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. 2002 Apr 15;363(Pt 2):403–409. doi: 10.1042/0264-6021:3630403

Probing the serpin structural-transition mechanism in ovalbumin mutant R339T by proteolytic-cleavage kinetics of the reactive-centre loop.

Yasuhiro Arii 1, Masaaki Hirose 1
PMCID: PMC1222492  PMID: 11931671

Abstract

A mutant ovalbumin (R339T), but not the wild-type protein, is transformed into the canonical loop-inserted, thermostabilized form after the P1-P1' cleavage [Yamasaki, Arii, Mikami and Hirose (2002) J. Mol. Biol. 315, 113-120]. The loop-insertion mechanism in the ovalbumin mutant was investigated by proteolytic-cleavage kinetics. The nature of the inserted loop prevented further cleavage of the P1-P1' pre-cleaved R339T mutant by subtilisin, which cleaved the second P8-P7 loop site in the P1-P1' pre-cleaved wild-type protein. After subtilisin proteolysis of the intact R339T, however, two final products that corresponded to the single P1-P1' and double P1-P1'/P8-P7 cleavages were generated with variable ratios depending on the proteolysis conditions. This was accounted for by the occurrence of two mutually competitive reactions: the loop-insertion reaction and the proteolytic cleavage of the second P8-P7 site in the immediate intermediate after the P1-P1' cleavage. The competitive nature of the two reactions enabled us to establish a kinetic method to determine the rate constants of the reactions. The first-order rate constant for the loop insertion was determined to be 4.0 x 10(-3)/s in the R339T mutant. The second-order rate constant for the P8-P7 cleavage in the immediate P1-P1' cleavage product for the R339T mutant was >10 times compared with that for its wild-type counterpart. This highly accessible loop nature may play a crucial role in the loop-insertion mechanism for R339T mutant ovalbumin.

