Crystal structure of an in vitro affinity- and specificity-matured anti-testosterone Fab in complex with testosterone

Jarkko Valjakka, Ari Hemminki, Seija Niemi, Hans Söderlund, Kristiina Takkinen, Juha Rouvinen

Research output: Contribution to journalArticleScientificpeer-review

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Abstract

A highly selective, high affinity recombinant anti-testosterone Fab fragment has been generated by stepwise optimization of the complementarity-determining regions (CDRs) by random mutagenesis and phage display selection of a monoclonal antibody (3-C4F5). The best mutant (77 Fab) was obtained by evaluating the additivity effects of different independently selected CDR mutations. The 77 Fab contains 20 mutations and has about 40-fold increased affinity (Kd = 3 × 1010 M) when compared with the wild-type (3-C4F5) Fab. To obtain structural insight into factors, which are needed to improve binding properties, we have determined the crystal structures of the mutant 77 Fab fragment with (2.15 Å) and without testosterone (2.10 Å) and compared these with previously determined wild-type structures. The overall testosterone binding of the 77 Fab is similar to that of the wild-type. The improved affinity and specificity of the 77 Fab fragment are due to more comprehensive packing of the testosterone with the protein, which is the result of small structural changes within the variable domains. Only one important binding site residue Glu-95 of the heavy chain CDR3 is mutated to alanine in the 77 Fab fragment. This mutation, originally selected from the phage library based on improved specificity, provides more free space for the testosterone D-ring. The light chain CDR1 of 77 Fab containing eight mutations has the most significant effect on the improved affinity, although it has no direct contact with the testosterone. The mutations of CDR-L1 cause a rearrangement in its conformation, leading to an overall fine reshaping of the binding site.
Original languageEnglish
Pages (from-to)44021-44027
JournalJournal of Biological Chemistry
Volume277
Issue number46
DOIs
Publication statusPublished - 2002
MoE publication typeA1 Journal article-refereed

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Testosterone
Immunoglobulin Fab Fragments
Crystal structure
Complementarity Determining Regions
Mutation
Bacteriophages
Binding Sites
Mutagenesis
Alanine
Conformations
In Vitro Techniques
Display devices
Monoclonal Antibodies
Light
Proteins

Keywords

  • antibody Fab'-fragment
  • Fab-fragment
  • testosterone
  • anti-doping

Cite this

Valjakka, Jarkko ; Hemminki, Ari ; Niemi, Seija ; Söderlund, Hans ; Takkinen, Kristiina ; Rouvinen, Juha. / Crystal structure of an in vitro affinity- and specificity-matured anti-testosterone Fab in complex with testosterone. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 46. pp. 44021-44027.
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abstract = "A highly selective, high affinity recombinant anti-testosterone Fab fragment has been generated by stepwise optimization of the complementarity-determining regions (CDRs) by random mutagenesis and phage display selection of a monoclonal antibody (3-C4F5). The best mutant (77 Fab) was obtained by evaluating the additivity effects of different independently selected CDR mutations. The 77 Fab contains 20 mutations and has about 40-fold increased affinity (Kd = 3 × 1010 M) when compared with the wild-type (3-C4F5) Fab. To obtain structural insight into factors, which are needed to improve binding properties, we have determined the crystal structures of the mutant 77 Fab fragment with (2.15 {\AA}) and without testosterone (2.10 {\AA}) and compared these with previously determined wild-type structures. The overall testosterone binding of the 77 Fab is similar to that of the wild-type. The improved affinity and specificity of the 77 Fab fragment are due to more comprehensive packing of the testosterone with the protein, which is the result of small structural changes within the variable domains. Only one important binding site residue Glu-95 of the heavy chain CDR3 is mutated to alanine in the 77 Fab fragment. This mutation, originally selected from the phage library based on improved specificity, provides more free space for the testosterone D-ring. The light chain CDR1 of 77 Fab containing eight mutations has the most significant effect on the improved affinity, although it has no direct contact with the testosterone. The mutations of CDR-L1 cause a rearrangement in its conformation, leading to an overall fine reshaping of the binding site.",
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Crystal structure of an in vitro affinity- and specificity-matured anti-testosterone Fab in complex with testosterone. / Valjakka, Jarkko; Hemminki, Ari; Niemi, Seija; Söderlund, Hans; Takkinen, Kristiina; Rouvinen, Juha.

