Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1997 Feb;6(2):263–274. doi: 10.1002/pro.5560060201

The internal thioester and the covalent binding properties of the complement proteins C3 and C4.

S K Law 1, A W Dodds 1
PMCID: PMC2143658  PMID: 9041627

Abstract

The covalent binding of complement components C3 and C4 is critical for their activities. This reaction is made possible by the presence of an internal thioester in the native protein. Upon activation, which involves a conformational change initiated by the cleavage of a single peptide bond, the thioester becomes available to react with molecules with nucleophilic groups. This description is probably sufficient to account for the binding of the C4A isotype of human C4 to amino nucleophiles. The binding of the C4B isotype, and most likely C3, to hydroxyl nucleophiles, however, involves a histidine residue, which attacks the thioester to form an intramolecular acyl-imidazole bond. The released thiolate anion then acts as a base to catalyze the binding of hydroxyl nucleophiles, including water, to the acyl function. This mechanism allows the complement proteins to bind to the hydroxyl groups of carbohydrates found on all biological surfaces, including the components of bacterial cell walls. In addition, the fast hydrolysis of the thioester provides a means to contain this very damaging reaction to the immediate proximity of the site of activation.

Full Text

The Full Text of this article is available as a PDF (2.5 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alcolea J. M., Antón L. C., Marqués G., Sánchez-Corral P., Vivanco F. Formation of covalent complexes between the fourth component of human complement and IgG immune aggregates. Complement. 1987;4(1):21–32. doi: 10.1159/000463004. [DOI] [PubMed] [Google Scholar]
  2. Antón L. C., Alcolea J. M., Sánchez-Corral P., Marqués G., Sánchez A., Vivanco F. C3 binds covalently to the C gamma 3 domain of IgG immune aggregates during complement activation by the alternative pathway. Biochem J. 1989 Feb 1;257(3):831–838. doi: 10.1042/bj2570831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Antón L. C., Ruiz S., Barrio E., Marqués G., Sánchez A., Vivanco F. C3 binds with similar efficiency to Fab and Fc regions of IgG immune aggregates. Eur J Immunol. 1994 Mar;24(3):599–604. doi: 10.1002/eji.1830240316. [DOI] [PubMed] [Google Scholar]
  4. Auerbach H. S., Burger R., Dodds A., Colten H. R. Molecular basis of complement C3 deficiency in guinea pigs. J Clin Invest. 1990 Jul;86(1):96–106. doi: 10.1172/JCI114721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Awdeh Z. L., Alper C. A. Inherited structural polymorphism of the fourth component of human complement. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3576–3580. doi: 10.1073/pnas.77.6.3576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Awdeh Z. L., Raum D., Yunis E. J., Alper C. A. Extended HLA/complement allele haplotypes: evidence for T/t-like complex in man. Proc Natl Acad Sci U S A. 1983 Jan;80(1):259–263. doi: 10.1073/pnas.80.1.259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Belt K. T., Carroll M. C., Porter R. R. The structural basis of the multiple forms of human complement component C4. Cell. 1984 Apr;36(4):907–914. doi: 10.1016/0092-8674(84)90040-0. [DOI] [PubMed] [Google Scholar]
  8. Campbell R. D., Gagnon J., Porter R. R. Amino acid sequence around the thiol and reactive acyl groups of human complement component C4. Biochem J. 1981 Nov 1;199(2):359–370. doi: 10.1042/bj1990359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Campbell R. D., Milner C. M. MHC genes in autoimmunity. Curr Opin Immunol. 1993 Dec;5(6):887–893. doi: 10.1016/0952-7915(93)90101-w. [DOI] [PubMed] [Google Scholar]
  10. Carroll M. C., Fathallah D. M., Bergamaschini L., Alicot E. M., Isenman D. E. Substitution of a single amino acid (aspartic acid for histidine) converts the functional activity of human complement C4B to C4A. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6868–6872. doi: 10.1073/pnas.87.17.6868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chu C. T., Pizzo S. V. alpha 2-Macroglobulin, complement, and biologic defense: antigens, growth factors, microbial proteases, and receptor ligation. Lab Invest. 1994 Dec;71(6):792–812. [PubMed] [Google Scholar]
  12. Chu C. T., Rubenstein D. S., Enghild J. J., Pizzo S. V. Mechanism of insulin incorporation into alpha 2-macroglobulin: implications for the study of peptide and growth factor binding. Biochemistry. 1991 Feb 12;30(6):1551–1560. doi: 10.1021/bi00220a016. [DOI] [PubMed] [Google Scholar]
