Current Science

Open Access Open Access  Restricted Access Subscription Access

Asp72 of Pro-Peptide is an Important pH Sensor in the Zymogen Activation Process of Papain:A Structural and Mechanistic Insight


Affiliations
1 Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700 064, India
 

The zymogen of papain contains a pro-peptide at the N-terminus of the catalytic domain. Pro-peptide contains residues which act as pH-sensors in zymogen activation cascade. To understand the influence of pro-peptide in the pH-induced zymogen activation process of the protease, we performed structural studies of the zymogen of papain at activation pH of 4.0. The X-ray structure of zymogen at acidic pH reveals that Asp72 of the pro-peptide, a highly conserved residue of the GXNXFXD motif, plays an important role in pH-induced conformational changes in the prodomain leading to the zymogen activation process. Far-UV circular dichroism spectrum of zymogen at pH 4.0 demonstrates loss of helical structure compared to that at pH 8.0. The structural observation is further corroborated by mutational studies, where D72A mutant is shown to undergo auto-activation at pH 5.0 compared to pH 4.0 for wild-type, though the general proteolytic activity of the D72A mutant remains similar to that of wild-type. Our findings indicate that the conserved Asp72 residue is an important pH sensor in the zymogen activation process and that the pro-peptide part can also be a useful target of protein engineering for altering the activation pH of a protease.

Keywords

Cysteine Protease, pH Regulation, Protein Engineering, Pro-Peptide, X-Ray Crystallography.
User
Notifications
Font Size

  • Rawlings, N. D. and Barrett, A. J., Families of cysteine peptidases. Meth. Enzymol., 1994, 244, 461–486.

  • Grzonka, Z., Kasprzykowski, F. and Wiczk, W., In Industrial Enzymes: Structure, Function and Applications (eds Polaina, J. and MacCabe, A. P.), Springer, The Netherlands, 2007, pp. 181–195.

  • Baker, E. N. and Drenth, J., In The Thiol Proteases: Structure and Mechanism in Biological Macromolecules and Assemblies (eds Jurnak, F. A. and McPherson, A.), Wiley, New York, USA, 1987, pp. 313–368.

  • Polgar, L., In Mechanisms of Protease Action, CRC Press, Boca Raton, FL, USA, 1989, pp. 43–76.

  • Drenth, J., Jansonius, J. N., Koekoek, R., Swen, H. M. and Wolthers, B. G., Structure of papain. Nature, 1968, 218, 929–932.

  • Rawlings, N. D., Barrett, A. J. and Bateman, A., MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucl. Acids Res., 2012, 40, D343–D350.

  • Dardenne, L. E., Werneck, A. S., deOliveira Neto, M. and Bisch, P. M., Electrostatic properties in the catalytic site of papain: a possible regulatory mechanism for the reactivity of the ion pair. Proteins, 2003, 52, 236–253.

  • Turk, V., Stok, V., Vasiljeva, O., Renko, M., Sun, T., Turk, B. and Turk, D., Cysteine cathepsins: from structure, function and regulation to new frontiers. Biochim. Biophys. Acta, 2012, 1824, 68–88.

  • Wiederanders, B., Kaulmann, G. and Schilling, K., Functions of propeptide parts in cysteine proteases. Curr. Protein Pept. Sci., 2003, 4, 309–326.

  • Khan, A. R. and James, M. N. G., Molecular mechanisms for the conversion of zymogens to active proteolytic enzymes. Protein Sci., 1998, 7, 815–836.

  • Cygler, M. and Mort, J. S., Proregion structure of members of the papain superfamily. Mode of inhibition of enzymatic activity. Biochimie, 1997, 79, 645–652.

  • Coulombe, R., Grochulski, P., Sivaraman, J., Menard, R., Mort, J. S. and Cygler, M., Structure of human procathepsin L reveals the molecular basis of inhibition by the prosegment. EMBO J., 1996, 15, 5492–5503.

  • Groves, M. R., Taylor, M. A., Scott, M., Cummings, N. J., Pickersgill, R. W. and Jenkins, J. A., The prosequence of procaricain forms an alpha-helical domain that prevents access to the substrate-binding cleft. Structure, 1996, 4, 1193–1203.

  • LaLonde, J. M. et al., The crystal structure of human procathepsin K. Biochemistry, 1999, 38, 862–869.

