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The amino acid sequence of ervatamin-C, a thermostable cysteine protease from a tropical plant, revealed an additional 24-amino-acid extension at its C-terminus (CT). The role of this extension peptide in zymogen activation, catalytic activity, folding and stability of the protease is reported. | IFEBS Journal C-Terminal extension of a plant cysteine protease modulates proteolytic activity through a partial inhibitory mechanism Sruti Dutta Debi Choudhury Jiban K. Dattagupta and Sampa Biswas Crystallography and Molecular Biology Division Saha Institute of Nuclear Physics Kolkata India Keywords C-terminalextension cysteine proteases modulation of proteolytic activity papain-like thermostable Correspondence S. Biswas Crystallography and Molecular Biology Division Saha Institute of Nuclear Physics 1 AF Bidhannagar Kolkata 700 064 India Fax 91 332 337 4637 Tel 91 332 337 5345 E-mail sampa.biswas@saha.ac.in Received 14 March 2011 revised 16 May 2011 accepted 22 June 2011 doi 10.1111 j.1742-4658.2011.08221.x The amino acid sequence of ervatamin-C a thermostable cysteine protease from a tropical plant revealed an additional 24-amino-acid extension at its C-terminus CT . The role of this extension peptide in zymogen activation catalytic activity folding and stability of the protease is reported. For this study we expressed two recombinant forms of the protease in Escherichia coli one retaining the CT-extension and the other with it truncated. The enzyme with the extension shows autocatalytic zymogen activation at a higher pH of 8.0 whereas deletion of the extension results in a more active form of the enzyme. This CT-extension was not found to be cleaved during autocatalysis or by limited proteolysis by different external proteases. Molecular modeling and simulation studies revealed that the CT-extension blocks some of the substrate-binding unprimed subsites including the specificity-determining subsite S2 of the enzyme and thereby partially occludes accessibility of the substrates to the active site which also corroborates the experimental observations. The CT-extension in the model structure shows tight packing with the catalytic domain of the enzyme mediated by strong hydrophobic and H-bond interactions thus restricting accessibility of its cleavage sites to the