  13. Czop J., Nussenzweig V. Studies on the mechanism of solubilization of immune precipitates by serum. J Exp Med. 1976 Mar 1;143(3):615–630. doi: 10.1084/jem.143.3.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dalmasso A. P., Müller-Eberhard H. J. Hemolytic activity of lipoprotein-depleted serum and the effect of certain anions on complement. J Immunol. 1966 Nov;97(5):680–685. [PubMed] [Google Scholar]
  15. Dalmasso A. P., Müller-Eberhard H. J. Physico-chemical characteristics of the third and fourth component of complement after dissociation from complement-cell complexes. Immunology. 1967 Sep;13(3):293–305. [PMC free article] [PubMed] [Google Scholar]
  16. Dodds A. W., Law S. K., Porter R. R. The origin of the very variable haemolytic activities of the common human complement component C4 allotypes including C4-A6. EMBO J. 1985 Sep;4(9):2239–2244. doi: 10.1002/j.1460-2075.1985.tb03920.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dodds A. W., Law S. K., Porter R. R. The purification and properties of some less common allotypes of the fourth component of human complement. Immunogenetics. 1986;24(5):279–285. doi: 10.1007/BF00395532. [DOI] [PubMed] [Google Scholar]
  18. Dodds A. W., Law S. K. Structural basis of the binding specificity of the thioester-containing proteins, C4, C3 and alpha-2-macroglobulin. Complement. 1988;5(2):89–97. doi: 10.1159/000463039. [DOI] [PubMed] [Google Scholar]
  19. Dodds A. W., Law S. K. The complement component C4 of mammals. Biochem J. 1990 Jan 15;265(2):495–502. doi: 10.1042/bj2650495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Erickson B. W., Khan S. A. Synthetic lactam and thiolactone models of protein metastable binding sites. Ann N Y Acad Sci. 1983;421:167–177. doi: 10.1111/j.1749-6632.1983.tb18107.x. [DOI] [PubMed] [Google Scholar]
  21. Gordon J., Whitehead H. R., Wormall A. The Action of Ammonia on Complement. The Fourth Component. Biochem J. 1926;20(5):1028–1035. doi: 10.1042/bj0201028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Harpel P. C., Hayes M. B., Hugli T. E. Heat-induced fragmentation of human alpha 2-macroglobulin. J Biol Chem. 1979 Sep 10;254(17):8669–8678. [PubMed] [Google Scholar]
  23. Harrison R. A., Thomas M. L., Tack B. F. Sequence determination of the thiolester site of the fourth component of human complement. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7388–7392. doi: 10.1073/pnas.78.12.7388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hostetter M. K., Thomas M. L., Rosen F. S., Tack B. F. Binding of C3b proceeds by a transesterification reaction at the thiolester site. Nature. 1982 Jul 1;298(5869):72–75. doi: 10.1038/298072b0. [DOI] [PubMed] [Google Scholar]
  25. Howard J. B. Reactive site in human alpha 2-macroglobulin: circumstantial evidence for a thiolester. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2235–2239. doi: 10.1073/pnas.78.4.2235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Isaac L., Isenman D. E. Structural requirements for thioester bond formation in human complement component C3. Reassessment of the role of thioester bond integrity on the conformation of C3. J Biol Chem. 1992 May 15;267(14):10062–10069. [PubMed] [Google Scholar]
  27. Isenman D. E., Kells D. I. Conformational and functional changes in the fourth component of human complement produced by nucleophilic modification and by proteolysis with C1s-. Biochemistry. 1982 Mar 16;21(6):1109–1117. doi: 10.1021/bi00535a001. [DOI] [PubMed] [Google Scholar]
  28. Isenman D. E., Kells D. I., Cooper N. R., Müller-Eberhard H. J., Pangburn M. K. Nucleophilic modification of human complement protein C3: correlation of conformational changes with acquisition of C3b-like functional properties. Biochemistry. 1981 Jul 21;20(15):4458–4467. doi: 10.1021/bi00518a034. [DOI] [PubMed] [Google Scholar]
  29. Isenman D. E., Young J. R. The molecular basis for the difference in immune hemolysis activity of the Chido and Rodgers isotypes of human complement component C4. J Immunol. 1984 Jun;132(6):3019–3027. [PubMed] [Google Scholar]
  30. Janatova J., Tack B. F., Prahl J. W. Third component of human complement: structural requirements for its function. Biochemistry. 1980 Sep 16;19(19):4479–4485. doi: 10.1021/bi00560a015. [DOI] [PubMed] [Google Scholar]
