Ravi Kant Pal

Unlike humans, Mycobacterium tuberculosis ( Mtb ), the causative agent of human tuberculosis, has a de novo histidine-biosynthesis pathway. The enzyme imidazole glycerol phosphate dehydratase (IGPD), which catalyses the conversion of imidazole glycerol phosphate to imidazole acetol phosphate, has been studied extensively from various organisms and has become a major target for the development of antibacterial, antiweed and antifungal small molecules. In our previous studies, we have shown that in crystals IGPD forms a 24-mer oligomeric state in which the monomers are arranged in 432 symmetry. In order to gain insights into the oligomeric state of Mtb IGPD in solution, we determined cryogenic sample electron microscopy (cryo-EM) structures of apo IGPD at 2.2 and 3.1 Å resolution. In addition, we also determined the cryo-EM structure of IGPD in the presence of 3-amino-1,2,4-triazole (ATZ) to 2.8 Å resolution. The results of this work, which corroborate those from the crystallographic studies, indicate that IGPD forms a homo-oligomeric structure in solution comprising of 24 subunits. ATZ binds in the active-site pocket of the enzyme, which is located at the interface of three monomers and tethers 24 ATZ molecules. The results of this study suggest that cryo-EM, in addition to being a rapidly evolving and complementary imaging technology for elucidating 3D structures of biological macromolecules, can be useful in pinpointing the mode of binding small molecules of low mass (here ∼85 Da) and mapping protein-ligand interactions, which could assist in the design of accurate (high-potency) inhibitors.

Enolase, a ubiquitous enzyme, catalyzes the reversible conversion of 2-phosphoglycerate (2PG) to phosphoenolpyruvate (PEP) in the glycolytic pathway of organisms of all three domains of life. The underlying mechanism of the 2PG to PEP conversion has been studied in great detail in previous work, however that of the reverse reaction remains to be explored. Here we present structural snapshots of Mycobacterium tuberculosis (Mtb) enolase in apo, PEP-bound and two 2PG-bound forms as it catalyzes the conversion of PEP to 2PG. The two 2PG-bound complex structures differed in the conformation of the bound product (2PG) viz the widely reported canonical conformation and a novel binding pose, which we refer to here as the alternate conformation. Notably, we observed two major differences compared with the forward reaction: the presence of Mg_B is non-obligatory for the reaction and 2PG assumes an alternate conformation that is likely to facilitate its dissociation from the active site. Molecular dynamics studies and binding free energy calculations further substantiate that the alternate conformation of 2PG causes distortions in both metal ion coordination and hydrogen-bonding interactions, resulting in an increased flexibility of the active-site loops and aiding product release. Taken together, this study presents a probable mechanism involved in PEP to 2PG catalysis that is likely to be mediated by the conformational change of 2PG at the active site.

Eukaryotic Rab5s are highly conserved small GTPase-family proteins that are involved in the regulation of early endocytosis. Leishmania donovani Rab5a regulates the sorting of early endosomes that are involved in the uptake of essential nutrients through fluid-phase endocytosis. Here, the 1.80 angstrom resolution crystal structure of the N-terminal GTPase domain of L. donovani Rab5a in complex with GDP is presented. The crystal structure determination was enabled by the design of specific single-site mutations and two deletions that were made to stabilize the protein for previous NMR studies. The structure of LdRab5a shows the canonical GTPase fold, with a six-stranded central mixed 13-sheet surrounded by five alpha-helices. The positions of the Switch I and Switch II loops confirm an open conformation, as expected in the absence of the y-phosphate. However, in comparison to other GTP-bound and GDP-bound homologous proteins, the Switch I region traces a unique disposition in LdRab5a. One magnesium ion is bound to the protein at the GTP-binding site. Molecular-dynamics simulations indicate that the GDP-bound structure exhibits higher stability than the apo structure. The GDP-bound LdRab5a structure presented here will aid in efforts to unravel its interactions with its regulators, including the guanine nucleotide-exchange factor, and will lay the foundation for a structure-based search for specific inhibitors