Biochemical characterization of C4 protein of Cotton Leaf Curl Kokhran Virus-Dabawali

• Published in: Biochimica et biophysica acta Vol. 1830; no. 6; pp. 3734 - 3744
• Main Authors: Guha, Debojit, Poornima Priyadarshini, C.G., Purakayastha, Arunima, Thippeswamy, R., Lakshmikanth, M., Savithri, H.S.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.06.2013
• Subjects: absorbance; Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - metabolism; adenosine triphosphate; adenosinetriphosphatase; Amino Acid Motifs; arginine; ATPase; Begomovirus; Begomovirus - enzymology; Begomovirus - genetics; Catalytic Domain; cysteine; dabs; enzyme activity; Geminivirus; hydrolysis; Inorganic pyrophosphatase; Inorganic Pyrophosphatase - chemistry; Inorganic Pyrophosphatase - genetics; Inorganic Pyrophosphatase - metabolism; Natively unfolded; phosphates; Phosphotyrosine phosphatase; Protein Folding; Protein–protein interaction; radiolabeling; sequence deletion; thin layer chromatography; viral proteins; Viral Proteins - chemistry; Viral Proteins - genetics; Viral Proteins - metabolism; virion; viruses; Phosphotyrosine phosphatase; Geminivirus; Inorganic pyrophosphatase; Natively unfolded; ATPase; Protein–protein interaction
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2013.02.026

Cotton leaf curl Kokhran Virus-Dabawali (CLCuKV-Dab) is a monopartite begomovirus encoding two proteins V1 and V2 in the virion sense and four proteins C1, C2, C3 and C4 in the complementary sense. The C4 protein of monopartite begomoviruses has been implicated to play a role in symptom determination and virus movement. The present work aims at the biochemical characterization of this protein. The C4 protein of CLCuKV-Dab was purified in fusion with GST and tested for the ability to hydrolyze ATP and other phosphate containing compounds. ATPase activity was assayed by using radiolabeled γ-[32P]-ATP and separating the product of reaction by thin layer chromatography. The hydrolysis of other compounds was monitored by the formation of a blue colored phosphomolybdate complex which was estimated by measuring the absorbance at 655nm. The purified GST-C4 protein exhibited metal ion dependent ATPase and inorganic pyrophosphatase activities. Deletion of a sequence resembling the catalytic motif present in phosphotyrosine phosphatases resulted in 70% reduction in both the activities. Mutational analysis suggested arginine 13 to be catalytically important for the ATPase and cysteine 8 for the pyrophosphatase activity of GST-C4. Interaction of V2 with GST-C4 resulted in an increase in both the enzymatic activities of GST-C4. The residues important for the enzymatic activities of GST-C4 are present in a motif different from the classical Walker motifs and the non-classical ATP binding motifs reported so far. The C4 protein of CLCuKV-Dab, a putative natively unfolded protein, exhibits enzymatic activities. •C4 of Cotton leaf curl Kokhran virus-Dabawali affinity purified in fusion with GST.•GST-C4 exhibited metal ion dependent ATPase and inorganic pyrophosphatase activities.•Catalytic motif resembling phosphotyrosine phosphatases found at the N terminus of C4.•Arginine 13 was found to be important for ATPase activity by mutational analysis.•Cysteine 8 was found to be important for pyrophosphatase activity by mutational analysis.