  31. Johnston R. B., Jr, Klemperer M. R., Alper C. A., Rosen F. S. The enhancement of bacterial phagocytosis by serum. The role of complement components and two cofactors. J Exp Med. 1969 Jun 1;129(6):1275–1290. doi: 10.1084/jem.129.6.1275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kan C. C., Solomon E., Belt K. T., Chain A. C., Hiorns L. R., Fey G. Nucleotide sequence of cDNA encoding human alpha 2-macroglobulin and assignment of the chromosomal locus. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2282–2286. doi: 10.1073/pnas.82.8.2282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Khan S. A., Erickson B. W. An equilibrium model of the metastable binding sites of alpha 2-macroglobulin and complement proteins C3 and C4. J Biol Chem. 1982 Oct 25;257(20):11864–11867. [PubMed] [Google Scholar]
  34. Kim Y. U., Carroll M. C., Isenman D. E., Nonaka M., Pramoonjago P., Takeda J., Inoue K., Kinoshita T. Covalent binding of C3b to C4b within the classical complement pathway C5 convertase. Determination of amino acid residues involved in ester linkage formation. J Biol Chem. 1992 Feb 25;267(6):4171–4176. [PubMed] [Google Scholar]
  35. Kinoshita T., Takata Y., Kozono H., Takeda J., Hong K. S., Inoue K. C5 convertase of the alternative complement pathway: covalent linkage between two C3b molecules within the trimolecular complex enzyme. J Immunol. 1988 Dec 1;141(11):3895–3901. [PubMed] [Google Scholar]
  36. Kozono H., Kinoshita T., Kim Y. U., Takata-Kozono Y., Tsunasawa S., Sakiyama F., Takeda J., Hong K., Inoue K. Localization of the covalent C3b-binding site on C4b within the complement classical pathway C5 convertase, C4b2a3b. J Biol Chem. 1990 Aug 25;265(24):14444–14449. [PubMed] [Google Scholar]
  37. 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]
  38. Lambris J. D., Lao Z., Pang J., Alsenz J. Third component of trout complement. cDNA cloning and conservation of functional sites. J Immunol. 1993 Dec 1;151(11):6123–6134. [PubMed] [Google Scholar]
  39. Law S. K., Dodds A. W., Porter R. R. A comparison of the properties of two classes, C4A and C4B, of the human complement component C4. EMBO J. 1984 Aug;3(8):1819–1823. doi: 10.1002/j.1460-2075.1984.tb02052.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Law S. K., Levine R. P. Interaction between the third complement protein and cell surface macromolecules. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2701–2705. doi: 10.1073/pnas.74.7.2701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Law S. K., Minich T. M., Levine R. P. Binding reaction between the third human complement protein and small molecules. Biochemistry. 1981 Dec 22;20(26):7457–7463. doi: 10.1021/bi00529a020. [DOI] [PubMed] [Google Scholar]
  42. Law S. K. Non-enzymic activation of the covalent binding reaction of the complement protein C3. Biochem J. 1983 May 1;211(2):381–389. doi: 10.1042/bj2110381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Müller-Eberhard H. J., Polley M. J., Calcott M. A. Formation and functional significance of a molecular complex derived from the second and the fourth component of human complement. J Exp Med. 1967 Feb 1;125(2):359–380. doi: 10.1084/jem.125.2.359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Nagase H., Harris E. D., Jr Ovostatin: a novel proteinase inhibitor from chicken egg white. II. Mechanism of inhibition studied with collagenase and thermolysin. J Biol Chem. 1983 Jun 25;258(12):7490–7498. [PubMed] [Google Scholar]
  45. Nagase H., Harris E. D., Jr, Woessner J. F., Jr, Brew K. Ovostatin: a novel proteinase inhibitor from chicken egg white. I. Purification, physicochemical properties, and tissue distribution of ovostatin. J Biol Chem. 1983 Jun 25;258(12):7481–7489. [PubMed] [Google Scholar]
  46. Nicholson A., Brade V., Lee G. D., Shin H. S., Mayer M. M. Kinetic studies of the formation of the properdin system enzymes on zymosan: evidence that nascent C3b controls the rate of assembly. J Immunol. 1974 Mar;112(3):1115–1123. [PubMed] [Google Scholar]
  47. Nielsen K. L., Sottrup-Jensen L. Evidence from sequence analysis that hen egg-white ovomacroglobulin (ovostatin) is devoid of an internal beta-Cys-gamma-Glu thiol ester. Biochim Biophys Acta. 1993 Mar 5;1162(1-2):230–232. doi: 10.1016/0167-4838(93)90153-i. [DOI] [PubMed] [Google Scholar]
  48. Nonaka M., Nakayama K., Yeul Y. D., Takahashi M. Complete nucleotide and derived amino acid sequences of the fourth component of mouse complement (C4). Evolutionary aspects. J Biol Chem. 1985 Sep 15;260(20):10936–10943. [PubMed] [Google Scholar]
