDAC to conform to a number of oligomeric species.^18,19 A recent AFM study, on freshly isolated OMM of potato tubers, revealed the most frequently identified VDAC oligomer to be hexamers that may serve as platforms for hexokinase binding to the mitochondria or result in mega-pore formation with pro-apoptotic proteins such as Bax or Bak resulting in the release of cytochrome c in the event of mitochondrial-dependent cell death.¹⁵

Greater insights into the interaction facilitating oligomerization of VDAC1 can be seen through packing analysis of mVDAC1 (PDB 3EMN) crystals, grown in a native-like bilayer environment using lipidic bicelles. A crystallographic-imposed antiparallel dimer formed by transmembrane β-strands 1, 2, 3, 4, 18 and 19 (Figure 1A) is observed. The dimer interface has a buried surface area of 2,376Ų composed of 127 inter-monomer contacts contributed by 36 residues per protomer.²⁰ All contacts are formed by van der Waals interactions with the exception of a lone hydrogen bond, situated in the middle of β-strand 2, between Ser-43 of each protomer (Figure 1A). The mVDAC1 antiparallel dimer represents a significantly tighter interface compared to the parallel dimer of hVDAC1 (550Ų),⁹ and furthermore resembles the values identified from known physiological dimers.^21,22

This tightly packed antiparallel dimer acts as a building block for additional protein-protein and protein-lipid interactions (Figure 1B), developing a hexamer arrangement that is nearly identical to those observed in EM and AFM studies on native OMM.^14,15 Figure 2 shows the mVDAC1 hexamer superimposed on the hexagonal lattice observed in EM images of Neurospora crassa OMM. Each dimer packs laterally within the bicelle membrane plane with a dimer-dimer buried surface area of 1,037Ų, suggesting that it is more dynamic than the individual dimers. This dimer-dimer interface is formed by 51 inter-dimer contacts (7 hydrogen bonds and 44 van der waals interactions), contributed by β-strands 7-10 from dimer-1 and 12-18 from dimer-2. Additionally, the lipid DMPC, sandwiched between the antiparallel dimers, stabilizes the interface by contributing 54 lipid-dimer contacts (31 lipid-dimer1 and 23 lipid-dimer2) formed by van der waals interactions and 3 hydrogen bonds. While lipids have already been shown to regulate VDAC activity, our crystal structure shows that they may also play a critical role in its oligomerization, as seen for other membrane proteins.^22-24

Is the asymmetry physiological?

It is intriguing to speculate as to whether the antiparallel dimer and the resulting hexamer formation seen in the mVDAC1 crystal packing are physiologically relevant. There are a number of comprehensive arguments in support of this scenario: First, mVDAC1 was crystallized in lipidic bicelles that mimic the physiological bilayer environment and possibly represent the native arrangement within the OMM. Second, the antiparallel mVDAC1 dimer interface is considerably tighter than the observed parallel interface of hVDAC1 dimer. Third, the hexamer packing identified in mVDAC1 crystals mimics the native arrangement observed by EM and AFM studies on the OMM.^14,15 Thus, an antiparallel arrangement of VDAC1 may be a physiological requirement for a compact dimer suitable for composing a hexameric arrangement seen in native membranes.

Further evidence from previous scientific reports suggests a dual-directionality of VDAC in the outer membrane of the mitochondria. Early studies from Pinto et al.¹¹ labeled intact mitochondria with antibodies generated against the N-terminus of VDAC indicating a cytoplasmic orientation while Stanley et al.¹² showed a contradictory result where the N-terminus faces the intermembrane space. It has been widely speculated that the differences between the studies were the result of improper handling and isolation of the mitochondria in the former study. A more recent study further complicates the issue where McDonald et al.¹³ probed the membrane orientation of yeast VDAC1 with FLAG-epitopes designed using the hVDAC1 and mVDAC1 structures. While epitopes FLAG1 and FLAG5 located on the N-terminal region and C-terminus respectively, suggested an orientation of VDAC with the C-terminus exposed to the cytosol; epitopes FLAG2 and FLAG3 located on loop regions facing opposite sides of the OMM, were more ambiguous (Figure 3). Both VDAC-FLAG2 and VDAC-FLAG3 bound antibody in intact mitochondria, but binding increased significantly upon membrane solubilization, suggesting that both sides of VDAC are exposed to the cytosol and the inter-membrane space. The authors attributed these conflicting results to dynamic behavior of this region formed by β-strands 1-6. It would appear that the topological orientation of VDAC in the OMM is still an ongoing debate.

Dual-topology membrane proteins

It is generally believed that integral membrane proteins have only a single orientation within the membrane. Recently, however, dual-topology proteins displaying opposite orientations in the membrane have been reported.^25-30 The most well documented of these is the Escherichia coli proton-coupled multidrug transporter EmrE. Supporting evidence for this observation came from structural studies on 2-D and 3-D crystals of EmrE that show an asymmetric dimer with the protomers in an antiparallel topological orientation.^25,26 Additionally, von Heijne’s group found using a comparative genomics approach that EmrE has a dual-topology.^27,28 Importantly, recent biochemical studies from the Schuldiner group show that EmrE displays unique promiscuous behavior where protomers can adopt different topologies relative to each other²⁹ and to the lipid bilayer (personal communication by Professor Schuldiner), and yet are still capable of transport. The authors show that two EmrE protomers can associate in a parallel C_in-C_in orientation, parallel C_out-C_out orientation or an antiparallel C_in-C_out orientation and remarkably all retain functionality as a homodimer.

In addition to EmrE, von Heijne’s group identified four other dual-topology candidates, SugE (multi-drug resistance protein), CrcB (camphor resistance protein), YdgC (associated with alginate biosynthesis) and YnfA.²⁸ It has been suggested that the flip-flopping of membrane proteins to adopt a mixture of topologies may serve as an effective strategy to satisfy functional requirements.³⁰ Furthermore, it may represent a stage in the topological evolution of membrane proteins resulting from gene duplications and mutations.²⁹ Although the list of identified dual-topology membrane proteins is small, the idea that proteins can adopt multiple topologies in the membrane has been established.

There is increasing evidence to support an antiparallel topology for VDAC in the mitochondria. A dual-topology model will help to explain the inconclusive results of topology studies and will link the hexamers observed in the OMM with those seen in mVDAC1 crystals grown in lipidic bicelles. The hypothesized bi-orientation of VDAC discussed here establishes a model to be tested biochemically.