A Hidden Active Site in the Potential Drug Target Mycobacterium tuberculosis dUTPase Is Accessible through Small Amplitude Protein Conformational Changes

Abstract

Edited by Norma AllewelldUTPases catalyze the hydrolysis of dUTP into dUMP and pyrophosphate to maintain the proper nucleotide pool for DNA metabolism. Recent evidence suggests that dUTPases may also represent a selective drug target in mycobacteria because of the crucial role of these enzymes in maintaining DNA integrity. Nucleotide-hydrolyzing enzymes typically harbor a buried ligand-binding pocket at interdomain or intersubunit clefts, facilitating proper solvent shielding for the catalyzed reaction. The mechanism by which substrate binds this hidden pocket and product is released in dUTPases is unresolved because of conflicting crystallographic and spectroscopic data. We sought to resolve this conflict by using a combination of random acceleration molecular dynamics (RAMD) methodology and structural and biochemical methods to study the dUTPase from Mycobacterium tuberculosis. In particular, the RAMD approach used in this study provided invaluable insights into the nucleotide dissociation process that reconciles all previous experimental observations. Specifically, our data suggest that nucleotide binding takes place as a small stretch of amino acids transiently slides away and partially uncovers the active site. The in silico data further revealed a new dUTPase conformation on the pathway to a relatively open active site. To probe this model, we developed the Trp 21 reporter and collected crystallographic, spectroscopic, and kinetic data that confirmed the interaction of Trp 21 with the active site shielding C-terminal arm, suggesting that the RAMD method is effective. In summary, our computational simulations and spectroscopic results support the idea that small loop movements in dUTPase allow the shuttling of the nucleotides between the binding pocket and the solvent.

The Role of a Key Amino Acid Position in Species-Specific Proteinaceous dUTPase Inhibition

“…In the equation F 0 is the unquenched and F is the quenched tryptophan fluorescence, Q is the quencher (acrylamide), K sv is the dynamic (bimolecular) quenching constant and V is the static (sphere of action) component of quenching [42,43,44].…”

Targeting a critical step in fungal hexosamine biosynthesis

“…However, when considering buried protein pockets and cavities in general, such seemingly trivial displacements may be of significance. Recent molecular dynamics simulations and experiments have suggested that large-scale side chain or secondary structure conformational changes are not a prerequisite for ligand binding to buried cavities but rather subtle and rapid movement of peptide structural elements can be sufficient to facilitate ligand ingress and egress (47)(48)(49)(50). Although beyond the scope of this work, we speculate that the effects of any subtle movement of structural elements may propagate within AfGna1 and be important for ligandbinding dynamics to the unusual buried dimer interface pocket.…”

Beyond Chelation: EDTA Tightly Binds Taq DNA Polymerase, MutT and dUTPase and Directly Inhibits dNTPase Activity

“…EDTA seems to preferably bind to one of the entrances of the polar central channel of hDUT (Figure 3 A–B) while it only weakly populates the corresponding apolar area in mtDUT in the docking runs (Figure 3 C–D). Considering the enzymatic mechanism, EDTA binding to the outer central channel may not have great or any effect on the enzymatic activity as this part have not shown significant conformational changes in previous studies [55,74]. On the other hand, EDTA may perturb the interaction of the C-terminal arm with the protein core as this contact surface is the first and second most populated EDTA binding site in mtDUT and hDUT, respectively, in the docking simulations (Figure 3).…”

“…On the other hand, EDTA may perturb the interaction of the C-terminal arm with the protein core as this contact surface is the first and second most populated EDTA binding site in mtDUT and hDUT, respectively, in the docking simulations (Figure 3). The intact C-terminal arm–protein core interaction is necessary for the hydrolysis to occur as this interaction provides the shielding of the active site from the solvent [52,57,74]. The differences in the predicted EDTA-protein interactions, especially at the C-terminal arm can explain the observed differences in EDTA-inhibited dUTPase activities between hDUT and mtDUT.…”