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High-affinity binding to staphylococcal protein A by an engineered dimeric Affibody molecule.

Authors
  • Lindborg, Malin
  • Dubnovitsky, Anatoly
  • Olesen, Kenneth
  • Björkman, Tomas
  • Abrahmsén, Lars
  • Feldwisch, Joachim
  • Härd, Torleif
Type
Published Article
Journal
Protein Engineering Design and Selection
Publisher
Oxford University Press
Publication Date
Oct 01, 2013
Volume
26
Issue
10
Pages
635–644
Identifiers
DOI: 10.1093/protein/gzt038
PMID: 23924760
Source
Medline
Keywords
License
Unknown

Abstract

Affibody molecules are engineered binding proteins, in which the three-helix bundle motif of the Z domain derived from protein A is used as a scaffold for sequence variation. We used phage display to select Affibody binders to staphylococcal protein A itself. The best binder, called ZpA963, binds with similar affinity and kinetics to the five homologous E, D, A, B and C domains of protein A, and to a five-domain protein A construct with an average dissociation constant, K(D), of ~20 nM. The structure of ZpA963 in complex with the Z domain shows that it interacts with a surface on Z that is identical in the five protein A domains, which explains the multi-domain affinity. This property allows for high-affinity binding by dimeric Affibody molecules that simultaneously engage two protein A domains in a complex. We studied two ZpA963 dimers in which the subunits were linked by a C-terminal disulfide in a symmetric dimer or head-to-tail in a fusion protein, respectively. The dimers both bind protein A with high affinity, very slow off-rates and with saturation-dependent kinetics that can be understood in terms of dimer binding to multiple sites. The head-to-tail (ZpA963)2htt dimer binds with an off-rate of k(off) ≤ 5 × 10(-6) s(-1) and an estimated K(D) ≤ 16 pM. The results illustrate how dimers of selected monomer binding proteins can provide an efficient route for engineering of high-affinity binders to targets that contain multiple homologous domains or repeated structural units.

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