Structural Prediction and Mutational Analysis of Rv3906c Gene of Mycobacterium tuberculosis H37Rv to Determine Its Essentiality in Survival

Abstract

The emergence of tuberculosis is at the peak; therefore to station it at its lower level we hereby try bioinformatics approach against Mycobacterium tuberculosis [M. tuberculosis] pathogenesis. Rv3906c is a conserved hypothetical gene of M. tuberculosis and contains many GTP binding protein motif DXXG which demonstrate that this gene might be processed in a GTP binding or in GTP hydrolyzing manner. This gene shows interaction with its adjacent genes as well as pcnA which is a polymerase and localized in the extracellular region and found to be a soluble protein. Rv3906c has binding pockets for calcium atom at various positions which prove that calcium might have some role during the process of this gene. GTP binding protein motif DXXG is present in various positions and calcium binds at this site with a C-score of 0.25. Mutational analysis on this motif shows the large decrease of stability after mutation of aspartate residue with glycine. Stress conditions like pH and temperature also change stability of the protein. A decrease in stability at this position might play a role in inhibition of survival of the pathogen. These computational studies of this gene might be a successful step towards drug development against tuberculosis.

Figure 1

Prediction of localization by LocTree3. LocTree3 tool used for determining protein subcellular localization which shows that it is an extracellular regional, secreted protein. The figure shows that 0.99% of this protein is a secretory protein which is present in extracellular region. 0.01% of this protein is present in transmembrane region.

Figure 2

Modelling of protein via I-TASSER. Rv3906c protein modelled via I-TASSER showing C-score -1.37, estimated RMSD 0.55±0.15, and estimated TM-score 7.9±4.4Å. (a) Cartoon model, (b) ribbon model, (c) sticks model, (d) mesh model, and (e) dot model of (f) surface model.

Figure 3

Prediction of calcium binding pocket in modelled protein by COACH server. The figure shows calcium ligand binding in pocket of Rv3906c protein. (a) Calcium binds to aspartate residue at 22, 24, 26, 33, and 35 positions with C-score of 0.25 (rank 1). (b) Calcium binds to aspartate residue at positions 122, 124, 126, and 131 with C-score of 0.23 (rank 2).

Figure 4

RAMPAGE analysis of modelled protein. RAMPAGE analysis shows that in favored region there were 67.7% of residues and in allowed region 23.4% of residues and outlier region 9.0% of residues were present.

Figure 5

Structure validation with ERRAT tool. The result of ERRAT tool shows that overall quality factor of the modelled protein based on various sorts of atoms were found to be 82.500 which are satisfactory.

Figure 6

PROVE analysis of protein. Z-score defined the energy separation between native fold and average of an ensemble of misfold unit. The average Z-score was 0.600 and the Z-score RMS was 1.681.

Figure 7

Analysis of 3D-1D score of modelled protein by Verify 3D. The figure shows that 74.56% of the residues had an average of 3D-1D score >=0.2 that is acceptable for our demonstrated protein.

Figure 8

Structure-based function prediction by cofactor server. The figure shows gene ontology term with molecular function and biological process. The whole procedure demonstrates the C-score of 0.99 with the cell metabolic process and 0.63 with translation, DNA templated. COFACTOR online server predicts templated protein with comparable binding site which comes at positions 22 and 24 additionally.

Figure 9

Prediction of stability change of protein by I-Mutant 3.0 suite. I-Mutant 3.0 suite predicts stability change upon single point mutation at Aspartate [D22] residue which is important residue for GTP binding activity. The figure emphasizes that changing of aspartate with glycine decreases the stability most with a DDG value of -1.2.

Figure 10

Difference in stability of the protein by changing position. The figure shows that aspartate of positions 22 and 24 is the most important site for GTP binding and hydrolyzing activity with a DDG value of -1.2.

Figure 11

Change in stability in stress condition. The figure shows change in stability in stress condition, i.e., pH and temperature. The blue color in figure shows three different pH, i.e., 5.6, 7, and 8 and three different temperatures, i.e., 25°C, 37°C, and 42°C. The graph demonstrates that pH does not affect the stability of the protein whereas by increasing temperature stability decreases by 21%.