Probing the Molecular Basis of Cofactor Affinity and Conformational Dynamics of Mycobacterium tuberculosis Elongation Factor Tu: An Integrated Approach Employing Steered Molecular Dynamics and Umbrella Sampling Simulations.

The emergence of multidrug-resistant and extensively drug-resistant tuberculosis strains is the reason that the infectious tuberculosis pathogen is still the most common cause of death. The quest for new antitubercular drugs that can fit into multidrug regimens, function swiftly, and overcome the ever-increasing prevalence of drug resistance continues. The crucial role of Mtb EF-Tu in translation and trans-translation processes makes it an excellent target for antitubercular drug design. In this study, the primary sequence of Mtb EF-Tu was used to model the three-dimensional structures of Mtb EF-Tu in the presence of GDP ("off" state) and GTP ("on" state). The binding free energy computed using both the molecular mechanics/Poisson-Boltzmann surface area and umbrella sampling approaches shows that GDP binds to Mtb EF-Tu with an ∼2-fold affinity compared to GTP. The steered molecular dynamics (SMD) and umbrella sampling simulation also shows that the dissociation of GDP from Mtb EF-Tu in the presence of Mg 2+ is a thermodynamically intensive process, while in the absence of Mg 2+ , the destabilized GDP dissociates very easily from the Mtb EF-Tu. Naturally, the dissociation of Mg 2+ from the Mtb EF-Tu is facilitated by the nucleotide exchange factor EF-Ts, and this prior release of magnesium makes the dissociation process of destabilized GDP easy, similar to that observed in the umbrella sampling and SMD study. The MD simulations of Mtb EF-Tu's "on" state conformation in the presence of GTP reveal that the secondary structure of switch-I and Mg 2+ coordination network remains similar to its template despite the absence of identity in the conserved region of switch-I. On the other hand, the secondary structure in the conserved region of the switch-I of Mtb EF-Tu unwinds from a helix to a loop in the presence of GDP. The major conformational changes observed in switch-I and the movement of Thr64 away from Mg 2+ mainly reflect essential conformational changes to make the shift of Mtb EF-Tu's "on" state to the "off" state in the presence of GDP. These obtained structural and functional insights into Mtb EF-Tu are pivotal for a better understanding of structural-functional linkages of Mtb EF-Tu, and these findings may serve as a basis for the design and development of Mtb EF-Tu-specific inhibitors.