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. 1999 Jan;8(1):253–258. doi: 10.1110/ps.8.1.253

Comparison of anionic and cationic trypsinogens: the anionic activation domain is more flexible in solution and differs in its mode of BPTI binding in the crystal structure.

A Pasternak 1, D Ringe 1, L Hedstrom 1
PMCID: PMC2144100  PMID: 10210204

Abstract

Unlike bovine cationic trypsin, rat anionic trypsin retains activity at high pH. This alkaline stability has been attributed to stabilization of the salt bridge between the N-terminal Ile16 and Asp194 by the surface negative charge (Soman K, Yang A-S, Honig B, Fletterick R., 1989, Biochemistry 28:9918-9926). The formation of this salt bridge controls the conformation of the activation domain in trypsin. In this work we probe the structure of rat trypsinogen to determine the effects of the surface negative charge on the activation domain in the absence of the Ile16-Asp194 salt bridge. We determined the crystal structures of the rat trypsin-BPTI complex and the rat trypsinogen-BPTI complex at 1.8 and 2.2 A, respectively. The BPTI complex of rat trypsinogen resembles that of rat trypsin. Surprisingly, the side chain of Ile16 is found in a similar position in both the rat trypsin and trypsinogen complexes, although it is not the N-terminal residue and cannot form the salt bridge in trypsinogen. The resulting position of the activation peptide alters the conformation of the adjacent autolysis loop (residues 142-153). While bovine trypsinogen and trypsin have similar CD spectra, the CD spectrum of rat trypsinogen has only 60% of the intensity of rat trypsin. This lower intensity most likely results from increased flexibility around two conserved tryptophans, which are adjacent to the activation domain. The NMR spectrum of rat trypsinogen contains high field methyl signals as observed in bovine trypsinogen. It is concluded that the activation domain of rat trypsinogen is more flexible than that of bovine trypsinogen, but does not extend further into the protein core.

