Structural basis of thermal stability of the tungsten cofactor synthesis protein MoaB from Pyrococcus furiosus

Abstract

Molybdenum and tungsten cofactors share a similar pterin-based scaffold, which hosts an ene-dithiolate function being essential for the coordination of either molybdenum or tungsten. The biosynthesis of both cofactors involves a multistep pathway, which ends with the activation of the metal binding pterin (MPT) by adenylylation before the respective metal is incorporated. In the hyperthermophilic organism Pyrococcus furiosus, the hexameric protein MoaB (PfuMoaB) has been shown to catalyse MPT-adenylylation. Here we determined the crystal structure of PfuMoaB at 2.5 Å resolution and identified key residues of α3-helix mediating hexamer formation. Given that PfuMoaB homologues from mesophilic organisms form trimers, we investigated the impact on PfuMoaB hexamerization on thermal stability and activity. Using structure-guided mutagenesis, we successfully disrupted the hexamer interface in PfuMoaB. The resulting PfuMoaB-H3 variant formed monomers, dimers and trimers as determined by size exclusion chromatography. Circular dichroism spectroscopy as well as chemical cross-linking coupled to mass spectrometry confirmed a wild-type-like fold of the protomers as well as inter-subunits contacts. The melting temperature of PfuMoaB-H3 was found to be reduced by more than 15 °C as determined by differential scanning calorimetry, thus demonstrating hexamerization as key determinant for PfuMoaB thermal stability. Remarkably, while a loss of activity at temperatures higher than 50 °C was observed in the PfuMoaB-H3 variant, at lower temperatures, we determined a significantly increased catalytic activity. The latter suggests a gain in conformational flexibility caused by the disruption of the hexamerization interface.

Figure 1. Adenylylation of the cofactor intermediate MPT catalyzed by hexameric MoaB (P. furiosus) and trimeric MogA (E. coli).

Figure 2. Crystal structure of PfuMoaB.

(A) Ribbon representation of PfuMoaB monomer, secondary structure elements, N- and C-termini are labelled; α-helices are coloured in cyan, β-sheets in magenta, 3₁₀-helices in green, loops in pink. (B) and (C) top and side view of the PfuMoaB hexamer, respectively. Subunits are shown in different colours. Zoom-in represent ionic interactions at the trimerization interface of PfuMoaB-WT. Residues mediating the contacts between subunits are shown in stick representation and are labelled. (D) Sulfate ion at the active site of PfuMoaB. The residues of the conserved Gly-Gly-Thr-Gly motif and the sulfate ion are shown superimposed with the experimentally phased electron density, contoured at 1 σ.

Figure 3. Hexamerization interface and structure-guided mutagenesis of PfuMoaB.

(A) Side view of PfuMoaB hexamer. Two trimers of PfuMoaB are depicted in grey and green. The conserved Gly-Gly-Thr-Gly motif within the active site is shown in the upper PfuMoaB monomer in red. The trimer-trimer interaction interface is boxed-in. α3-helix of the upper monomer is depicted in orange. The zoom-in shows labelled residues at the interface in stick representation. As the interface is build up by identical surfaces of each subunit, the hydrophobic residues of the bottom subunit (Ile59, Leu62, Ile63, Phe66, Ile69, Leu97) are not shown for the sake of clarity. (B) Structure-guided mutagenesis of PfuMoaB. Sequences of α3-helix of PfuMoaB-WT and EcoMogA are coloured in black, flanking residues of the helices are represented in grey. Residues are numbered accordingly to the PfuMoaB sequence. Sequences of the variants PfuMoaB I59R/I63R/F66R and PfuMoaB-H3 are shown. Arginine residues of PfuMoaB I59R/I63R/F66R are depicted in bold. Residues of PfuMoaB-H3, which were not exchanged accordingly to the EcoMogA sequence, are underlined.

Figure 4. Multiple sequence alignment of MPT-adenylyl-transferases from different organisms.

Corresponding MPT-adenylyl-transferases are abbreviated as follows: PfuMoaB, Pyrococcus furious; StoMoaB, Sulfolobus tokodaii; BceMoaB, Bacillus cereus; EcoMogA and EcoMoaB, Escherichia coli; TthMogA, Thermus thermophilus; AaeMogA, Aquifex aeolicus; Arabidopsis thaliana; HsaGephG, Homo sapiens. Secondary structure elements of PfuMoaB are shown. The conserved MPT-binding motif GGTG is highlighted with a red box, the conserved aspartate residue coordinating Mg²⁺- ion with a green box , residues of PfuMoaB α3-helix with a blue box. Highly conserved residues are depicted in white letters and black background; semi-conserved residues are shadowed in grey. Consensus threshold was set to 0.8. Sequences were aligned with Clustal Omega ,and modified with BoxShade server (Swiss Institute of Bioinformatics).

Figure 5. Biochemical characterization of the PfuMoaB-H3 variant in comparison to PfuMoaB-WT.

(A) 15% Coomassie-Blue-stained SDS polyacrylamide gel showing 200 pmol of Ni-NTA-purified PfuMoaB-WT and PfuMoaB-H3. (B) Far-UV CD spectra of Ni-NTA purified PfuMoaB-WT (solid line) and PfuMoaB-H3 (dotted line). (C) Size exclusion chromatography of Ni-NTA purified PfuMoaB-WT and PfuMoaB-H3. 5 nmol of WT and 10 nm of PfuMoaB-H3 were applied on a Superdex 200 10/300 column. Peaks referring to the different oligomerization states of both proteins are labelled. Molecular masses were determined using protein standards. Elution of PfuMoaB-WT is shown as solid line, the PfuMoaB-H3 variant as dotted line. (D–E) SDS-PAGE of cross-linked PfuMoaB-WT and PfuMoaB-H3 with BS³ (D) and EDC (E). Samples without addition of cross-linkers were used as control (“–”). Observed oligomeric forms of both proteins are labelled. The cross-linked protein bands with a size corresponding to the trimers (designated with *) were further subjected to mass spectrometry analysis. (F) Differential scanning calorimetry of MPT-adenylyl-transferases. Melting curves of PfuMoaB-WT, PfuMoaB-H3, EcoMogA, EcoMoaB and AthCnx1G recorded by DSC. The maximum of each peak represents the respective T _m value. Average T _m values for each protein are summarized in the Table 2. Measurements were performed in duplicate for each experiment.

Figure 6. In vitro adenylylation of MPT by PfuMoaB-WT and PfuMoaB-H3 variant.

Adenylylation rates were determined for both proteins at 25, 35, 50, 65 and 80°C by monitoring formation of MTP-AMP over time. Initial velocities of PfuMoaB-WT and the H3 variant at different temperatures are depicted as solid and dotted lines, respectively. Error bars represent the standard deviation of data obtained in at least two independent experiments.

Figure 7. Active site of PfuMoaB-WT.

Two PfuMoaB subunits at the hexamerization interface are shown as ribbon in green and grey, respectively. The conserved Asp56 residue coordinating Mg²⁺ (pink) is shown in sticks. MPT-AMP in the active site is derived from a superimposition with the structure of the PfuMoaB homologue A. thaliana Cnx1G (1UUY). The Mg²⁺-ion derived from a superimposition with the homologues sub-domain 3 of E. coli MoeA (1FC5) , .