  • Kaulmann, G., Palm, G. J., Schilling, K., Hilgenfeld, R. and Wiederanders, B., The crystal structure of a Cys25 Ala mutant of human procathepsin S elucidates enzyme prosequence interactions. Protein Sci., 2006, 15, 2619–2629.

  • Roy, S., Choudhury, D., Aich, P., Dattagupta, J. K. and Biswas, S., The structure of a thermostable mutant of pro-papain reveals its activation mechanism. Acta Cryst. Section D., Biol. Crystallogr., 2012, 68, 1591–1603.

  • Choudhury, D., Biswas, S., Roy, S. and Dattagupta, J. K., Improving thermostability of papain through structure-based protein engineering. Protein Eng. Des. Sel., 2010, 23, 457–467.

  • Roy, S., Choudhury, D., Chakrabarti, C., Biswas, S. and Dattagupta, J. K., Crystallization and preliminary X-ray diffraction studies of the precursor protein of a thermostable variant of papain. Acta Crystallogr., Sect. F, 2011, 67, 634–636.

  • Otwinowski, Z. and Minor, W., Processing of X-ray diffraction data collected in oscillation mode. Meth. Enzymol., 1997, 276, 307–326.

  • McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. and Read, R. J., Phaser crystallographic software. J. Appl. Crystallogr., 2007, 40, 658–674.

  • Adams, P. D. et al., PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr., Sect. D, 2002, 58, 1948–1954.

  • Emsley, P. and Cowtan, K., COOT: model-building tools for molecular graphics. Acta Crystallogr., Sect. D, 2004, 60, 2126–2132.

  • Murshudov, G. N., Vagin, A. A. and Dodson, E. J., Refinement of macromolecular structures by the maximum-likelihood method. Acta. Crystallogr., Sect. D, 1997, 53, 240–255.

  • Laskowski, R. A., MacArthur, M. W., Moss, D. S. and Thornton, J. M., PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr., 1993, 26, 283–291.

  • Choudhury, D., Roy, S., Chakrabarti, C., Biswas, S. and Dattagupta, J. K., Production and recovery of recombinant propapain with high yield. Phytochemistry, 2009, 70, 465–472.

  • Whitmore, L. and Wallace, B. A., DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucl. Acids Res., 2004, 32, 668–673.

  • McGrath, M. E., The lysosomal cysteine proteases. Annu. Rev. Biophys. Biomol. Struct., 1999, 28, 181–204.

  • Vernet, T. et al., Processing of the papain precursor. The ionization state of a conserved amino acid motif within the Pro region participates in the regulation of intramolecular processing. J. Biol. Chem., 1995, 270, 10838–10846.


Abstract Views: 248

PDF Views: 76




  • Asp72 of Pro-Peptide is an Important pH Sensor in the Zymogen Activation Process of Papain:A Structural and Mechanistic Insight

Abstract Views: 248  |  PDF Views: 76

Authors

Sumana Roy
Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700 064, India
Sampa Biswas
Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700 064, India

Abstract


The zymogen of papain contains a pro-peptide at the N-terminus of the catalytic domain. Pro-peptide contains residues which act as pH-sensors in zymogen activation cascade. To understand the influence of pro-peptide in the pH-induced zymogen activation process of the protease, we performed structural studies of the zymogen of papain at activation pH of 4.0. The X-ray structure of zymogen at acidic pH reveals that Asp72 of the pro-peptide, a highly conserved residue of the GXNXFXD motif, plays an important role in pH-induced conformational changes in the prodomain leading to the zymogen activation process. Far-UV circular dichroism spectrum of zymogen at pH 4.0 demonstrates loss of helical structure compared to that at pH 8.0. The structural observation is further corroborated by mutational studies, where D72A mutant is shown to undergo auto-activation at pH 5.0 compared to pH 4.0 for wild-type, though the general proteolytic activity of the D72A mutant remains similar to that of wild-type. Our findings indicate that the conserved Asp72 residue is an important pH sensor in the zymogen activation process and that the pro-peptide part can also be a useful target of protein engineering for altering the activation pH of a protease.

Keywords


Cysteine Protease, pH Regulation, Protein Engineering, Pro-Peptide, X-Ray Crystallography.

References





DOI: https://doi.org/10.18520/cs%2Fv114%2Fi11%2F2356-2362