Albany 2013: Book of Abstracts
June 11-15 2013
©Adenine Press (2012)
Investigating the mode of action of an essential OBG GTPase, CgtA in bacteria
CgtA is an essential OBG GTPase (Trach and Hoch, 1989) highly conserved from bacteria to eukaryotes. It is a multifunctional protein, involved in DNA replication, chromosome partitioning (Slominska et al., 2002), nutritional stress response, initiation of sporulation, ribosome maturation, etc. Despite being a multifunctional essential protein, its mode of action is not well characterized and key question remains: how does this protein work in wide varieties of cellular function? The expression of cgtA-mRNA increases on the onset of nutritional stress. Purified CgtA protein shows increased GTPase activity in the presence of ribosome. Our experiment with Thiostrepton reveals that, site of GTPase inducing activity is different from other regular translation factors like EF-G, that uses GTP. For structure function study we have generated an energy minimized homology model of the V. cholerae CgtA protein, which reveals two large domains, an OBG fold and a GTP- hydrolysis domain, with an extended C-terminal part. We compared the amino acid sequence of CgtA across various species in the database, and found that it’s Glycine98 and the Tyrosine195 residues are 100% conserved in prokaryotes. These amino acids are highly conserved in eukaryotes as well. Gly98 and Tyr195 are located on the hinge region of CgtA comprising of portions of the OBG and the GTP- hydrolysis domains respectively. To decipher the mode of actions of CgtA and the role of the conserved Gly98 residue, we have replaced the Gly with a relatively larger amino acid, i.e., Asp. Our study reveals that the mutant CgtA(G98D) shows a reduced GTPase activity in presence of ribosome compared to the wild type. This indicates a restricted inter domain movement of CgtA due to the above point mutation. To understand this phenomenon we are using MD simulations. We will discuss results from MD simulations and other mutation studies as well. Our results indicate that ribosome acts as a modulator for increasing the GTPase activity of CgtA. The perfect conservation of G98 residue is important for the proper functionality of CgtA.
Our lab’s research activity is currently supported by IISER-K and DBT
Slominska M, Konopa G, Webgrzyn G and Czyz A (2002) Impaired chromosome partitioning and synchronization of DNA replication initiation in an insertional mutant in the Vibrio harveyi cgtA gene coding for a common GTP-binding protein. Biochem. J. 362: 579-584.
Department of Biological Science