The CoxD protein, a novel AAA+ ATPase involved in metal cluster assembly: hydrolysis of nucleotide-triphosphates and oligomerization

Abstract

CoxD of the α-proteobacterium Oligotropha carboxidovorans is a membrane protein which is involved in the posttranslational biosynthesis of the [CuSMoO₂] cluster in the active site of the enzyme CO dehydrogenase. The bacteria synthesize CoxD only in the presence of CO. Recombinant CoxD produced in E. coli K38 pGP1-2/pETMW2 appeared in inclusion bodies from where it was solubilized by urea and refolded by stepwise dilution. Circular dichroism spectroscopy revealed the presence of secondary structural elements in refolded CoxD. CoxD is a P-loop ATPase of the AAA-protein family. Refolded CoxD catalyzed the hydrolysis of MgATP yielding MgADP and inorganic phosphate at a 1∶1∶1 molar ratio. The reaction was inhibited by the slow hydrolysable MgATP-γ-S. GTPase activity of CoxD did not exceed 2% of the ATPase activity. Employing different methods (non linear regression, Hanes and Woolf, Lineweaver-Burk), preparations of CoxD revealed a mean K(M) value of 0.69±0.14 mM ATP and an apparent V(max) value of 19.3±2.3 nmol ATP hydrolyzed min⁻¹ mg⁻¹. Sucrose density gradient centrifugation and gel filtration showed that refolded CoxD can exist in various multimeric states (2-mer, 4-mer or 6-mer), preferentially as hexamer or dimer. Within weeks the hexamer dissociates into the dimer, a process which can be reversed by MgATP or MgATP-γ-S within hours. Only the hexamers and the dimers exhibited MgATPase activity. Transmission electron microscopy of negatively stained CoxD preparations revealed distinct particles within a size range of 10-16 nm, which further corroborates the oligomeric organization. The 3D structure of CoxD was modeled with the 3D structure of BchI from Rhodobacter capsulatus as template. It has the key elements of an AAA+ domain in the same arrangement and at same positions as in BchI and displays the characteristic inserts of the PS-II-insert clade. Possible functions of CoxD in [CuSMoO₂] cluster assembly are discussed.

Figure 1. Sequence similarity between CoxD of Oligotropha carboxidovorans and BchI of the Rhodobacter capsulatus Mg-chelatase complex (top) along with secondary structural elements and AAA+ motifs in CoxD and BchI (bottom).

Panel (A) compares the sequences of CoxD (295 aa) of O. carboxidovorans and BchI (350 aa) of R. capsulatus employing the UVa FASTA Server (fasta.bioch.virginia.edu/fasta_www2/fasta_list2.shtml). Colons and dots indicate identity or similarity, respectively. The secondary structures shown in panel (B) have been adopted from the crystal structure of BchI and the amino acid sequence of CoxD , , , . The N-terminus of CoxD contains the N-linker showing the characteristic motif consisting of a hydrophobic amino acid (A¹⁷) and glycine (G¹⁸). The P-loop of the Walker A motif lies between strand β1 and helix α2 and contains the consensus G⁴³XXGXG⁴⁸KT⁵⁰, whereas V¹³⁰LLIDE¹³⁵ on β3 of CoxD is the Walker B motif (hhhhDE, h stands for hydrophobic aa) which interacts with ATP. The loop between β2 and α2 is the suggested pore loop of CoxD. The conserved aromatic-hydrophobic-glycine pore loop motif of AAA unfoldases/translocases that resides between β2 and α2 of the AAA+ core is modified in BchI to PLG. The second region of homology (SRH) is considered to coordinate nucleotide hydrolysis and conformational changes between subunits of the AAA ATPase oligomer . SRH of CoxD (aa 175–189) consists of part of β4 and all of α4 and contains sensor 1 (N¹⁷⁷) and arginine fingers (R¹⁸⁸R¹⁸⁹) at the end of α4. Sensor 2 (R²⁵⁹) of CoxD resides near the N-terminal end of α7 and is as in BchI part of the VWA-binding motif (R²⁵⁹AE²⁶¹). The numbers preceding the motifs in panel (B) refer to the position of the first amino acid. Blue rectangles refer to α-helices, and green arrows indicate β-strands.

Figure 2. Detection of CoxD in subcellular fractions of O. carboxidovorans cultivated under different conditions.

O. carboxidovorans OM5 (lanes A and B) and the insertional mutants coxD (lane D), coxE (lane E), and coxF (lane F) were grown under different metabolic conditions: lane A, chemolithoautotrophically with CO (45 CO, 5 CO₂, 50 air; all values in % v/v); all other lanes, chemolithoautotrophically with H₂ in the presence of CO (30 H₂, 5 CO₂, 30 CO, 35 air) to induce the transcription of cox-genes. In order to separate cytoplasmic fractions (S, supernatant) and membrane fractions (P, pellet), crude extracts prepared from the bacteria were subjected to ultracentrifugation (100,000× g, 2 h, and 4°C). The distribution of CoxD in the supernatant or the pellet was analyzed by applying 50 µg of protein from S or P to SDS-PAGE. Prior to PAGE all samples were boiled in 1% SDS as described in the methods section. CoxD was identified by Western blotting employing IgG antibodies raised against recombinant CoxD. The grey bands apparent from lanes A, B, E, and F originate from the CoxD protein.