In: Journal of Biological Chemistry, Vol. 277, No. 46, 2002, p. 44021-44027.

Research output: Contribution to journalArticleScientificpeer-review

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T1 - Crystal structure of an in vitro affinity- and specificity-matured anti-testosterone Fab in complex with testosterone

AU - Valjakka, Jarkko

AU - Hemminki, Ari

AU - Niemi, Seija

AU - Söderlund, Hans

AU - Takkinen, Kristiina

AU - Rouvinen, Juha

PY - 2002

Y1 - 2002

N2 - A highly selective, high affinity recombinant anti-testosterone Fab fragment has been generated by stepwise optimization of the complementarity-determining regions (CDRs) by random mutagenesis and phage display selection of a monoclonal antibody (3-C4F5). The best mutant (77 Fab) was obtained by evaluating the additivity effects of different independently selected CDR mutations. The 77 Fab contains 20 mutations and has about 40-fold increased affinity (Kd = 3 × 1010 M) when compared with the wild-type (3-C4F5) Fab. To obtain structural insight into factors, which are needed to improve binding properties, we have determined the crystal structures of the mutant 77 Fab fragment with (2.15 Å) and without testosterone (2.10 Å) and compared these with previously determined wild-type structures. The overall testosterone binding of the 77 Fab is similar to that of the wild-type. The improved affinity and specificity of the 77 Fab fragment are due to more comprehensive packing of the testosterone with the protein, which is the result of small structural changes within the variable domains. Only one important binding site residue Glu-95 of the heavy chain CDR3 is mutated to alanine in the 77 Fab fragment. This mutation, originally selected from the phage library based on improved specificity, provides more free space for the testosterone D-ring. The light chain CDR1 of 77 Fab containing eight mutations has the most significant effect on the improved affinity, although it has no direct contact with the testosterone. The mutations of CDR-L1 cause a rearrangement in its conformation, leading to an overall fine reshaping of the binding site.

AB - A highly selective, high affinity recombinant anti-testosterone Fab fragment has been generated by stepwise optimization of the complementarity-determining regions (CDRs) by random mutagenesis and phage display selection of a monoclonal antibody (3-C4F5). The best mutant (77 Fab) was obtained by evaluating the additivity effects of different independently selected CDR mutations. The 77 Fab contains 20 mutations and has about 40-fold increased affinity (Kd = 3 × 1010 M) when compared with the wild-type (3-C4F5) Fab. To obtain structural insight into factors, which are needed to improve binding properties, we have determined the crystal structures of the mutant 77 Fab fragment with (2.15 Å) and without testosterone (2.10 Å) and compared these with previously determined wild-type structures. The overall testosterone binding of the 77 Fab is similar to that of the wild-type. The improved affinity and specificity of the 77 Fab fragment are due to more comprehensive packing of the testosterone with the protein, which is the result of small structural changes within the variable domains. Only one important binding site residue Glu-95 of the heavy chain CDR3 is mutated to alanine in the 77 Fab fragment. This mutation, originally selected from the phage library based on improved specificity, provides more free space for the testosterone D-ring. The light chain CDR1 of 77 Fab containing eight mutations has the most significant effect on the improved affinity, although it has no direct contact with the testosterone. The mutations of CDR-L1 cause a rearrangement in its conformation, leading to an overall fine reshaping of the binding site.

KW - antibody Fab'-fragment

KW - Fab-fragment

KW - testosterone

KW - anti-doping

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M3 - Article

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EP - 44027

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

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ER -