Home Modeling the 3D structure of wheat subtilisin/chymotrypsin inhibitor (WSCI). Probing the reactive site with two susceptible proteinases by time-course analysis and molecular dynamics simulations
Article
Licensed
Unlicensed Requires Authentication

Modeling the 3D structure of wheat subtilisin/chymotrypsin inhibitor (WSCI). Probing the reactive site with two susceptible proteinases by time-course analysis and molecular dynamics simulations

  • Angelo M. Facchiano , Susan Costantini , Antimo Di Maro , Daniela Panichi , Angela Chambery , Augusto Parente , Simone Di Gennaro and Elia Poerio
Published/Copyright: July 20, 2006
Biological Chemistry
From the journal Volume 387 Issue 7

Abstract

Comparative modeling and time-course hydrolysis experiments have been applied to investigate two enzyme-inhibitor complexes formed between the wheat subtilisin-chymotrypsin inhibitor (WSCI) and two susceptible proteinases. WSCI represents the first case of a wheat protein inhibitor active against animal chymotrypsins and bacterial subtilisins. The model was created using as template structure that of the CI-2A inhibitor from barley (PDB code: 2CI2), which shares 87% sequence identity with WSCI. Under these conditions of high similarity, the comparative modeling approach can be successfully applied. We predicted the WSCI 3D model and used it to investigate enzyme-inhibitor complex systems. Experimental observations indicated that chymotrypsin, but not subtilisin, in addition to cleavage at the primary reactive site Met48-Glu49, is able to hydrolyze a second peptide bond between Phe58 and Val59. Here, we report on cleavage of the peptide bond at the inhibitor's reactive site (Met48-Glu49) determined using time-course hydrolysis experiments; the same event was investigated for both subtilisin/WSCI and chymotrypsin/WSCI complexes using molecular dynamics simulations. The molecular details of the initial inhibitor-enzyme interactions, as well as of the changes observed during the simulations, allow us to speculate on the different fates of the two WSCI-proteinase complexes.

:

Corresponding author

References

Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D.J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res.25, 3389–3402.10.1093/nar/25.17.3389Search in Google Scholar

Birk, Y. (2003). Plant protease inhibitors: significance in nutrition, plant protection, cancer prevention, and genetic engineering (Berlin, Germany: Springer-Verlag).Search in Google Scholar

Cai, M., Gong, Y., Kao, J.L., and Krishnamoorthi, R. (1995). Three-dimensional solution structure of Cucurbita maxima trypsin inhibitor-V determined by NMR spectroscopy. Biochemistry34, 5201–5211.10.1021/bi00015a034Search in Google Scholar

Caporale, C., Caruso, C., Facchiano, A., Nobile, M., Leonardi, L., Bertini, L., Colonna, G., and Buonocore, V. (1999). Probing the modelled structure of Wheatwin1 by controlled proteolysis and sequence analysis of unfractionated digestion mixtures. Proteins36, 192–204.10.1002/(SICI)1097-0134(19990801)36:2<192::AID-PROT5>3.0.CO;2-LSearch in Google Scholar

Costantini, S., Rossi, M., Colonna, G., and Facchiano, A.M. (2005). Modelling of HLA-DQ2 and its interaction with gluten peptides to explain molecular recognition in celiac disease. J. Mol. Graph. Model.23, 419–431.10.1016/j.jmgm.2004.12.002Search in Google Scholar

Di Gennaro, S., Ficca, A.G., Panichi, D., and Poerio, E. (2005). cDNA cloning and heterologous expression of a wheat proteinase inhibitor of subtilisin and chymotrypsin (WSCI) that interferes with digestive enzymes of insect pests. Biol. Chem.386, 383–389.10.1515/BC.2005.046Search in Google Scholar

Facchiano, A.M., Stiuso, P., Chiusano, M.L., Caraglia, M., Giuberti, G., Marra, M., Abruzzese, A., and Colonna, G. (2001). Homology modelling of the human eukaryotic initiation factor 5A (eIF-5A). Protein Eng.14, 881–890.10.1093/protein/14.11.881Search in Google Scholar

Hilder, V.A. and Boulter, D. (1999). Genetic engineering of crop plants for insect resistance – a critical review. Crop Prot.18, 177–191.10.1016/S0261-2194(99)00028-9Search in Google Scholar

Hubbard, S.J., Campbell, S.F., and Thornton, J.M. (1991). Molecular recognition. Conformational analysis of limited proteolytic sites and serine proteinase protein inhibitors. J. Mol. Biol.220, 507–530.10.1016/0022-2836(91)90027-4Search in Google Scholar

Jonassen, I.B. and Svendsen, I.B. (1982). Identification of the reactive sites in two homologous serine proteinase inhibitors isolated from barley. Carlsberg Res. Commun.47, 199–203.10.1007/BF02907871Search in Google Scholar

Jones, S. and Thornton, J.M. (1996). Principles of protein-protein interactions derived from structural studies. Proc. Natl. Acad. Sci. USA93, 13–20.10.1073/pnas.93.1.13Search in Google Scholar

Jongsma, M.A. and Bolter, C. (1997). The adaptation of insect to plant protease inhibitors. J. Insect Physiol.43, 885–895.10.1016/S0022-1910(97)00040-1Search in Google Scholar

Jouanin, L., Bonadé-Bottino, M., Girard, C., Morrot, G., and Giband, M. (1998). Transgenic plants for insect resistance. Plant Sci.131, 1–11.10.1016/S0168-9452(97)00239-2Search in Google Scholar

Kabsch, W. and Sander, C. (1983). Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers22, 2577–2637.10.1002/bip.360221211Search in Google Scholar

Koiwa, H., Bressan, R.A., and Hasegawa, P.M. (1997). Regulation of protease inhibitors and plant defense. Trends Plant Sci.2, 379–384.10.1016/S1360-1385(97)90052-2Search in Google Scholar

Laskowski, R.A., MacArthur, M.W., Moss, D.S., and Thornton, J.M. (1993). PROCHECK – a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr.26, 283–291.10.1107/S0021889892009944Search in Google Scholar

Lawrence, P.K. and Koundal, K.R. (2002). Plant protease inhibitors in control of phytophagous insects. Electron. J. Biotechnol.5, 93–109.10.2225/vol5-issue1-fulltext-3Search in Google Scholar

Marabotti, A. and Facchiano, A.M. (2005). Homology modeling studies on human galactose-1-phosphate uridylyltransferase and on its galactosemia-related mutant Q188R provide an explanation of molecular effects of the mutation on homo- and heterodimers. J. Med. Chem.48, 773–779.10.1021/jm049731qSearch in Google Scholar PubMed

Marabotti, A., D'Auria, S., Rossi, M., and Facchiano, A.M. (2004). Theoretical model of the three-dimensional structure of a sugar-binding protein from Pyrococcus horikoshii: structural analysis and sugar-binding simulations. Biochem. J.380, 677–684.10.1042/bj20031876Search in Google Scholar PubMed PubMed Central

McPhalen, C.A. and James, M.N.G. (1987). Crystal and molecular structure of the serine proteinase inhibitor CI-2 from barley seeds. Biochemistry26, 261–269.10.1021/bi00375a036Search in Google Scholar PubMed

Moult, J., Fidelis, K., Zemla, A., and Hubbard, T. (2003). Critical assessment of methods of protein structure prediction (CASP)-round V. Proteins53 (Suppl. 6), 334–339.10.1002/prot.10556Search in Google Scholar PubMed

Parente, A., Verde, C., Malorni, A., Montecucchi, P., Aniello, F., and Geraci, G. (1993). Amino-acid sequence of the cooperative dimeric myoglobin from the radular muscles of the marine gastropod Nassa mutabilis. Biochim. Biophys. Acta1162, 1–9.10.1016/0167-4838(93)90120-GSearch in Google Scholar

Poerio, E., Di Gennaro, S., Di Maro, A., Farisei, F., Ferranti, P., and Parente, A. (2003). Primary structure and reactive site of a novel wheat proteinase inhibitor of subtilisin and chymotrypsin. Biol. Chem.384, 295–304.10.1515/BC.2003.033Search in Google Scholar

Rashid, A., Khurshid, R., Begum, M., Gul-e-Raana, Latif, M., and Salim, A. (2004). Modeling the mutational effects on calmodulin structure: prediction of alteration in the amino acid interactions. Biochem. Biophys. Res. Commun.317, 363–369.10.1016/j.bbrc.2004.03.051Search in Google Scholar

Ryan, C.A. (1990). Protease inhibitors in plants: genes for improving defenses against insects and pathogens. Annu. Rev. Phytophatol.28, 425–449.10.1146/annurev.py.28.090190.002233Search in Google Scholar

Sali, A. and Blundell, T.L. (1993). Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol.234, 779–815.10.1006/jmbi.1993.1626Search in Google Scholar

Sattar, R., Ali, S.A., Kamal, M., Khan, A.A., and Abbasi, A. (2004). Molecular mechanism of enzyme inhibition: prediction of the three-dimensional structure of the dimeric trypsin inhibitor from Leucaena leucocephala by homology modelling. Biochem. Biophys. Res. Commun.314, 755–765.10.1016/j.bbrc.2003.12.177Search in Google Scholar

Schägger, H. and von Jagow, G. (1987). Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem.166, 368–379.10.1016/0003-2697(87)90587-2Search in Google Scholar

Schuler, T.H., Poppy, G.M., Kerry, B.R., and Denholm, I. (1998). Insect-resistant transgenic plants. Trends Biotechnol.16, 168–175.10.1016/S0167-7799(97)01171-2Search in Google Scholar

Shewry, P.R. and Lucas, J.A. (1997). Plant proteins that confer resistance to pests and pathogens. In: Advances in Botanical Research, vol. 26, J. Callow ed. (London, UK: Academic Press), pp. 135–192.10.1016/S0065-2296(08)60120-2Search in Google Scholar

Sippl, M. (1993). Recognition of errors in three-dimensional structures of proteins. Proteins27, 355–362.10.1002/prot.340170404Search in Google Scholar PubMed

Venclovas, C. (2001). Comparative modeling of CASP4 target proteins: combining results of sequence search with three-dimensional structure assessment. Proteins45 (Suppl. 5), 47–54.10.1002/prot.10008Search in Google Scholar PubMed

Venclovas, C. (2003). Comparative modeling in CASP5: progress is evident, but alignment errors remain a significant hindrance. Proteins53 (Suppl. 6), 380–388.10.1002/prot.10591Search in Google Scholar PubMed

Wallner, B. and Elofsson, A. (2005). All are not equal: a benchmark of different homology modeling programs. Protein Sci.14, 1315–1327.10.1110/ps.041253405Search in Google Scholar PubMed PubMed Central

Yamada, T., Ohta, H., Shinohara, A., Iwamatsu, A., Shimada, H., Tsuchiya, T., Masuda, T., and Takamiya, K. (2000). A cysteine protease from maize isolated in a complex with cystatin. Plant Cell. Physiol.41, 185–191.10.1093/pcp/41.2.185Search in Google Scholar PubMed

Yamauchi, Y., Ejiri, Y., Sugimoto, T., Sueyoshi, K., Oji, Y., and Tanaka, K. (2001). A high molecular weight glutamyl endopeptidase and its endogenous inhibitors from cucumber leaves. J. Biochem. (Tokyo)130, 257–261.10.1093/oxfordjournals.jbchem.a002980Search in Google Scholar PubMed

Published Online: 2006-07-20
Published in Print: 2006-07-01

©2006 by Walter de Gruyter Berlin New York

Articles in the same Issue

  1. 4th General Meeting of the International Proteolysis Society/International Conference on Protease Inhibitors
  2. Extracellular granzymes: current perspectives
  3. Impact of the N-terminal amino acid on targeted protein degradation
  4. Structural aspects of recently discovered viral deubiquitinating activities
  5. Cysteine cathepsins and caspases in silicosis
  6. The proprotein convertases and their implication in sterol and/or lipid metabolism
  7. PREPL: a putative novel oligopeptidase propelled into the limelight
  8. Human cathepsin L rescues the neurodegeneration and lethality in cathepsin B/L double-deficient mice
  9. Helicobacter pylori-induced downregulation of the secretory leukocyte protease inhibitor (SLPI) in gastric epithelial cell lines and its functional relevance for H. pylori-mediated diseases
  10. Increased local levels of granulocyte colony-stimulating factor are associated with the beneficial effect of pre-elafin (SKALP/trappin-2/WAP3) in experimental emphysema
  11. Interaction of a novel form of Pseudomonas aeruginosa alkaline protease (aeruginolysin) with interleukin-6 and interleukin-8
  12. Analysis of aldosterone-induced differential receptor-independent protein patterns using 2D-electrophoresis and mass spectrometry
  13. Modeling the 3D structure of wheat subtilisin/chymotrypsin inhibitor (WSCI). Probing the reactive site with two susceptible proteinases by time-course analysis and molecular dynamics simulations
  14. A stable analogue of glucose-dependent insulinotropic polypeptide, GIP(LysPAL16), enhances functional differentiation of mouse embryonic stem cells into cells expressing islet-specific genes and hormones
  15. Transcription factor FOXM1c is repressed by RB and activated by cyclin D1/Cdk4
  16. Despite its strong transactivation domain, transcription factor FOXM1c is kept almost inactive by two different inhibitory domains
  17. Inhibition of calcineurin by infusion of CsA causes hyperphosphorylation of tau and is accompanied by abnormal behavior in mice
  18. Isolation and properties of extracellular proteinases of Penicillium marneffei
  19. Isolation and comparative characterization of Ki-67 equivalent antibodies from the HuCAL® phage display library
Downloaded on 14.9.2025 from https://www.degruyterbrill.com/document/doi/10.1515/BC.2006.117/html
Scroll to top button