Dr. Ravishankar joined CSIR-CDRI at the end of 2002. His group elucidates molecular mechanisms underlying key pathways/proteins from human pathogens using a combination of X-ray crystallography, biochemical/biophysical studies, NMR, and rational structure-based computational analyses. The basic knowledge is exploited for the translational development of inhibitors with therapeutic potential collaboratively with geneticists, medicinal chemists, and others. Several proteins/components that have been characterised are recognised as new, validated and druggable targets. Consequently, inhibitors developed against these targets have new modes of action with potential to overcome several resistance issues. These are being explored under initiatives like the CSIR-Open Source Drug Discovery (OSDD), and other initiatives.

Detailed list of publications with citation statistics can be found at
http://scholar.google.com/citations?user=CNHV6coAAAAJ

• DNA repair and recombination are essential processes of an organism. We have structurally and functionally characterized fundamentally important mycobacterial components from these processes like MtbLigA and Mtbβ-clamp.  Further, we demonstrated that these proteins do not exhibit direct interactions in mycobacteria unlike in humans and even in bacteria like E. coli; instead, they interact through other factors. The results identify mechanistic differences in these fundamental processes in mycobacteria. The studies have also led to the identification of new classes of compounds that exhibit anti-bacterial specificity and distinguish the human enzyme several fold, both in vitro and in LigA deficient strains. The results have paved the way for the development of inhibitors with new mode(s) of action and have been actively taken up by the ‘Open source drug discovery’ project of CSIR and other programmes. PCT and Indian patents have also been filed.

• Mycobacteria possess ESX/Type-VII secretion systems that export essential virulence factors like the ESAT-6 and CFP10. Very little is known about the molecular details and mechanisms of the components of these secretion systems. We have characterized proteins from this system, particularly EccA1 and its homologs without which the virulence factors are unable to be secreted. We demonstrated that EccA1 is a member of the earlier uncharacterized CbbX/Cfqx family of AAA-ATPases, and suggested how these thermostable ATPases can transfer energy to other components of the secretion system. In the translational context, ongoing work will lead to the identification of inhibitors with a new mode of action that does not allow the pathogen to secrete essential virulence factors.

• Many global and local regulatory proteins are involved in controlling the changes to the lifestyle of M. tuberculosis. Feast/famine regulatory proteins are thought to be important for the switch from 'feasting' to 'famine' state and contain a small RAM (Regulator of Amino acid Metabolism) domain. The domain consist of about 80 amino acids, exhibit a ßaßßaß topology and are found in proteins with diverse functions. 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. Our work on the mycobacterial Feast/famine regulatory proteins has identified that these proteins can adopt the rare ‘open’ quaternary structures in response to changes to the ligand binding site; eg. those that occur upon ligand binding. In these studies, the oligomeric symmetry that is normally strictly preserved, is disrupted to presumably enable the protein to bind to non-symmetric target DNA binding sites in response to ligand binding events, other than to symmetric regulatory sites. The studies also suggest how these proteins can form nucleosome-like particles like those observed through earlier electron-microscopy experiments and how ligand binding events could trigger specific regulatory outcomes by changing the oligomeric state of the protein and thereby enabling it to apparently bind to different target sites.

• HIV-1 Nef is known to play a pivotal role in HIV pathogenesis and binds to different protein partners in lower and higher oligomeric states respectively. Our structural studies on HIV-1 Nef, a collaboration with Dr. R.K. Tripathi's lab, has led to interesting results. We demonstrated crystallographically that the protein forms higher order oligomers like tetramers with C4/ 4-fold symmetry. Normally a protein that exists as a dimer associates as a dimer-of-dimers, i.e with 222-symmetry, to form a tetramer. HIV-1 Nef has revealed the only known instance of a transition from a dimer to a tetramer with 4-fold symmetry. The functional implications of this unique transition are in an elegant mechanism where HIV-1 Nef in the dimeric state binds to a set of protein partners that are subsequently excluded from associating with the protein at higher oligomeric states due to the change in the subunit association. The binding site(s) that was available to partner proteins in the dimer are sterically inaccessible in the tetrameric state, where presumably Nef can interact with a different set of protein partners to result in varied functional consequences. The work has also been exploited through the rational identification of inhibitors with new mode of action that works by disrupting specific HIV-1 Nef –co-protein interactions [PCT and Indian patents filed].

• The apicoplast is an essential relict plastid in P. falciparum. Many of the original proteins of the apicoplast are now nuclear encoded. This means that over 500 potential NEAT (Nuclear Encoded Apicoplast Targeted) proteins have to interact with partners in the apicoplast and/or have to be transported to the plastid. Our studies on P. falciparum chaperones like Hsp70 that exhibit important interactions with NEAT proteins establishes methods for the analysis of potential substrate proteins with these chaperones using carefully designed peptides as probes and are important for designing inhibitors that disrupt essential interactions of NEAT proteins.

• Sigma factors are an important family of regulatory proteins. Work on mycobacterial sigma factors and associated proteins like UsfX, SigF and RsfA demonstrates that novel properties of these mycobacterial factors can form a framework for new therapy development.