Structural Basis for a Unique ATP Synthase Core Complex from Nanoarcheaum equitans

Abstract

ATP synthesis is a critical and universal life process carried out by ATP synthases. Whereas eukaryotic and prokaryotic ATP synthases are well characterized, archaeal ATP synthases are relatively poorly understood. The hyperthermophilic archaeal parasite, Nanoarcheaum equitans, lacks several subunits of the ATP synthase and is suspected to be energetically dependent on its host, Ignicoccus hospitalis. This suggests that this ATP synthase might be a rudimentary machine. Here, we report the crystal structures and biophysical studies of the regulatory subunit, NeqB, the apo-NeqAB, and NeqAB in complex with nucleotides, ADP, and adenylyl-imidodiphosphate (non-hydrolysable analog of ATP). NeqB is ∼20 amino acids shorter at its C terminus than its homologs, but this does not impede its binding with NeqA to form the complex. The heterodimeric NeqAB complex assumes a closed, rigid conformation irrespective of nucleotide binding; this differs from its homologs, which require conformational changes for catalytic activity. Thus, although N. equitans possesses an ATP synthase core A3B3 hexameric complex, it might not function as a bona fide ATP synthase.

Keywords: ATP synthase; Nanoarchaeum equitans; archaea; catalytic core; crystal structure; evolution; protein complex.

FIGURE 1.

Schematic representation of the putative N. equitans ATP synthase. The N. equitans ATP synthase is made up of five subunits as follows: A, B, D, I, and c (proteolipid). The putative N. equitans ATP synthase model was generated based on the model of A₁A₀-ATP synthase of M. jannaschii.

FIGURE 2.

Conserved sequences and the phylogenetic analysis. a shows the sequence alignment of the Walker A and Walker B regions of NeqA and NeqB with homologs. The functionally important and conserved Glu-256 of NeqA is marked with an asterisk. NeqB was compared with the F-type ATP synthase subunit β and the homologous Walker regions, as highlighted. b and c, phylogenetic tree shows the evolutionary distance between NeqA and NeqB and their homologs. The numbers in red indicate the confidence levels obtained from bootstrapping for that particular node.

FIGURE 3.

Structure of N. equitans subunits. a, bar diagram representing conserved motifs on the NeqA and NeqB subunits. b, crystal structure of NeqB (in corresponding colors). The Walker homologous and GVP motif are labeled.

FIGURE 4.

Interactions of NeqA and NeqB with nucleotides. Representative ITC profiles for binding of NeqA and NeqB are shown. The upper part of each panel shows the thermogram after baseline correction, and the lower part show the fit of the data to a model, taking into consideration a single class of binding sites. a, ITC profiles representing moderate affinity binding between Mg-ATP (300 μm) and NeqA (10 μm). The c value is 1.9. b, low affinity binding between Mg-ATP (1 mm) and NeqB (10 μm). The c value is 0.16. The table represents the derived thermodynamic parameters.

FIGURE 5.

Interaction of NeqA and NeqB to form hexameric complex. a, AUC profile representing the formation of the catalytic core (A₃B₃) hexamer (∼300 kDa). b, NeqAB complex was compared with monomeric NeqA and NeqB using blue native gels to show formation of an ∼300-kDa hexamer in the NeqAB complex. c, gel filtration profile of 15 mg/ml purified NeqAB complex overlaid with standard calibration proteins from GE Healthcare; ferritin, 440 kDa; conalbumin, 75 kDa. The red peak at 11.75 ml represents the AB complex at ∼300 kDa.

FIGURE 6.

Interactions between NeqA and NeqB. a, calorimetric titrations corresponding to the interaction between NeqA and NeqB in the absence of nucleotide (left, 100 μm NeqA titrated into 10 μm NeqB). The c value is 0.90. b, in the presence (right, 100 μm NeqB titrated into 10 μm NeqA) of nucleotide, Mg-ADP. The c value is 89. An increase in binding affinity between NeqA and NeqB is observed in the presence of Mg-ADP. The table represents the derived thermodynamic parameters.

FIGURE 7.

Structure of NeqAB. a, structure of apo-NeqAB showing NeqA (magenta) and NeqB (cyan) in cartoon representation. The three major domains have been labeled. The P-loop is indicated in black. b, Cα trace of superimposition of NeqB (cyan) from the apo-NeqAB complex and independent NeqB (blue) shows the conformational differences in the N-terminal regions.

FIGURE 8.

Cα trace representation of the bulge region on NeqA. NeqA (magenta) overlaid with a. F-type β (yellow), and b, V-type A subunit (green) shows the Bulge region that is typical of the V/A-type ATPase A subunits.

FIGURE 9.

Structure of NeqAB complex and its closed conformation. Structure of NeqAB complex and its closed conformation. The apo-NeqAB complex (NeqA in magenta and NeqB in cyan) overlaid with (left) nucleotide-free (open) and (right) nucleotide-bound (closed) T. thermophilus V₁ complex AB dimers (A in green; B in gold color). This figure shows that the apo-NeqAB is similar to nucleotide-bound AB dimers of T. thermophilus. The black arrows indicate the displacement between the C-terminal helices of subunits A and B of T. thermophilus. Only the C-terminal regions of the structures are shown. The helix numbers are labeled.

FIGURE 10.

Superimposition of Cα trace representation of independent NeqB with homologs. a, subunit B from T. thermophilus V₁ complex (PDB code 3GQB) (green) versus NeqB (marine blue). b, independent M. mazei subunit B (PDB code 2C61) (dark red) versus NeqB (marine blue). This comparison shows the conformational change taking place in the N terminus in response to the binding of subunit A.

FIGURE 11.

Interaction of nucleotides with NeqA and NeqB. Stereo diagrams are as follows: a, side chains of NeqA (magenta) and NeqB (cyan) interacting with Mg-ADP (green); b, side chains of NeqA and NeqB interacting with Mg-AMP-PNP (blue). In both images, the P-loop motif (Walker A) residues of NeqA in its interaction with the ligand are shown in a cartoon loop representation, and the Arg-326 residue from NeqB is highlighted in red. The final electron density map (2F_o − F_c map, contoured at 1.0σ) is shown for both ADP and AMP-PNP. c, nucleotide binding pocket of NeqA is shown in electrostatic surface representation, with AMP-PNP in stick representation (green). The inset shows the orientation of AMP-PNP on the surface of NeqA. For clarity, NeqB is not shown.

FIGURE 12.

Comparison of apo-NeqAB with its nucleotide-bound forms. Superimposition of NeqAB_apo with a. NeqAB_ADP and b with NeqAB_AMP-PNP.

FIGURE 13.

Hexameric catalytic core complex A₃B₃ hexamer. a, ribbon representation displaying the top-down view of the central cavity of AMP-PNP-bound A₃B₃ hexameric core of the V₁-ATPase from E. hirae (PDB code 3VR3). A_E and B_E denote the open or empty forms, whereas A_T/T′ and B_T/T′ denote the tight or closed form. The open and close dimers have also been indicated. b, NeqAB catalytic core complex (A₃B₃) was generated using symmetry-related molecules displaying a top-down view of the central cavity of AMP-PNP-bound NeqAB hexameric core complex (A₃B₃).

FIGURE 14.

Schematic model of the inactive mechanism of the N. equitans core complex system. The nucleotide-induced conformational changes as seen in the asymmetric states of the active T. thermophilus A₃B₃ complex (PDB code 3W3A) are represented in the left panel (ADP in blue solid circles), and the right panel shows the lack of conformational changes in NeqAB dimer in the presence of nucleotide, AMP-PNP (black-yellow circle) and ADP.