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Selected References

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  1. Bennett W. S., Huber R. Structural and functional aspects of domain motions in proteins. CRC Crit Rev Biochem. 1984;15(4):291–384. doi: 10.3109/10409238409117796. [DOI] [PubMed] [Google Scholar]
  2. Bode W., Schwager P., Huber R. The transition of bovine trypsinogen to a trypsin-like state upon strong ligand binding. The refined crystal structures of the bovine trypsinogen-pancreatic trypsin inhibitor complex and of its ternary complex with Ile-Val at 1.9 A resolution. J Mol Biol. 1978 Jan 5;118(1):99–112. doi: 10.1016/0022-2836(78)90246-2. [DOI] [PubMed] [Google Scholar]
  3. Bolognesi M., Gatti G., Menagatti E., Guarneri M., Marquart M., Papamokos E., Huber R. Three-dimensional structure of the complex between pancreatic secretory trypsin inhibitor (Kazal type) and trypsinogen at 1.8 A resolution. Structure solution, crystallographic refinement and preliminary structural interpretation. J Mol Biol. 1982 Dec 25;162(4):839–868. doi: 10.1016/0022-2836(82)90550-2. [DOI] [PubMed] [Google Scholar]
  4. Brünger A. T., Kuriyan J., Karplus M. Crystallographic R factor refinement by molecular dynamics. Science. 1987 Jan 23;235(4787):458–460. doi: 10.1126/science.235.4787.458. [DOI] [PubMed] [Google Scholar]
  5. Craik C. S., Roczniak S., Largman C., Rutter W. J. The catalytic role of the active site aspartic acid in serine proteases. Science. 1987 Aug 21;237(4817):909–913. doi: 10.1126/science.3303334. [DOI] [PubMed] [Google Scholar]
  6. Fehlhammer H., Bode W., Huber R. Crystal structure of bovine trypsinogen at 1-8 A resolution. II. Crystallographic refinement, refined crystal structure and comparison with bovine trypsin. J Mol Biol. 1977 Apr 25;111(4):415–438. doi: 10.1016/s0022-2836(77)80062-4. [DOI] [PubMed] [Google Scholar]
  7. Fersht A. R. Conformational equilibria in -and -chymotrypsin. The energetics and importance of the salt bridge. J Mol Biol. 1972 Mar 14;64(2):497–509. doi: 10.1016/0022-2836(72)90513-x. [DOI] [PubMed] [Google Scholar]
  8. Hedstrom L., Lin T. Y., Fast W. Hydrophobic interactions control zymogen activation in the trypsin family of serine proteases. Biochemistry. 1996 Apr 9;35(14):4515–4523. doi: 10.1021/bi951928k. [DOI] [PubMed] [Google Scholar]
  9. Hedstrom L., Szilagyi L., Rutter W. J. Converting trypsin to chymotrypsin: the role of surface loops. Science. 1992 Mar 6;255(5049):1249–1253. doi: 10.1126/science.1546324. [DOI] [PubMed] [Google Scholar]
  10. Jones T. A., Zou J. Y., Cowan S. W., Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A. 1991 Mar 1;47(Pt 2):110–119. doi: 10.1107/s0108767390010224. [DOI] [PubMed] [Google Scholar]
  11. Kerr M. A., Walsh K. A., Neurath H. Catalysis by serine proteases and their zymogens. A study of acyl intermediates by circular dichroism. Biochemistry. 1975 Nov 18;14(23):5088–5094. doi: 10.1021/bi00694a010. [DOI] [PubMed] [Google Scholar]
  12. Pasternak A., Liu X., Lin T. Y., Hedstrom L. Activating a zymogen without proteolytic processing: mutation of Lys15 and Asn194 activates trypsinogen. Biochemistry. 1998 Nov 17;37(46):16201–16210. doi: 10.1021/bi980951d. [DOI] [PubMed] [Google Scholar]
  13. Perkins S. J., Wüthrich K. Conformational transition from trypsinogen to trypsin. 1H nuclear magnetic resonance at 360 MHz and ring current calculations. J Mol Biol. 1980 Mar 25;138(1):43–64. doi: 10.1016/s0022-2836(80)80004-0. [DOI] [PubMed] [Google Scholar]
  14. Perona J. J., Tsu C. A., Craik C. S., Fletterick R. J. Crystal structures of rat anionic trypsin complexed with the protein inhibitors APPI and BPTI. J Mol Biol. 1993 Apr 5;230(3):919–933. doi: 10.1006/jmbi.1993.1210. [DOI] [PubMed] [Google Scholar]
  15. Perona J. J., Tsu C. A., McGrath M. E., Craik C. S., Fletterick R. J. Relocating a negative charge in the binding pocket of trypsin. J Mol Biol. 1993 Apr 5;230(3):934–949. doi: 10.1006/jmbi.1993.1211. [DOI] [PubMed] [Google Scholar]
  16. Soman K., Yang A. S., Honig B., Fletterick R. Electrical potentials in trypsin isozymes. Biochemistry. 1989 Dec 26;28(26):9918–9926. doi: 10.1021/bi00452a007. [DOI] [PubMed] [Google Scholar]
  17. Sprang S., Standing T., Fletterick R. J., Stroud R. M., Finer-Moore J., Xuong N. H., Hamlin R., Rutter W. J., Craik C. S. The three-dimensional structure of Asn102 mutant of trypsin: role of Asp102 in serine protease catalysis. Science. 1987 Aug 21;237(4817):905–909. doi: 10.1126/science.3112942. [DOI] [PubMed] [Google Scholar]
  18. Thanki N., Thornton J. M., Goodfellow J. M. Distributions of water around amino acid residues in proteins. J Mol Biol. 1988 Aug 5;202(3):637–657. doi: 10.1016/0022-2836(88)90292-6. [DOI] [PubMed] [Google Scholar]
  19. Vincent J. P., Lazdunski M. Trypsin-pancreatic trypsin inhibitor association. Dynamics of the interaction and role of disulfide bridges. Biochemistry. 1972 Aug 1;11(16):2967–2977. doi: 10.1021/bi00766a007. [DOI] [PubMed] [Google Scholar]
  20. Weinreb P. H., Zhen W., Poon A. W., Conway K. A., Lansbury P. T., Jr NACP, a protein implicated in Alzheimer's disease and learning, is natively unfolded. Biochemistry. 1996 Oct 29;35(43):13709–13715. doi: 10.1021/bi961799n. [DOI] [PubMed] [Google Scholar]
  21. Wolfenden R. V., Cullis P. M., Southgate C. C. Water, protein folding, and the genetic code. Science. 1979 Nov 2;206(4418):575–577. doi: 10.1126/science.493962. [DOI] [PubMed] [Google Scholar]

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