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Selected References

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  1. Arii Y., Takahashi N., Tatsumi E., Hirose M. Structural properties of recombinant ovalbumin and its transformation into a thermostabilized form by alkaline treatment. Biosci Biotechnol Biochem. 1999 Aug;63(8):1392–1399. doi: 10.1271/bbb.63.1392. [DOI] [PubMed] [Google Scholar]
  2. Carrell R. W., Stein P. E., Fermi G., Wardell M. R. Biological implications of a 3 A structure of dimeric antithrombin. Structure. 1994 Apr 15;2(4):257–270. doi: 10.1016/s0969-2126(00)00028-9. [DOI] [PubMed] [Google Scholar]
  3. Chaillan-Huntington C. E., Gettins P. G., Huntington J. A., Patston P. A. The P6-P2 region of serpins is critical for proteinase inhibition and complex stability. Biochemistry. 1997 Aug 5;36(31):9562–9570. doi: 10.1021/bi970651g. [DOI] [PubMed] [Google Scholar]
  4. Davis A. E., 3rd, Aulak K., Parad R. B., Stecklein H. P., Eldering E., Hack C. E., Kramer J., Strunk R. C., Bissler J., Rosen F. S. C1 inhibitor hinge region mutations produce dysfunction by different mechanisms. Nat Genet. 1992 Aug;1(5):354–358. doi: 10.1038/ng0892-354. [DOI] [PubMed] [Google Scholar]
  5. DelMar E. G., Largman C., Brodrick J. W., Geokas M. C. A sensitive new substrate for chymotrypsin. Anal Biochem. 1979 Nov 1;99(2):316–320. doi: 10.1016/s0003-2697(79)80013-5. [DOI] [PubMed] [Google Scholar]
  6. Engh R. A., Huber R., Bode W., Schulze A. J. Divining the serpin inhibition mechanism: a suicide substrate 'springe'? Trends Biotechnol. 1995 Dec;13(12):503–510. doi: 10.1016/S0167-7799(00)89013-7. [DOI] [PubMed] [Google Scholar]
  7. Gettins P., Patston P. A., Schapira M. The role of conformational change in serpin structure and function. Bioessays. 1993 Jul;15(7):461–467. doi: 10.1002/bies.950150705. [DOI] [PubMed] [Google Scholar]
  8. Hood D. B., Huntington J. A., Gettins P. G. Alpha 1-proteinase inhibitor variant T345R. Influence of P14 residue on substrate and inhibitory pathways. Biochemistry. 1994 Jul 19;33(28):8538–8547. doi: 10.1021/bi00194a020. [DOI] [PubMed] [Google Scholar]
  9. Huber R., Carrell R. W. Implications of the three-dimensional structure of alpha 1-antitrypsin for structure and function of serpins. Biochemistry. 1989 Nov 14;28(23):8951–8966. doi: 10.1021/bi00449a001. [DOI] [PubMed] [Google Scholar]
  10. Huntington J. A., Fan B., Karlsson K. E., Deinum J., Lawrence D. A., Gettins P. G. Serpin conformational change in ovalbumin. Enhanced reactive center loop insertion through hinge region mutations. Biochemistry. 1997 May 6;36(18):5432–5440. doi: 10.1021/bi9702142. [DOI] [PubMed] [Google Scholar]
  11. Huntington J. A., Read R. J., Carrell R. W. Structure of a serpin-protease complex shows inhibition by deformation. Nature. 2000 Oct 19;407(6806):923–926. doi: 10.1038/35038119. [DOI] [PubMed] [Google Scholar]
  12. Kaslik G., Kardos J., Szabó E., Szilágyi L., Závodszky P., Westler W. M., Markley J. L., Gráf L. Effects of serpin binding on the target proteinase: global stabilization, localized increased structural flexibility, and conserved hydrogen bonding at the active site. Biochemistry. 1997 May 6;36(18):5455–5464. doi: 10.1021/bi962931m. [DOI] [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Lee C., Park S. H., Lee M. Y., Yu M. H. Regulation of protein function by native metastability. Proc Natl Acad Sci U S A. 2000 Jul 5;97(14):7727–7731. doi: 10.1073/pnas.97.14.7727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lee K. N., Im H., Kang S. W., Yu M. H. Characterization of a human alpha1-antitrypsin variant that is as stable as ovalbumin. J Biol Chem. 1998 Jan 30;273(5):2509–2516. doi: 10.1074/jbc.273.5.2509. [DOI] [PubMed] [Google Scholar]
  16. Loebermann H., Tokuoka R., Deisenhofer J., Huber R. Human alpha 1-proteinase inhibitor. Crystal structure analysis of two crystal modifications, molecular model and preliminary analysis of the implications for function. J Mol Biol. 1984 Aug 15;177(3):531–557. [PubMed] [Google Scholar]
  17. McCarthy B. J., Worrall D. M. Analysis of serpin inhibitory function by mutagenesis of ovalbumin and generation of chimeric ovalbumin/PAI-2 fusion proteins. J Mol Biol. 1997 Apr 4;267(3):561–569. doi: 10.1006/jmbi.1996.0909. [DOI] [PubMed] [Google Scholar]
