Our results show that deletion

Our results show that deletion of the first loop of Obg does not abolish the augmentation of the GTPase activity of CgtAvc, revealing that the first loop is mainly responsible for the anchoring of the protein in the 50S and the transient interaction with ribosome is enough for augmentation of GTPase activity, whereas, deletions of loop 2 and loop 3 do not completely abolish the ribosome binding ability but affect ribosome mediated augmentation in GTPase activity. Thus, it suggests that loop 2 and loop 3 are involved in passing on the effects of “Ribosome-CgtA” interactions to its GTPase domain upon ribosome binding. Since CgtA interacts with other proteins, thus, it is possible that the effect of the interaction of other cellular components with the Obg domain will also influence the GTPase domain through the inter-domain linker region. Since the linker region is single chain, most likely it has the capcity to allow flexible interdomain movement between the Obg and the GTPase domain. Although deletion of the CTD does not affect in ribosome binding ability but it significantly increases ribosome associated GTPase activity of CgtAvc. Therefore, it can be concluded that the CTD of CgtAvc plays a negative regulatory role during ribosome associated GTPase activity of CgtAvc. Furthermore, it is predicted from our CgtA interacting proteins network study from different organisms that, though, it interacts with few conserved ribosomal proteins, ribosome maturation factors, and elongation factors, it also interacts with other proteins that are organism specific (see Fig. 6A). Although CgtA is considered a universally conserved GTPase, its CTD varies widely in length, amino CCG-100602 sequences and property wise. Sequence alignment of the CTD of CgtA from different prokaryotic organisms shows that the CTD varies greatly in size and sequence (Fig. 6B, Fig. S1 and Table S1). In fact, in some organisms, like Chlamydia abortus, the C-terminal part is even missing, however, that does not affect the ribosome binding property of the organism [32]. On the contrary, it does not help to complement the temperature sensitive mutants in E. coli [32]. In gram positive bacteria, like B. subtilis, it was found that deletion of nine amino acids from the C-terminal end affects the initiation of sporulation whereas in gram negative bacteria, like E. coli and Vibrio sp., deletion of 22 amino acids from the C-terminal end cause reduction in cell elongation property during over-expression of CgtA protein [9,33]. From a comparative sequence analysis we found that the CTD is longer in gram positive bacteria that ranges from 89 to 148 amino acids and they have slightly basic pI (pI > 5–6) compared to the gram negative bacteria whose CTD ranges from 33 to 59 amino acids long and pI ranges between 3.5 and 4.5 (Table S1). Also the CTD of gram-positive bacteria are more hydrophobic having hydrophobicity between −0.196 and −0.99, whereas in gram-negative bacteria, it ranges from −1.388 to −1.776, suggesting that the role played by the CTD of the protein is not conserved. Since CTD varies widely both in length and amino acid sequences (see Fig. 6B and Fig. S1) it indicates the structural variability of the CTD portion of CgtA. Therefore, it can be suggested that since CTD is the only non-conserved domain of CgtA, this portion of CgtA might play important role in recognition of the organism specific factors.
Author contributions
Funding The authors acknowledge DST-SERB of India, (File No.- EMR/2015/002473), for providing research fund to Dr. Partha Pratim Datta.
Acknowledgements Authors acknowledge Dr. Rupak K. Bhadra from the Indian Institute of Chemical Biology, Kolkata for providing the V. cholerae N16961 strain, and IISER-Kolkata for providing instrument facility. Ananya Chatterjee and Sagarika Das are supported by a PhD fellowship from IISER-K. Arita Acharjee is supported by a PhD fellowship from CSIR.