Current Research
Our lab focuses on understanding structural and mechanistic aspects of proteins belonging to the class of HAS-GTPases and Protein Kinases. The following projects are currently being investigated in the lab.
1. Structural biology of HAS-GTPases
GTP-binding proteins or GTPases function as molecular switches cycling between GDP-bound 'OFF'- and GTP-bound 'ON'- states. Hydrolysis of the bound GTP returns them to the GDP-bound 'OFF'-state; thereby completing the GTPase cycle . Two commonly known regulators, Guanine Nucleotide Exchange Factors (GEFs) and GTPase Activating Proteins (GAPs) play key roles in this cycle. GEFs facilitate exchange of the bound GDP and increase the nucleotide dissociation rates by several orders of magnitude. GTPases, as the name suggests, do hydrolyse GTP, albeit slowly. The acceleration of this hydrolysis by orders of magnitude is due to GAPs , which are specific for the respective GTP-binding protein. Once the protein gets loaded with GTP, it enables them to interact with effectors, which are by definition ‘proteins that bind tightly and specifically to the ON-state'. It is by means of this interaction that a signal transduction cascade begins or carries on. The importance of proper functioning of the GTPase cycle is demonstrated by the occurrence of diseases associated with either mutations in the GTP-binding proteins, which then function as oncoproteins, or by mutations in GAPs, GEFs and effectors. Ras superfamily GTP-binding proteins regulate important signaling events in the cell. Ras, which often serves as a prototype, efficiently hydrolyses GTP in conjunction with its regulator GTPase Activating Protein (GAP). A conserved glutamine plays a vital role in GTP hydrolysis in most GTP binding proteins. Mutating this glutamine in Ras has oncogenic effects since it disrupts GTP hydrolysis.
We have analysed an important and emerging set of GTP binding proteins that lack this key catalytic Glutamine, which is usually well conserved. We have termed these proteins HAS-GTPases (Hydrophobic Amino acid Substituted GTPases) and divided them into several sub-families for analysis. The sequence analysis reveals that there are prominent insertions in all the sub-families and homology modelling suggests that they could have specific roles. Our work aims at understanding how these proteins hydrolyse GTP in the absence of the catalytic glutamine. An interesting observation is that the hydrophobic residue in switch II, that replaces the catalytic Glutamine of Ras , is drawn away from the catalytic site as it is not required for catalysis.
We call this a retracted conformation, which is further stabilized by a neighbouring hydrophobic pocket: the residues constituting this pocket are strictly hydrophobic in HAS-GTPases. These features that seem to be tailored to HAS-GTPases in turn suggest that the catalytic machinery in GTPases could arrive not only from the well known Switch II, but also from neighbouring regions. Overall, we believe HAS-GTPases invoke appealing variations in the known catalytic machinery to hydrolyse GTP. ( To learn more about it see Mishra, et.al . Proteins: Structure, Function and Bioinformatics (2005) 59, 332-338 . )
We are beginning to realize that a large number of HAS-GTPases are involved in ribosome assembly. The broader goal is to understand the catalytic mechanism prevalent in these proteins: for this, we employ x-ray crystallographic techniques combined with bioinformatics and bioinformatics.
2. Circularly Permuted GTPases
Recently, we began working on a special set of GTP binding proteins that we call ‘Circularly Permuted GTPases' or cpGTPases in short, since they carry a circular permutation in their primary sequence while retaining the overall structural features displayed by GTPases.
This work based on sequence and structural analysis of these multi-domain GTPases leads us to conclude that they bind RNA and raises an interesting possibility that RNA binding is perhaps coupled to GTP binding/hydrolysis. These inferences are also in line with experimental reports on a few cpGTPases that suggest the interaction of cpGTPases with the ribosome.
Here, we began to explore all possible circular permutations in nature using profile based sequence analysis of artificially permuted sets of classical GTPase sequences. It identified that apart from classical GTPases, only one circular permutation prevails in nature and it was possible to find a structural rationale for the absence of other permutations. Furthermore, we could infer that the structural design of such a circular permutation puts forward a natural and interesting restraint that cpGTPases, unlike some of their classical counterparts like Ras GTPases, cannot exist as single domain proteins and would need at least an additional C-terminal domain to stabilize the GTP binding domain. A data base search reveals that this is indeed the case for the four sub-families of cpGTPases, with three of them containing an addition N-terminal domain as well. Domain recognition analysis indicate that they bind RNA and the experimental reports available for some of these proteins, show that the additional N and C terminal domains indeed bind RNA. Interestingly, we could also find that these proteins have a close evolutionary relationship to a set of ancient bacterial ribosome binding GTPases, some of which are also HAS-GTPases.
There are currently very few reports on cpGTPases and their functions still need to be elucidated in detail. This work, aimed at understanding cpGTPases, leads us to deduce that cpGTPases would bind RNA or more precisely the ribosome. We believe that our work would draw the interest of a large number of researchers working in the area of GTP binding/RNA binding proteins and would stimulate thought and rigorous experimentation to understand the function of these important proteins. ( To learn more about it, see Anand et.al., Nucleic Acids Research. (2006) 34, 2196-2205 )
3. Structural and Biochemical investigations to determine the roles of eukaryotic-like serine/threonine protein kinases (STPKs) in M. tuberculosis.
This Project is being pursued in collaboration with Dr. Vinay Kumar Nandicoori at the National Institute of Immunology, New Delhi.
In eukaryotes, signals are transduced mainly by phosphorylation of proteins on serine, threonine or tyrosine residues and this is coupled to dephosphorylation reactions carried out by protein phosphatases. Protein phosphorylation events also play regulatory roles in events such as chemotaxis, bacteriophage infection, nutrient uptake and gene transcription in prokaryotes. However, kinases involved in these regulatory functions are either 1) protein histidine kinases 2) phosphotransferases or 3) protein serine kinases. In 1994, eukaryotic-like serine/threonine protein phosphorylations were detected for the first time in M. tuberculosis . Using various techniques, eight eukaryotic-like protein kinases were later identified in M. tuberculosis . Analysis of the M. tuberculosis genome sequence suggested the presence of 11 putative eukaryotic-like protein kinases and four protein phosphatases. Nine of these kinases contain a putative transmembrane domain, suggesting their localization to the cell membrane. PknG and PknK kinases do not have any apparent transmembrane domain. Eight kinases have been biochemically characterized. They are PknA, PknB, PknD, PknE, PknF, PknI, PknH and PknG. Substrates of PknA, PknB, PknF and PknH have been identified.
Tuberculosis (TB) infection is a major cause of death globally. Macrophages are the primary hosts for clinically important mycobacteria, which can colonize and grow within these cells. Once phagocytosed by macrophages, mycobacteria reside and replicate in phagosomes by actively blocking macrophage maturation. M. tuberculosis STPKs are shown to have a role in modulating M. tuberculosis cell shape and possibly its cell division. Some were shown to play a role in the survival of the pathogen in host macrophages by modulating phagosome-lysosome fusion (lysosomal transfer) after macrophages phagocytose the mycobacterium.