Sequence and biochemical analysis of vaccinia virus A32 protein: Implications for in vitro stability and coiled-coil motif mediated regulation of the DNA-dependent ATPase activity

Abstract

Nucleocytoplasmic large DNA viruses (NCLDVs) have massive genome and particle sizes compared to other known viruses. NCLDVs, including poxviruses, encode ATPases of the FtsK/HerA superfamily to facilitate genome encapsidation. However, their biochemical and structural characteristics are yet to be discerned. In this study, we demonstrate that the viral ATPases are significantly shorter than their bacterial homologs, representing the minimal ATPase core of the FtsK/HerA superfamily. We analysed the sequence and secondary structural features of the vaccinia virus A32 protein and determined their roles in the protein's ATPase activity. We sought to purify A32 by various techniques and noted that recombinant A32 expressed in E. coli is highly insoluble and unstable in solution. N-terminal fusion with the thioredoxin solubility tag could alleviate this issue to some extent, but subsequent tag cleavage results in increased susceptibility to precipitation and degradation. We have also predicted a highly conserved coiled-coil motif (CCM) towards the C-terminus of vaccinia virus A32. ATPase activity of A32 is known to increase in the presence of DNA. Comparative analysis of the wildtype protein versus its CCM mutants suggests that this DNA dependence of A32's ATPase activity is likely regulated by the CCM. We demonstrate that oligomerization of A32, mediated by the CCM, is required for its DNA-binding but is not dependent on ATP- or DNA-binding. Our findings suggest a key role of the CCM, and thus, higher-order structure formation in the regulated ATPase activity of A32, providing new opportunities for further detailed characterization of the poxvirus genome packaging process.

Fig 1. Sequence and structure analysis of viral FtsK-like genome packaging ATPases.

(a) Multiple sequence alignment of putative genome packaging ATPases of NCLDVs, PRD1 lineage (membrane-containing dsDNA viruses) and Inoviridae family (ssDNA filamentous phages). Green- strictly conserved identical residues; yellow- similar residues (b) Structural alignment of representative ATPases of each virus group using AlphaFold2-predicted structures. E- β strand (blue), L- loop (green), H- α helix (red). NCLDVs representative- Vaccinia virus A32 (yellow), membrane-containing dsDNA viruses’ representative- PRD1 P9 (pink), ssDNA filamentous phages representative- M13 gp1 (orange) (c) secondary structure elements and domain organization of representative viral ATPases. WA- Walker A motif, WB- Walker B motif, R- arginine finger, Q- sensor residue (red), TM- transmembrane domain (brown), CCM- coiled-coil motif (pink). Blue- β strands, yellow- α helices. Conserved ATPase core is highlighted in green. Scale bar: 10 aa. Accession no- Vaccinia virus: YP_233037.1, Organic Lake phycodnavirus 1: ADX05856.1, African swine fever virus: YP_009702812.1, M13 phage- NP_510893.1, PRD1 phage- AAX45927.1, FtsK_CΔγ PDB ID- 2IUT.

Fig 2. Purification and ATPase activity of A32_WT.

(a) Purified A32_WT protein. M-marker, A32_WT- Purified and concentrated A32 protein after Heparin and Ni²⁺-NTA affinity chromatography (b) (i) bar graph and (ii) autoradiograph showing ATPase activity in the presence/absence of DNA as a function of A32_WT concentration. Trx-10 denotes 10 μM thioredoxin control. Values represent mean of duplicates with standard deviation, normalized with no protein control.

Fig 3. Assessment of coiled-coil motif in A32.

(a) Probability plot for coiled-coil motif prediction by PCOILS in (i) A32_WT (ii) A32_L234K and (iii) A32_{L234K_Q237A}. The default output of probabilities in the scanning windows of 14 (green), 21 (blue) and 28 (purple) aa residues are shown. (b) The predicted probability of heptad repeats between aa residue 231 to 237 for the scanning windows of 21 or 28. (c) Multiple sequence alignment of coiled-coil region in A32 homologs of Chordopoxvirinae subfamily using MUSCLE. Red- strictly conserved residues; yellow-conserved amino acids in majority of the sequences (d) Bit map image for the conservation of coiled-coil motif in Chordopoxvirinae.

Fig 4

Regulation of ATPase activity by coiled-coil motif (a) Purified proteins. Trx- Thioredoxin, A32_WT- A32 wild type, A32_K31A- Walker A motif mutant, A32_L234K- coiled-coil motif mutant 1, A32_{L234K_Q237A} - coiled-coil motif mutant 2, M- marker. (b) (i) bar graph and (ii) autoradiograph of comparative ATPase activities of wildtype A32_WT and its mutants. Trx denotes 10 μM thioredoxin control. Lanes from original image have been rearranged for representation. Values represent mean of duplicates with standard deviation, normalized with no protein control. (c) Steady- state kinetics analysis of (i) A32_WT (ii) A32_K31A and (iii) A32_{L234K_Q237A}. Values represent mean of duplicates with standard deviation, normalized with no ATP control.

Fig 5. Higher order structure formation and DNA-binding by A32.

(a) Native PAGE comparison of A32 or its mutants in the presence or absence of linear dsDNA or ATP. (b) AlphaFold2-predicted dimeric structure of A32, with the predicted coiled-coil motif highlighted in red. (c) EMSA with increasing concentration of (i) wildtype A32_WT (ii) Walker A motif mutant A32_K31A and (iii) CCM mutant A32_L234K. (d) EMSA of 10 μM wildtype A32_WT in the presence or absence of 1 mM ATP. Trx denotes 10 μM purified thioredoxin control.