The VKOR homolog from Synechococcus sp.,
naturally fused to a Trx-like domain, was expressed in E. coli
and purified in the detergent n-dodecyl-β-
D-maltoside (DDM). The purified protein contained bound endogenous quinones, primarily ubiquinone (mass spectrometry data not shown), and showed a characteristic UV absorption at 400nm 13
. Because the wild type protein did not yield high-quality crystals, we tested mutants, in which cysteines were changed to alanines or serines to stabilize an interdomain disulfide bridge 14
. The VKOR domain mutant C56S gave crystals that belonged to the P61
space group (Table S1
). Experimental phases with a maximum resolution of 3.6Å were obtained by the multiple isomorphous replacement method and were dramatically improved by solvent flattening due to the 80% solvent content. The membrane helices were well resolved in the resulting electron density map (Fig. S3
), likely because they are packed side by side (Fig. S4
). A model could be built with confidence, and the register was confirmed by the positions of aromatic residues and by specific binding sites of heavy atoms (Fig. S5a
). In addition, the positions of all methionines were confirmed by crystallizing seleno-methionine substituted protein (Fig. S5b
). The Trx-like domain was also crystallized by itself; these crystals diffracted to 1.7Å (Table S1
). The phases were obtained by the single anomalous diffraction method using a mercury derivative and a model was built automatically (Fig. S6
). The structure of the Trx-like domain docked well into the map of the full-length protein. The model of the complex of VKOR and its redox partner was refined to Rwork
factors of 24.6% and 30.5%, respectively. The model lacks the 15 N-terminal and 5 C-terminal residues of the full-length protein and has uncertainties in the region encompassing amino acids 53 to 55.
The protein consists of the membrane-embedded domain containing VKOR, connected via a linker segment to the extracellular Trx-like domain (; see also Movie S1
). The membrane-embedded part contains five transmembrane helices (TM) (). TMs 1-4 are homologous to human VKOR (Fig. S2
; ~24% identical amino acids). Consistent with in vivo
, both termini and a loop between TMs 2 and 3 are located in the cytosol. The helices form a four-helix bundle with a quinone in its interior. The quinone is close to the periplasmic side of the membrane, in proximity to the CXXC active site motif in TM4 (formed by Cys130 and Cys133). TM5 passes through the membrane to connect with the fused periplasmic Trx-like domain. TM5 is located on the outside of the four-helix bundle, contacting TM3 () and the loop between TM1 and 2.
Architecture of Synechococcus VKOR in complex with its redox partner
The longest loop between TMs is on the periplasmic side of the VKOR homolog, between TM1 and TM2. This 1/2-loop contains an extended N-terminal part (called the “1/2-segment”), a short helix (called the “1/2-helix”), and a C-terminal segment that contacts TM5 (). The 1/2-segment contains a pair of cysteines (Cys50 and Cys56), which are present in all VKORs. The 1/2-helix begins after Cys56 and ends with the conserved serine/threonine residue (Ser62). In most species, there are five residues between the second cysteine and the conserved serine/threonine (Fig. S2
), suggesting that the 1/2-helix is a common feature. The 1/2-helix forms a lid on the four-helix bundle, shielding the quinone from the periplasmic space. The helix is amphipathic and likely lies on top of the lipid surface, with the well-conserved Trp64 as a major anchor point.
Consistent with the introduced mutation Cys56Ser, an inter-domain disulfide bridge with strong density at 3.5σ is observed between Cys50 in the VKOR domain and Cys209 in the CXXC motif of the Trx-like domain. Formation of the inter-domain disulfide bridge is facilitated by the insertion of the 1/2-segment of VKOR into the active site groove of the Trx-like domain (Fig. S7
). This groove is formed by several loops between the secondary structure elements, has a hydrophobic surface, and contains the active site CXXC motif. The groove likely also interacts with newly synthesized protein substrates, allowing disulfide exchanges. The Trx-like domain also contains two other cysteines (Cys231 and Cys244), which are disulfide bonded and likely play a structural role.
The plane of the quinone ring is clearly defined (; see also Fig. S8
and Movie S2
), tilted by 70° with respect to the membrane surface. Much of the isoprenyl tail of the ubiquinone can also be seen, intercalated into the V-shaped cleft between TMs 2 and 3 (). The location of the isoprenyl tail establishes the positions of the substituents on the quinone ring (; for chemical structure, see Fig. S1b
). Strong, continuous density is observed in the ~2Å gap between the C1 position of ubiquinone and Cys133 in the CXXC motif of VKOR, suggesting that they are linked by a covalent bond. Cys130 and Cys133 are located at the side of the quinone ring and there is strong density between the two sulfur atoms. It thus appears that the structure corresponds to a situation in which electrons are shared between the disulfide bridge in the CXXC motif and the quinone ring.