Figure 3. Analysis of recombinant CoxD by SDS-PAGE.

The formation of CoxD in E. coli K38 pGP1-2/pETMW2 was induced with 1 mM IPTG followed by a heat shock (42°C). Intact bacterial cells or washed inclusion bodies suspended in Tris/HCl supplied with SDS and β-mercaptoethanol were boiled for 4 min as detailed in Materials and Methods. Lanes 1 and 3 received 30 µg of bacterial cell protein or 5 µg of inclusion body protein, respectively. Protein was stained with Coomassie Brilliant Blue. The program ImageJ (lanes 2 and 4) was employed for densitometry . Lane 5 represents the same experiment as in lane 1, except that 5 µg of bacterial cell protein was applied and analysis was by Western-blotting employing IgG antibodies directed against CoxD.

Figure 4. Absorption spectra (A) and circular dichroism (CD) spectra of refolded CoxD kept in the absence (B) or presence of ATP-γ-S (C).

Solubilized CoxD (5.5 mg ml⁻¹) was refolded by rapid dilution (50-fold) in ice cold aqueous Tris (20 mM) under magnetic stirring. The solution was immediately adjusted to pH 9.0 (A, dashed line) and subsequently brought to pH 8.0 (A, dotted line) in pH increments of 0.2 per 24 h as specified in Materials and Methods. Then the protein was concentrated to 1.04 mg ml⁻¹ by ultra-filtration (A, solid line). The CD spectra of CoxD (0.471 mg ml⁻¹; 10 mM KH₂PO₄/KOH, pH 8.0) are shown in (B). The CD spectra of CoxD (0.537 mg ml⁻¹; 10 mM KH₂PO₄/KOH, pH 8.0) in the presence of 0.1 mM MgATP-γ-S are shown in (C). After 2 h of incubation with MgATP-γ-S, excess nucleotides were removed by gel filtration on Sephadex G-25. All data were recorded at 20°C. Raw data are shown in black. The smoothed data used for secondary structure estimation and the back-calculated CD-spectrum based on deconvolution with the CDSSTR algorithm is depicted in red and blue, respectively. For further details refer to Materials and Methods.

Figure 5. Hydrolysis of NTPs by refolded CoxD.

(A) Release of P_i from ATP (1 mM Na₂ATP in 100 mM Tris/acetate, pH 8.0) catalyzed by CoxD (0.22 mg protein ml⁻¹) in the presence (•) or absence (○) of 1 mM Mg²⁺-acetate. (B) Formation of P_i (•, ○) and ADP (▪, □) in the presence (•, ▪) or absence (○, □) of CoxD; assays contained 1 mM Mg²⁺-acetate. (C) Correlation of ATPase activity and amount of CoxD (•); assays contained 1 mM Mg²⁺-acetate; incubations were for 60 min. (D) Dependence of the CoxD ATPase activity (0.11 mg protein ml⁻¹) on the concentration of ATP in the assay (•). The dotted lines indicate the calculated K_M and 0.5 V_max. (E) Formation of P_i from GTP (1 mM Na₂GTP in 100 mM Tris/acetate, pH 8.0; assays contained 1 mM Mg²⁺-acetate) in the presence (•) or absence (○) of CoxD (0.22 mg protein ml⁻¹). (F) Effect of ATP-γ-S (○) or GTP (•) on the hydrolysis of ATP (1 mM) by CoxD. Assays contained 1 mM Mg²⁺-acetate. An activity of 100% corresponds to 9.37 nmol ADP released min⁻¹ mg⁻¹ (○) or 8.72 nmol ADP released min⁻¹ mg⁻¹ (•). See Material and Methods for experimental details. All experiments were at 25°C. Experimental errors are indicated by vertical bars.

Figure 6. Analysis of oligomerization of refolded CoxD protein by sucrose density gradient centrifugation.