  18. Mottonen J., Strand A., Symersky J., Sweet R. M., Danley D. E., Geoghegan K. F., Gerard R. D., Goldsmith E. J. Structural basis of latency in plasminogen activator inhibitor-1. Nature. 1992 Jan 16;355(6357):270–273. doi: 10.1038/355270a0. [DOI] [PubMed] [Google Scholar]
  19. OTTESEN M. The transformation of ovalbumin into plakalbumin; a case of limited proteolysis. C R Trav Lab Carlsberg Chim. 1958;30(14):211–270. [PubMed] [Google Scholar]
  20. Olson S. T., Swanson R., Day D., Verhamme I., Kvassman J., Shore J. D. Resolution of Michaelis complex, acylation, and conformational change steps in the reactions of the serpin, plasminogen activator inhibitor-1, with tissue plasminogen activator and trypsin. Biochemistry. 2001 Oct 2;40(39):11742–11756. doi: 10.1021/bi0107290. [DOI] [PubMed] [Google Scholar]
  21. Patston P. A., Gettins P. G. Significance of secondary structure predictions on the reactive center loop region of serpins: a model for the folding of serpins into a metastable state. FEBS Lett. 1996 Mar 25;383(1-2):87–92. doi: 10.1016/0014-5793(96)00231-1. [DOI] [PubMed] [Google Scholar]
  22. Patston P. A., Gettins P., Beechem J., Schapira M. Mechanism of serpin action: evidence that C1 inhibitor functions as a suicide substrate. Biochemistry. 1991 Sep 10;30(36):8876–8882. doi: 10.1021/bi00100a022. [DOI] [PubMed] [Google Scholar]
  23. Shore J. D., Day D. E., Francis-Chmura A. M., Verhamme I., Kvassman J., Lawrence D. A., Ginsburg D. A fluorescent probe study of plasminogen activator inhibitor-1. Evidence for reactive center loop insertion and its role in the inhibitory mechanism. J Biol Chem. 1995 Mar 10;270(10):5395–5398. doi: 10.1074/jbc.270.10.5395. [DOI] [PubMed] [Google Scholar]
  24. Stein P. E., Carrell R. W. What do dysfunctional serpins tell us about molecular mobility and disease? Nat Struct Biol. 1995 Feb;2(2):96–113. doi: 10.1038/nsb0295-96. [DOI] [PubMed] [Google Scholar]
  25. Stein P. E., Leslie A. G., Finch J. T., Carrell R. W. Crystal structure of uncleaved ovalbumin at 1.95 A resolution. J Mol Biol. 1991 Oct 5;221(3):941–959. doi: 10.1016/0022-2836(91)80185-w. [DOI] [PubMed] [Google Scholar]
  26. Stein P. E., Leslie A. G., Finch J. T., Turnell W. G., McLaughlin P. J., Carrell R. W. Crystal structure of ovalbumin as a model for the reactive centre of serpins. Nature. 1990 Sep 6;347(6288):99–102. doi: 10.1038/347099a0. [DOI] [PubMed] [Google Scholar]
  27. Stein P. E., Tewkesbury D. A., Carrell R. W. Ovalbumin and angiotensinogen lack serpin S-R conformational change. Biochem J. 1989 Aug 15;262(1):103–107. doi: 10.1042/bj2620103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Takahashi N., Tatsumi E., Orita T., Hirose M. Role of the intrachain disulfide bond of ovalbumin during conversion into S-ovalbumin. Biosci Biotechnol Biochem. 1996 Sep;60(9):1464–1468. doi: 10.1271/bbb.60.1464. [DOI] [PubMed] [Google Scholar]
  29. Tatsumi E., Takahashi N., Hirose M. Denatured state of ovalbumin in high concentrations of urea as evaluated by disulfide rearrangement analysis. J Biol Chem. 1994 Nov 11;269(45):28062–28067. [PubMed] [Google Scholar]
  30. Whisstock J., Skinner R., Lesk A. M. An atlas of serpin conformations. Trends Biochem Sci. 1998 Feb;23(2):63–67. doi: 10.1016/s0968-0004(97)01172-9. [DOI] [PubMed] [Google Scholar]
  31. Wright H. T. Ovalbumin is an elastase substrate. J Biol Chem. 1984 Dec 10;259(23):14335–14336. [PubMed] [Google Scholar]
  32. Wright H. T., Qian H. X., Huber R. Crystal structure of plakalbumin, a proteolytically nicked form of ovalbumin. Its relationship to the structure of cleaved alpha-1-proteinase inhibitor. J Mol Biol. 1990 Jun 5;213(3):513–528. doi: 10.1016/s0022-2836(05)80212-8. [DOI] [PubMed] [Google Scholar]
  33. Wright H. T., Scarsdale J. N. Structural basis for serpin inhibitor activity. Proteins. 1995 Jul;22(3):210–225. doi: 10.1002/prot.340220303. [DOI] [PubMed] [Google Scholar]
  34. Yamasaki Masayuki, Arii Yasuhiro, Mikami Bunzo, Hirose Masaaki. Loop-inserted and thermostabilized structure of P1-P1' cleaved ovalbumin mutant R339T. J Mol Biol. 2002 Jan 11;315(2):113–120. doi: 10.1006/jmbi.2001.5056. [DOI] [PubMed] [Google Scholar]

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