  49. O'Neill G. J., Yang S. Y., Dupont B. Two HLA-linked loci controlling the fourth component of human complement. Proc Natl Acad Sci U S A. 1978 Oct;75(10):5165–5169. doi: 10.1073/pnas.75.10.5165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. PILLEMER L., LEPOW I. H., BLUM L. The requirement for a hydrazine-sensitive serum factor and heat-labile serum factors in the inactivation of human C'3 by zymosan. J Immunol. 1953 Nov;71(5):339–345. [PubMed] [Google Scholar]
  51. Pangburn M. K., Müller-Eberhard H. J. Relation of putative thioester bond in C3 to activation of the alternative pathway and the binding of C3b to biological targets of complement. J Exp Med. 1980 Oct 1;152(4):1102–1114. doi: 10.1084/jem.152.4.1102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Pangburn M. K. Spontaneous thioester bond formation in alpha 2-macroglobulin, C3 and C4. FEBS Lett. 1992 Aug 24;308(3):280–282. doi: 10.1016/0014-5793(92)81293-u. [DOI] [PubMed] [Google Scholar]
  53. Porter R. R. Complement polymorphism, the major histocompatibility complex and associated diseases: a speculation. Mol Biol Med. 1983 Jul;1(1):161–168. [PubMed] [Google Scholar]
  54. Ren X. D., Dodds A. W., Enghild J. J., Chu C. T., Law S. K. The effect of residue 1106 on the thioester-mediated covalent binding reaction of human complement protein C4 and the monomeric rat alpha-macroglobulin alpha 1 I3. FEBS Lett. 1995 Jul 10;368(1):87–91. doi: 10.1016/0014-5793(95)00606-a. [DOI] [PubMed] [Google Scholar]
  55. Ren X. D., Dodds A. W., Law S. K. The thioester and isotypic sites of complement component C4 in sheep and cattle. Immunogenetics. 1993;37(2):120–128. doi: 10.1007/BF00216835. [DOI] [PubMed] [Google Scholar]
  56. Sahu A., Kozel T. R., Pangburn M. K. Specificity of the thioester-containing reactive site of human C3 and its significance to complement activation. Biochem J. 1994 Sep 1;302(Pt 2):429–436. doi: 10.1042/bj3020429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Sahu A., Pangburn M. K. Covalent attachment of human complement C3 to IgG. Identification of the amino acid residue involved in ester linkage formation. J Biol Chem. 1994 Nov 18;269(46):28997–29002. [PubMed] [Google Scholar]
  58. Sahu A., Pangburn M. K. Investigation of mechanism-based inhibitors of complement targeting the activated thioester of human C3. Biochem Pharmacol. 1996 Mar 22;51(6):797–804. doi: 10.1016/0006-2952(95)02398-4. [DOI] [PubMed] [Google Scholar]
  59. Sahu A., Pangburn M. K. Tyrosine is a potential site for covalent attachment of activated complement component C3. Mol Immunol. 1995 Jul;32(10):711–716. doi: 10.1016/0161-5890(95)98933-f. [DOI] [PubMed] [Google Scholar]
  60. Sepp A., Dodds A. W., Anderson M. J., Campbell R. D., Willis A. C., Law S. K. Covalent binding properties of the human complement protein C4 and hydrolysis rate of the internal thioester upon activation. Protein Sci. 1993 May;2(5):706–716. doi: 10.1002/pro.5560020502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Sim R. B., Sim E. Autolytic fragmentation of complement components C3 and C4 under denaturing conditions, a property shared with alpha 2-macroglobulin. Biochem J. 1981 Jan 1;193(1):129–141. doi: 10.1042/bj1930129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Stossel T. P., Field R. J., Gitlin J. D., Alper C. A., Rosen F. S. The opsonic fragment of the third component of human complement (C3). J Exp Med. 1975 Jun 1;141(6):1329–1347. doi: 10.1084/jem.141.6.1329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Tack B. F., Harrison R. A., Janatova J., Thomas M. L., Prahl J. W. Evidence for presence of an internal thiolester bond in third component of human complement. Proc Natl Acad Sci U S A. 1980 Oct;77(10):5764–5768. doi: 10.1073/pnas.77.10.5764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Theofilopoulos A. N., Dixon F. J., Bokisch V. A. Binding of soluble immune complexes to human lymphoblastoid cells. I. Characterization of receptors for IgG Fc and complement and description of the binding mechanism. J Exp Med. 1974 Oct 1;140(4):877–894. doi: 10.1084/jem.140.4.877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Venkatesh Y. P., Levine R. P. The esterase-like activity of covalently bound human third complement protein. Mol Immunol. 1988 Sep;25(9):821–828. doi: 10.1016/0161-5890(88)90118-6. [DOI] [PubMed] [Google Scholar]
  66. de Bruijn M. H., Fey G. H. Human complement component C3: cDNA coding sequence and derived primary structure. Proc Natl Acad Sci U S A. 1985 Feb;82(3):708–712. doi: 10.1073/pnas.82.3.708. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES