Binding studies between cytosinpeptidemycin and the superfamily 1 helicase protein of tobacco mosaic virus

Abstract

Tobacco mosaic virus (TMV) helicases play important roles in viral multiplication and interactions with host organisms. They can also be targeted by antiviral agents. Cytosinpeptidemycin has a good control effect against TMV. However, the mechanism of action is unclear. In this study, we expressed and purified TMV superfamily 1 helicase (TMV-Hel) and analyzed its three-dimensional structure. Furthermore, the binding interactions of TMV-Hel and cytosinpeptidemycin were studied. Microscale thermophoresis and isothermal titration calorimetry experiments showed that cytosinpeptidemycin bound to TMV-Hel with a dissociation constant of 0.24-0.44 μM. Docking studies provided further insights into the interaction of cytosinpeptidemycin with the His375 of TMV-Hel. Mutational and Microscale thermophoresis analyses showed that cytosinpeptidemycin bound to a TMV-Hel mutant (H375A) with a dissociation constant of 14.5 μM. Thus, His375 may be the important binding site for cytosinpeptidemycin. The data are important for designing and synthesizing new effective antiphytoviral agents.

Fig. 1. Construction of TMV-Hel in pMCSG19 plasmid. (A) The PCR-amplified and 1% agarose gel electrophoresis results showed that the length of TMV-Hel segments was 1353 bp, M was the DNA marker, 1 and 2 was two repeats. (B) M was marker, 1 was the false positive monoclonal of pMCSG19-TMV-Hel, 2 was the positive monoclonal of pMCSG19-TMV-Hel with 1353 bp TMV-Hel segments in pMCSG19. (C) The verification of pMCSG19-TMV-Hel by restriction enzyme Not I and Xho I, M is marker, 1 was the pMCSG19 segments with 6441 bp and TMV-Hel segments with 1353 bp.

Fig. 2. Purification and characterization of TMV-Hel. (A) TMV-Hel protein with MBP Tag was determined by 12% SDS-PAGE analysis with 89 kDa in lane 2, M was the protein marker, 1 was control. (B) TMV-Hel protein with MBP Tag was purified in lane 1 by Ni-NT affinity chromatography, M was the protein marker. (C) TMV-Hel protein with MBP Tag was digested by TEV protease to product TMV-Hel protein by 12% SDS-PAGE analysis with 49 kDa in lane 1, M was the protein marker. (D) TMV-Hel protein eluted under the corresponding position of 67 kDa in 10 mM Tris/HCl, 150 mM sodium chloride (pH 7.5) by using SEC analysis with about 49 kDa.

Fig. 3. Interactions between cytosinpeptidemycin and TMV-Hel measured by MST and ITC methods. (A) MST measurements showed that cytosinpeptidemycin bound to TMV-Hel with a dissociation constant (K_d) of 0.44 μM. (B) ITC results showed that one TMV-Hel combined three cytosinpeptidemycin molecule with a binding affinity (K) of 4.16 × 10⁶ M⁻¹, and its corresponding K_d is 0.24 μM, which similar to MST measurements.

Fig. 4. Alignment of the amino acid sequences of TMV-Hel and ToMV-Hel. Secondary structural elements were indicated schematically under the X-ray crystal structure of ToMV-Hel. There was 89.6% homology between TMV-Hel and ToMV-Hel, the residue His375 has highly conserved regions in both TMV-Hel and ToMV-Hel. Secondary structural elements were indicated schematically under the sequences, generated by EMBL-EBI sequence alignment programs server, and indicated as difference residues (blue box) or red cylinder (α helices) or green arrows (β sheets), the nucleic acid binding motifs of TMV-Hel was labelled by yellow box.

Fig. 5. Based on the X-ray crystal structure of ToMV-Hel (PDB code: 3VKW), the TMV-Hel was modeled. (A) TMV-Hel; (B) was rotate 180 degrees of (A). The figures for structural representation of TMV-Hel were generated on pyMOL software.

Fig. 6. Docking analysis of the interactions between cytosinpeptidemycin and TMV-Hel. (A) The binding-sites between cytosinpeptidemycin and TMV-Hel by using Discovery studio 4.5; (B) was a partial enlarged detail of (A), two binding-sites, CO⋯H–N = 3.12 Å and H–N⋯H–N = 3.73 Å, were found in the representative conformation. The stick model stands for cytosinpeptidemycin and His375.

Fig. 7. MST studies of cytosinpeptidemycin and TMV-Hel mutant. MST results showed that the K_d of cytosinpeptidemycin and TMV-Hel mutant (H375A) was 14.5 μM. It was 32 times of the K_d of cytosinpeptidemycin and TMV-Hel.