In the human genome, three isoforms of the voltage-dependent anion-channel are known (hvdac1–3
). However, the most abundant protein in the mitochondrial outer membrane of mammals is also the currently best described form in the literature and is termed isoform I or HVDAC1 protein (Gonçalves et al.
). This isoform was therefore used as a basis for further structural analysis. It has previously been shown that HVDAC1 protein was able to form highly ordered protein arrays in two dimensions that were appropriate for electron microscopy (Dolder et al.
In order to obtain diffracting crystals suitable for structure determination of VDAC, we started with a vector encoding the human mitochondrial isoform I. We expressed the human protein with a C-terminal His tag in E. coli
. This approach resulted in inclusion-body formation of multimilligram quantities of HVDAC1, in analogy to previous studies of related proteins (Koppel et al.
; Shi et al.
; Engelhardt et al.
). After solubilization, purification and refolding, the recovery of HVDAC1 from 1 l of cell culture yielded approximately 50 mg.
Successful refolding of the protein was confirmed by far-UV CD spectroscopy in 0.2% LDAO at pH 7 (Fig. 1). The spectra recorded, which have an intercept in ellipticity (λcrossover
) of 204 nm and a broad minimum of ellipticity (λmin
) around 215 nm, resemble the data for mitochondrial VDAC proteins from other organisms (Shao et al.
). Spectral deconvolution of the recorded spectrum predicted a fold with ~12% α-helical, ~26% β-sheet and ~15% β-turn structure, which is in agreement with previous data for the same VDAC analyzed by FTIR (Engelhardt et al.
) and for scVDAC from Saccharomyces cerevisiae
(35% β-sheet, 20% α-helix, 17% β-turn) which was critically dependent on pH values and the detergent used (Koppel et al.
Figure 1 CD spectrum of HVDAC1-His6 after refolding in C8E4. The intercept in ellipticity (λcrossover) of 204 nm and the ellipticity minimum (λmin) at 215 nm correspond almost ideally to the values for other mitochondrial VDAC proteins (more ...)
The large amount of HVDAC1 obtained by the method described facilitated the use of a variety of detergents for crystallization. The refolding process was induced in the presence of LDAO, while exchange against OPOE was achieved during column chromatography. On screening different detergents, pH values, precipitating agents and buffers against 5400 different crystallization conditions, HVDAC1 produced crystals under 84 different conditions (Table 1). Crystals typically appeared after 7–14 d over a broad range of detergent classes. For the crystallographic data shown here, crystals obtained using Cymal-5 were used. These crystals exhibited a rhombohedric morphology (Figs. 2
a and 2
b). Although most of the crystals obtained appeared similar in shape and size, only a very few were suitable for data collection. A resolution of 3.6 Å was achieved from Cymal-5-solubilised protein (Figs. 2
b and 3). The content of these crystals could be verified by SDS–PAGE and N-terminal sequencing of dissolved crystals (Fig. 1
c). Analysis of the diffraction data revealed the crystal form to be one of the trigonal space groups, with systematic absences (0, 0, 3n). The unit-cell parameters are a = 78.9, c = 165.7 Å. α = β = 90, γ = 120°. Scaling of the collected data indicated an R
merge of 10.8%, a mean I/σ(I) of 11.08 and a solvent content of 65% assuming the presence of one monomer and an unknown number of detergent molecules in the asymmetric unit. While the R
merge value for the outer shell is rather high, we are proceeding for now at 3.6 Å resolution and will not reduce the resolution until the experiments show that reduction is needed.
Figure 2 Pictures of the rhombohedral HVDAC1-His6 crystals with dimensions of 0.2 × 0.1 × 0.1 mm in the crystallization drop (a) and in a loop (b). (c) Representation of a Coomassie-stained 15% SDS–PAGE gel loaded with dissolved (more ...)
Diffraction pattern of the HVDAC1-His6 crystal shown in Fig. 2 recorded on beamline PX10 at SLS.
The structure of the protein will be pursued using two different approaches: (i) introduction of additional methionines into the wild-type protein sequence to improve the anomalous signal during data collection for MAD/SAD and (ii) analysis of NMR data of the protein to produce a NMR model to use in combination with experimental and model phases in phasing programs.
The results presented here support the successful crystallization of a mitochondrial porin. The structures of proteins from the outer membrane of mitochondria are not known and the structure of VDAC may form the first example of this class of proteins. Insights gained from a VDAC structure will be valuable with respect to structure comparisons of the bacterial homologues. Moreover, HVDACs have been described as potential targets for pharmaceutical research and a structure of this human protein at higher resolution may provide an ideal basis for modelling studies. Complex structures with proteins involved in VDAC regulation, i.e. tBid, are planned in order to study the structural differences known to occur upon complex formation.