Two conserved amino acids of juxtaposed domains of a ribosomal maturation protein CgtA sustain its optimal GTPase activity.
CgtA is a highly conserved ribosome binding protein involved in ribosome biogenesis and associated with stringent response. It is a 55 KDa GTPase protein consisting of GTPase, Obg and C-terminal domains. The function of the latter two domains was not clear and despite the importance, the mode of action of CgtA is still largely unknown. Knocking out of CgtA gene is lethal and mutations lead to growth, sporulation and developmental defects in bacteria. It was found that a growth defect and pinhole size colony morphology of Bacillus subtilis was associated with a Gly92Asp point mutation on the Obg domain of its CgtA protein, instead of its GTPase domain. CgtA is an important and essential protein of the deadly diarrhea causing bacteria Vibrio cholerae and in order to investigate the mode of action of the V. cholerae CgtA we have utilized this information. We measured the GTPase activity of V. cholerae CgtA (CgtAvc) protein in the presence of purified ribosome. Our results showed 5-fold increased GTP hydrolysis activity compared to its intrinsic activity. Then we explored the GTPase activity of the mutated CgtAvc (Gly98Asp) located at the Obg domain, which reduced the GTP hydrolysis rate to half. The double point mutations (Gly98Asp, and Tyr194Gly) encompassing another conserved residue, Tyr194, located at the diagonally opposite position in the GTPase domain largely restored (about 82%) the reduced GTPase activity, revealing a fine-tuned inter-domain movement readily associated with the GTPase activity of CgtA and thus maintaining the proper functioning of the CgtA protein.
Department of Biological Sciences, Indian Institute of Science Education and Research, Kolkata, Mohanpur, Nadia, West Bengal, India.
Ananya
A
Chatterjee
Department of Biological Sciences, Indian Institute of Science Education and Research, Kolkata, Mohanpur, Nadia, West Bengal, India. Electronic address: partha_datta@iiserkol.ac.in.
Partha P
PP
Datta
D000595:Amino Acid Sequence; D000596:Amino Acids; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D017124:Conserved Sequence; D004789:Enzyme Activation; D020558:GTP Phosphohydrolases / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014734:Vibrio cholerae / Q000378:metabolism*
D000596:Amino Acids; D001426:Bacterial Proteins; D012269:Ribosomal Proteins; C108621:CgtA protein, bacteria; D020558:GTP Phosphohydrolases; D020559:Monomeric GTP-Binding Proteins
CgtA; GTPase; Obg; Point mutation; Ribosome