Bajpai, U. | Search bioRxiv

bioRxiv2021

Functional characterization of a DNA-dependent AAA ATPase in SimranZ1, a F cluster mycobacteriophage

Mycobacteriophages are viruses of Mycobacterium spp. with promising diagnostic and therapeutic potential. Phage genome exploration and characterization of their proteomes are essential to gain a better understanding of their role in phage biology. So far, about 2014 mycobacteriophages have been genomically defined and 1563 phage protein families (phamilies) are identified. However, the function of only a fraction (about 15%) is known and a majority of ORFs in phage genomes are hypothetical proteins. In this study, from the annotated genome of a F1 cluster mycobacteriophage SimranZ1, a putative AAA ATPase (Gp65, Pham 9410) is characterized as a DNA-dependent ATPase. Sequence-based functional annotation predicted Gp65 to belong to the P-loop NTPase superfamily, having AAA_24 and RecA/RadA domains which are known to be involved in ATP-dependent DNA repair/maintenance mechanism. On molecular docking, Gly21 and Ser23 of Gp65 showed specific binding with ATP. Using a microtiter plate assay, ATPase activity of Gp65 was experimentally verified which was found to increase in the presence of dsDNA. Gel electrophoresis under non-denaturing condition showed the oligomeric states of Gp65 and Transmission Electron Microscopy revealed it to exist as a hexamer having a prominent central pore with a diameter of 1.9 nm. In summary, functional characterization of Gp65 as a DNA dependent AAA ATPase indicates its role in DNA repair/maintenance mechanism in mycobacteriophages.

biochemistry↗

bioRxiv2021

Computational identification and characterization of antigenic properties of Rv3899c of Mycobacterium tuberculosis and its interaction with Human leukocyte antigen (HLA)

Tuberculosis (TB) is the second largest infectious disease that kills 1.2 million people annually worldwide. A rise in drug-resistant TB cases demands continued efforts towards the discovery & development of drugs and vaccines. In the recent past, though novel drugs have been added to the current TB regimen [1], research on new vaccine candidates needs a greater thrust. Secretory proteins of H37Rv are frequently studied for their antigenicity and their scope as protein subunit vaccines decrees further analysis. In this study, Rv3899c of H37Rv emerges as a potential vaccine candidate on its evaluation by several bioinformatics tools. It is a non-toxin, secretory protein with an immunoglobulin-like fold which does not show similarity with a human protein. We found Rv3899c homologs in several mycobacterial species and its antigenic score (0.54) to compare well with the known immunogens such as ESAT-6 (0.56) and Rv1860 (0.52). Structural examination of Rv3899c predicted ten antigenic peptides, an accessibility profile of the antigenic determinants constituting B-cell epitope rich regions and a low Abundance of Antigenic Regions (AAR) value. Significantly, our study shows ESX-2 secretion system proteins and antigenic PE/PPE proteins of H37Rv as the interacting partners of Rv3899c. Further, molecular docking predicted Rv3899c to interact with human leukocyte antigen HLA-DRB1*04:01 through its antigenically conserved motif (RAAEQQRLQRIVDAVARQEPRISWAAGLRDDGTT). Interestingly, the binding affinity was observed to increase on citrullation of its Arg1 residue. Taken together, the computational characterization and predictive information suggest Rv3899c to be a promising TB vaccine candidate, which should be examined and validated experimentally.

immunology↗