elucidating the crystal structures of proteins from pathogens like M. tuberculosis andP. falciparum, Our work encompasses cloning, expression, purification and biochemical/functional studies on proteins, crystallization and 3D structure elucidation by x-ray crystallography approaches. Subsequently the structures are analyzed and novel inhibitors identified using state-of-the-art techniques like virtual and high-throughput screening. The lab is well equipped and has state-of-the-art instruments for protein purification and characterization in addition to a modern x-ray data collection facility. We also have several graphics workstations and servers for molecular modeling and analysis of protein structures. The projects in the lab are funded by CSIR and Department of Biotechnology, India.
Nucleic acid ligases
Nucleic acid ligases are important proteins which primarily join breaks in DNA or RNA. We have initiated a long-term project to elucidate the crystal structures of nucleic acid ligases and have cloned and expressed several ligases, primarily from M. tuberculosis.
We recently elucidated the 3D structure of the ~35 kD adenylation domain of the NAD+ -dependent DNA ligase (MtbLigA) from the pathogen with bound AMP co-factor. LigA is an ~70 kD multi-domain enzyme which seals nicks in dsDNA. The enzyme is recognized as a novel target as it is found exclusively in bacteria and some viral sources. We have now generated several mutants of the enzyme and are probing various aspects of the enzyme mechanism. We use the structure in virtual screening approaches and have identified novel inhibitors with diverse scaffolds. These are being optimized in collaboration with medicinal chemistry groups.
ACT/RAM domain containing regulatory proteins
Many global and local regulatory proteins are involved in controlling the changes to the lifestyle of M. tuberculosis. The RAM domain (Regulator of Amino acid Metabolism) and ACT domains share structural homology while ligand binding sites occur in different regions. They consist of about 80 amino acids and exhibit a ßaßßaß topology. Proteins with this domain appear to be involved in amino acid regulation and interact with amino acids. Many aspects of their mechanism are unclear. How ACT/RAM domains can control the function of a variety of proteins? The way by which they translate a ligand-binding event to the protein-DNA binding site/active site of enzymes is largely unknown. Some proteins are inhibited by the ligand-binding event while others are activated.
We have identified about 16 proteins with this domain from the analysis of the M. tuberculosis genome sequence. Some of them had been found earlier to be essential forin vitro/ in vivo survival of the pathogen. We have cloned several proteins containing this domain and have solved the crystal structure of one of them.
Proteins involved in tuberculosis persistence
M. tuberculosis is a successful pathogen primarily because of its ability to persist in the human host for years avoiding the host-immune system. We have chosen to work on many proteins which are up-regulated in tuberculosis models designed to mimic the persistent state.
Recently we elucidated the crystal structure of Lysine e-aminotransferase (MtbLAT), an enzyme which is up-regulated over 40-fold in a nutrient starvation model of tuberculosis. We elucidated structurally how the enzyme can distinguish between structurally similar substrates through an elegant mechanism involving a glutamate ‘switch’. We are dissecting aspects of the enzyme action through a series of structures as also a mutational analysis. Other proteins are in various stages of the structure elucidation pipeline.
P. falciparum proteins
The apicoplast is a relic plastid of plant origin which is an essential organelle ofP.falciparum. We are in the process of elucidating structures of proteins which are coded for by the apicoplast or which help nuclear encoded proteins to interact with the organelle.
Virtual Screening
The 3D structure of a protein is a powerful tool which helps in the design of novel inhibitors; much the same as a wax-imprint of the keyhole of a lock helps the design of a duplicate key.
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