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. 1996 Jun 11;93(12):5688–5692. doi: 10.1073/pnas.93.12.5688

Minimizing a binding domain from protein A.

A C Braisted 1, J A Wells 1
PMCID: PMC39121  PMID: 8650153

Abstract

We present a systematic approach to minimizing the Z-domain of protein A, a three-helix bundle (59 residues total) that binds tightly (Kd = 10 nM) to the Fc portion of an immunoglobin IgG1. Despite the fact that all the contacts seen in the x-ray structure of the complex with the IgG are derived from residues in the first two helices, when helix 3 is deleted, binding affinity is reduced > 10(5)-fold (Kd > 1 mM). By using structure-based design and phage display methods, we have iteratively improved the stability and binding affinity for a two-helix derivative, 33 residues in length, such that it binds IgG1, with a Kd of 43 nM. This was accomplished by stepwise selection of random mutations from three regions of the truncated Z-peptide: the 4 hydrophobic residues from helix 1 and helix 2 that contacted helix 3 (the exoface), followed by 5 residues between helix 1 and helix 2 (the intraface), and lastly by 19 residues at or near the interface that interacts with Fc (the interface). As selected mutations from each region were compiled (12 in total), they led to progressive increases in affinity for IgG, and concomitant increases in alpha-helical content reflecting increased stabilization of the two-helix scaffold. Thus, by sequential increases in the stability of the structure and improvements in the quality of the intermolecular contacts, one can reduce larger binding domains to smaller ones. Such mini-protein binding domains are more amenable to synthetic chemistry and thus may be useful starting points for the design of smaller organic mimics. Smaller binding motifs also provide simplified and more tractable models for understanding determinants of protein function and stability.

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Selected References

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

  1. Cedergren L., Andersson R., Jansson B., Uhlén M., Nilsson B. Mutational analysis of the interaction between staphylococcal protein A and human IgG1. Protein Eng. 1993 Jun;6(4):441–448. doi: 10.1093/protein/6.4.441. [DOI] [PubMed] [Google Scholar]
  2. Chang C. N., Rey M., Bochner B., Heyneker H., Gray G. High-level secretion of human growth hormone by Escherichia coli. Gene. 1987;55(2-3):189–196. doi: 10.1016/0378-1119(87)90279-4. [DOI] [PubMed] [Google Scholar]
  3. Clackson T., Wells J. A. In vitro selection from protein and peptide libraries. Trends Biotechnol. 1994 May;12(5):173–184. doi: 10.1016/0167-7799(94)90079-5. [DOI] [PubMed] [Google Scholar]
  4. Cunningham B. C., Lowe D. G., Li B., Bennett B. D., Wells J. A. Production of an atrial natriuretic peptide variant that is specific for type A receptor. EMBO J. 1994 Jun 1;13(11):2508–2515. doi: 10.1002/j.1460-2075.1994.tb06540.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Deisenhofer J. Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9- and 2.8-A resolution. Biochemistry. 1981 Apr 28;20(9):2361–2370. [PubMed] [Google Scholar]
  6. Djojonegoro B. M., Benedik M. J., Willson R. C. Bacteriophage surface display of an immunoglobulin-binding domain of Staphylococcus aureus protein A. Biotechnology (N Y) 1994 Feb;12(2):169–172. doi: 10.1038/nbt0294-169. [DOI] [PubMed] [Google Scholar]
  7. Doolittle R. F. Stein and Moore Award address. Reconstructing history with amino acid sequences. Protein Sci. 1992 Feb;1(2):191–200. doi: 10.1002/pro.5560010201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Epand R. M., Scheraga H. A. The influence of long-range interactions on the structure of myoglobin. Biochemistry. 1968 Aug;7(8):2864–2872. doi: 10.1021/bi00848a024. [DOI] [PubMed] [Google Scholar]
  9. Gish G., McGlone M. L., Pawson T., Adams J. A. Bacterial expression, purification and preliminary kinetic description of the kinase domain of v-fps. Protein Eng. 1995 Jun;8(6):609–614. doi: 10.1093/protein/8.6.609. [DOI] [PubMed] [Google Scholar]
  10. Gouda H., Torigoe H., Saito A., Sato M., Arata Y., Shimada I. Three-dimensional solution structure of the B domain of staphylococcal protein A: comparisons of the solution and crystal structures. Biochemistry. 1992 Oct 13;31(40):9665–9672. doi: 10.1021/bi00155a020. [DOI] [PubMed] [Google Scholar]
  11. Huston J. S., Cohen C., Maratea D., Fields F., Tai M. S., Cabral-Denison N., Juffras R., Rueger D. C., Ridge R. J., Oppermann H. Multisite association by recombinant proteins can enhance binding selectivity. Preferential removal of immune complexes from serum by immobilized truncated FB analogues of the B domain from staphylococcal protein A. Biophys J. 1992 Apr;62(1):87–91. doi: 10.1016/S0006-3495(92)81788-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Johnsson B., Löfås S., Lindquist G. Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors. Anal Biochem. 1991 Nov 1;198(2):268–277. doi: 10.1016/0003-2697(91)90424-r. [DOI] [PubMed] [Google Scholar]
  13. Karlsson R., Michaelsson A., Mattsson L. Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system. J Immunol Methods. 1991 Dec 15;145(1-2):229–240. doi: 10.1016/0022-1759(91)90331-9. [DOI] [PubMed] [Google Scholar]
  14. Kunkel T. A., Bebenek K., McClary J. Efficient site-directed mutagenesis using uracil-containing DNA. Methods Enzymol. 1991;204:125–139. doi: 10.1016/0076-6879(91)04008-c. [DOI] [PubMed] [Google Scholar]
  15. Li B., Tom J. Y., Oare D., Yen R., Fairbrother W. J., Wells J. A., Cunningham B. C. Minimization of a polypeptide hormone. Science. 1995 Dec 8;270(5242):1657–1660. doi: 10.1126/science.270.5242.1657. [DOI] [PubMed] [Google Scholar]
  16. Nilsson B., Moks T., Jansson B., Abrahmsén L., Elmblad A., Holmgren E., Henrichson C., Jones T. A., Uhlén M. A synthetic IgG-binding domain based on staphylococcal protein A. Protein Eng. 1987 Feb-Mar;1(2):107–113. doi: 10.1093/protein/1.2.107. [DOI] [PubMed] [Google Scholar]
  17. Provencher S. W., Glöckner J. Estimation of globular protein secondary structure from circular dichroism. Biochemistry. 1981 Jan 6;20(1):33–37. doi: 10.1021/bi00504a006. [DOI] [PubMed] [Google Scholar]
  18. Smith G. P. Surface presentation of protein epitopes using bacteriophage expression systems. Curr Opin Biotechnol. 1991 Oct;2(5):668–673. doi: 10.1016/0958-1669(91)90032-z. [DOI] [PubMed] [Google Scholar]
  19. Tsou C. L. Conformational flexibility of enzyme active sites. Science. 1993 Oct 15;262(5132):380–381. doi: 10.1126/science.8211158. [DOI] [PubMed] [Google Scholar]
  20. Uhlén M., Forsberg G., Moks T., Hartmanis M., Nilsson B. Fusion proteins in biotechnology. Curr Opin Biotechnol. 1992 Aug;3(4):363–369. doi: 10.1016/0958-1669(92)90164-e. [DOI] [PubMed] [Google Scholar]

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