Abstract
MCoTI-I and MCoTI-II from the seeds of Momordica cochinchinensis are inhibitors of trypsin-like proteases and the only known members of the large family of squash inhibitors that are cyclic and contain an additional loop connecting the amino- and the carboxy-terminus. To investigate the contribution of macrocycle formation to biological activity, we synthesized a set of open-chain variants of MCoTI-II that lack the cyclization loop and contain various natural and non-natural amino acid substitutions in the reactive-site loop. Upon replacement of P1 lysine residue #10 within the open-chain variant of MCoTI-II by the non-natural isosteric nucleo amino acid AlaG [β-(guanin-9-yl)-L-alanine], a conformationally restricted arginine mimetic, residual inhibitory activity was detected, albeit reduced by four orders of magnitude. While the cyclic inhibitors MCoTI-I and MCoTI-II were found to be very potent trypsin inhibitors, with picomolar inhibition constants, the open-chain variants displayed an approximately 10-fold lower affinity. These data suggest that the formation of a circular backbone in the MCoTI squash inhibitors results in enhanced affinity and therefore is a determinant of biological activity.
References
Albericio, F., Annis, I., Royo, M., and Barany, G. (2000). Preparation and handling of peptides containing methionine and cysteine. In: Fmoc Solid Phase Peptide Synthesis. A Practical Approach, W.C. Chan and P.D. White, eds. (New York, USA: Oxford University Press), pp. 81–91.Search in Google Scholar
Avrutina, O., Schmoldt, H.U., Kolmar, H., and Diederichsen, U. (2004). Fmoc-assisted synthesis of a 29-residue cystine-knot trypsin inhibitor containing a guaninyl amino acid at the P1-position. Eur. J. Org. Chem.2004, 4931–4935.10.1002/ejoc.200400440Search in Google Scholar
Barry, D.G., Daly, N.L., Clark, R.J., Sando, L., and Craik, D.J. (2003). Linearization of a naturally occurring circular protein maintains structure but eliminates hemolytic activity. Biochemistry42, 6688–6695.10.1021/bi027323nSearch in Google Scholar
Bieth, J.G. (1980). Pathophysiological interpretation of kinetic constants of protease inhibitors. Bull. Eur. Physiopathol. Respir.16 (Suppl.), 183–197.Search in Google Scholar
Bode, W., Greyling, H.J., Huber, R., Otlewski, J., and Wilusz, T. (1989). The refined 2.0 Å X-ray crystal structure of the complex formed between bovine β-trypsin and CMTI-I, a trypsin inhibitor from squash seeds (Cucurbita maxima). Topological similarity of the squash seed inhibitors with the carboxypeptidase A inhibitor from potatoes. FEBS Lett.242, 285–292.Search in Google Scholar
Chase, T. Jr. and Shaw, E. (1970). Titration of trypsin, plasmin, and thrombin with p-nitrophenyl-p′-guanidinobenzoate HCl. In: Methods in Enzymology, G.E. Perlmann, and L. Lorand, eds. (New York, USA: Academic Press), pp. 20–27.10.1016/0076-6879(70)19004-5Search in Google Scholar
Chiche, L., Heitz, A., Gelly, J.C., Gracy, J., Chau, P.T., Ha, P.T., Hernandez, J.F., and Le-Nguyen, D. (2004). Squash inhibitors: from structural motifs to macrocyclic knottins. Curr. Protein Pept. Sci.5, 341–349.10.2174/1389203043379477Search in Google Scholar
Colgrave, M.L. and Craik, D.J. (2004). Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: the importance of the cyclic cystine knot. Biochemistry43, 5965–5975.10.1021/bi049711qSearch in Google Scholar
Craik, D.J. (2001). Plant cyclotides: circular, knotted peptide toxins. Toxicon39, 1809–1813.10.1016/S0041-0101(01)00129-5Search in Google Scholar
Daly, N.L., Love, S., Alewood, P.F., and Craik, D.J. (1999). Chemical synthesis and folding pathways of large cyclic polypeptides: studies of the cystine knot polypeptide kalata B1. Biochemistry38, 10606–10614.10.1021/bi990605bSearch in Google Scholar PubMed
Delano, W.L. (2002). The PyMOL Molecular Graphics System on World Wide Web, www.pymol.org (San Carlos, USA: DeLano Scientific).Search in Google Scholar
Domingo, G.J., Leatherbarrow, R.J., Freeman, N., Patel, S., and Weir, M. (1995). Synthesis of a mixture of cyclic peptides based on the Bowman-Birk reactive site loop to screen for serine protease inhibitors. Int. J. Pept. Protein Res.46, 79–87.10.1111/j.1399-3011.1995.tb00585.xSearch in Google Scholar PubMed
Favel, A., Mattras, H., Coletti-Previero, M.A., Zwilling, R., Robinson, E.A., and Castro, B. (1989). Protease inhibitors from Ecballium elaterium seeds. Int. J. Pept. Protein Res.33, 202–208.10.1111/j.1399-3011.1989.tb00210.xSearch in Google Scholar PubMed
Felizmenio-Quimio, M.E., Daly, N.L., and Craik, D.J. (2001). Circular proteins in plants: solution structure of a novel macrocyclic trypsin inhibitor from Momordica cochinchinensis. J. Biol. Chem.276, 22875–22882.10.1074/jbc.M101666200Search in Google Scholar PubMed
Gustafson, K.R., McKee, T.C., and Bokesch, H.R. (2004). Anti-HIV cyclotides. Curr. Protein Pept. Sci.5, 331–340.10.2174/1389203043379468Search in Google Scholar
Heitz, A., Hernandez, J.F., Gagnon, J., Hong, T.T., Pham, T.T., Nguyen, T.M., Le-Nguyen, D., and Chiche, L. (2001). Solution structure of the squash trypsin inhibitor MCoTI-II. A new family for cyclic knottins. Biochemistry40, 7973–7983.Search in Google Scholar
Hernandez, J.F., Gagnon, J., Chiche, L., Nguyen, T.M., Andrieu, J.P., Heitz, A., Trinh Hong, T., Pham, T.T., and Le Nguyen, D. (2000). Squash trypsin inhibitors from Momordica cochinchinensis exhibit an atypical macrocyclic structure. Biochemistry39, 5722–5730.10.1021/bi9929756Search in Google Scholar
Jennings, C., West, J., Waine, C., Craik, D., and Anderson, M. (2001). Biosynthesis and insecticidal properties of plant cyclotides: the cyclic knotted proteins from Oldenlandia affinis. Proc. Natl. Acad. Sci. USA98, 10614–10619.10.1073/pnas.191366898Search in Google Scholar
Kokko, K.P., Arrigoni, C.E., and Dix, T.A. (2001). Selectivity enhancement induced by substitution of non-natural analogues of arginine and lysine in arginine-based thrombin inhibitors. Bioorg. Med. Chem. Lett.11, 1947–1950.10.1016/S0960-894X(01)00328-6Search in Google Scholar
Krätzner, R., Debreczeni, J.É., Pape, T., Schneider, T.R., Wentzel, A., Kolmar, H., Sheldrick, G.M., and Uson, I. (2005). Structure of Ecballium elaterium trypsin inhibitor II (EETI-II): a rigid molecular scaffold. Acta Cryst.D61, 1255–1262.Search in Google Scholar
Lorenz, K.B. and Diederichsen, U. (2003). Nucleo amino acids as arginine mimetics in cyclic peptides. Lett. Pept. Sci.10, 111–117.10.1023/B:LIPS.0000032374.98648.7bSearch in Google Scholar
McBride, J.D. and Leatherbarrow, R.J. (2001). Synthetic peptide mimics of the Bowman-Birk inhibitor protein. Curr. Med. Chem.8, 909–917.10.2174/0929867013372832Search in Google Scholar
Morrison, J.F. (1969). Kinetics of the reversible inhibition of enzyme-catalysed reactions by tight-binding inhibitors. Biochim. Biophys. Acta185, 269–286.10.1016/0005-2744(69)90420-3Search in Google Scholar
Schechter, I. and Berger, A. (1967). On the size of the active site in proteases. I. Papain. Biochem. Biophys. Res. Commun.27, 157–162.10.1016/S0006-291X(67)80055-XSearch in Google Scholar
Tam, J.P., Lu, Y.A., Yang, J.L., and Chiu, K.W. (1999). An unusual structural motif of antimicrobial peptides containing end-to-end macrocycle and cystine-knot disulfides. Proc. Natl. Acad. Sci. USA96, 8913–8918.10.1073/pnas.96.16.8913Search in Google Scholar
Trabi, M. and Craik, D.J. (2002). Circular proteins – no end in sight. Trends Biochem. Sci.27, 132–138.10.1016/S0968-0004(02)02057-1Search in Google Scholar
Witherup, K.M., Bogusky, M.J., Anderson, P.S., Ramjit, H., Ransom, R.W., Wood, T., and Sardana, M. (1994). Cyclopsychotride A, a biologically active, 31-residue cyclic peptide isolated from Psychotria longipes. J. Nat. Prod.57, 1619–1625.10.1021/np50114a002Search in Google Scholar PubMed
©2005 by Walter de Gruyter Berlin New York