CoxD in refolding buffer was subjected to sucrose density gradient centrifugation, and fractions were analyzed as indicated. Coomassie Brilliant Blue was employed for protein estimation. CO dehydrogenase (□) was included as a reference for molecular mass (277,074 Da). For details refer to Materials and Methods. (A) Oligomerization of CoxD and ATPase activity. Sucrose gradients received 1 ml of CoxD (0.5 mg). After centrifugation and fractionation, CoxD protein was determined (•); a, b, and c refer to different oligomers of CoxD, which are discussed in the text. ATPase activity was assayed by following the hydrolysis of MgATP to MgADP (▵) as detailed in the methods section. (B) Stability of CoxD hexamers in diluted and concentrated solution. Sucrose gradients received the following samples: Freshly refolded CoxD (0.01 mg ml⁻¹) was concentrated by ultra-filtration (0.9 mg ml⁻¹). From this solution, 1.5 mg protein was subjected to sucrose gradient centrifugation (○). Concentrated CoxD was kept for 28 d at 4°C and analyzed again (▵). Freshly refolded CoxD (0.01 mg ml⁻¹) was also kept for 28 d at 4°C, concentrated (0.82 mg ml⁻¹), and 1.5 mg were subjected to sucrose gradient centrifugation (•). (C) Recentrifugation of hexameric CoxD. Refolded CoxD was concentrated (2.13 mg ml⁻¹), and 1.5 mg were applied to sucrose density gradient centrifugation (○). The peak fractions representing the hexamer (19.6% to 22.1% sucrose) of five experiments were pooled. After removal of sucrose by gel filtration employing 100 mM Tris/acetate and concentration (0.21 mg ml⁻¹), 0.4 mg CoxD was recentrifuged (•). (D) Impact of ATP on dimeric CoxD. Dimeric CoxD was prepared from the hexamer as described in (B), concentrated (1.5 mg ml⁻¹), and 1 mg was subjected to sucrose gradient centrifugation (○). The effect of ATP was examined in samples of 0.5 mg CoxD in 73 mM Tris/acetate (pH 8.0) supplemented with 0.1 mM MgATP. Assays were kept for 2 h at 4°C and then supplied to sucrose density gradients which were amended with 0.1 mM MgATP (•). (E) Impact of slow hydrolysable ATP-γ-S on dimeric CoxD. Conditions and symbols are as in (D) with the exception that the CoxD sample contained 0.1 mM MgATP-γ-S, and the sucrose gradient was devoid of nucleotides. (F) Gel filtration of refolded CoxD (6 mg) on Sephacryl S-300 (•); Blue Dextran (□) served as a void volume marker in a separate run.

Figure 7. TEM analysis of recombinant CoxD.

(A) TEM image of CoxD negatively stained with uranyl acetate. Distinct particles are clearly visible. Most particles exhibit a size of 10–16 nm. Additionally, some smaller fragments and some elongated particles were also present. Scale bar: 25 nm. (B) Initial class averages of CoxD calculated from 19,091 raw particles. Reproducible substructures can be identified. Closer inspection of the classes revealed, that classes showing elongated features (marked with an asterisk) were clearly heterogeneous and were thus rejected from further analysis. (C) Final class averages calculated from 15,876 selected raw particles. Reproducible structure is clearly enhanced, revealing two types of particle views. The larger type of views comprises particles with a length of about 16 nm with a narrow top and wider end, respectively. The second type extents to only 10–11 nm. However, at this level of 2D analysis, it is impossible to assign the views to complexes with different sizes, e.g. to dimers, tetramers and hexamers, respectively, and/or different orientation of the complexes on the grid. (D) Representative class averages (left) and representation of randomly selected members from these classes (right). Scale bar (B–D): 15 nm.

Figure 8. Homology-built model of CoxD (top) in ribbon representation and the sequence alignment (CoxD*) from which the model was build (bottom).

The following adjustments were introduced into the amino acid sequence of CoxD to account for differences between CoxD (295 aa) and BchI (350 aa) which interfered with model building by the program ESyPred3D. The N-terminus of CoxD was extended with the sequence M¹TTAVAR⁷ of BchI (cf. Fig. 1). With the exception of the C-terminus ³²⁰T to ³⁵⁰P, all gaps in CoxD relative to BchI were filled up with corresponding BchI sequences (cf. Fig. 1): ³⁹T⁴⁰A, ⁸⁷MIP⁹⁰D, ⁹²ATVLS⁹⁷T, ¹⁰⁴TPVV¹⁰⁸D, ²⁰²RPQ²⁰⁵L, ²¹⁹R, ²³²D, ²⁴⁷PKDMDIRN²⁵⁵Q. The following deletions were made in CoxD to account for gaps in BchI: ⁵⁸QAN⁶¹G, ¹²²AI¹²⁴R, ¹⁵⁶V, ²⁴²E, ²⁶²P, ²⁷⁴K, ²⁸⁶VSD²⁸⁹R. The modified sequence of CoxD is being referred to as CoxD*. The 3D structure of CoxD was modeled with the automated web surfer ESyPred3D employing the CoxD* sequence shown and the 3D structure of BchI (1G8P) from Rhodobacter capsulatus as template. (A) Structure of CoxD showing the N-terminal Rossmann-fold domain (orange) with central β-sheet core (green) and the C-terminal α-helical domain (blue). (B) Positions of AAA+ domain key elements on CoxD: Walker A (red), Walker B (blue), sensor 1 (magenta), arginine finger (orange), sensor 2 (green), VWA-binding motif (cyan). (C) Inserts of the PS-II-insert clade in CoxD; helix-2-insert (orange), PS-I-insert (blue), PS-II-insert (green). (D) A model of the BchI hexamer of the Mg-chelatase complex (pdb 2X31) showing the inserts of the PS-II-insert clade. The program CHIMERA was employed to show the different views and color coding .