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

Journal article
Authors M. Lindborg
A. Dubnovitsky
Kenneth Olesen
T. Bjorkman
L. Abrahmsen
J. Feldwisch
Torleif Härd
Published in Protein Engineering Design & Selection
Volume 26
Issue 10
Pages 635-644
ISSN 1741-0126
Publication year 2013
Published at Institute of Biomedicine
Pages 635-644
Language en
Keywords molecular recognition, phage display, protein engineering, proteinprotein interactions, protein, BACTERIAL RECEPTOR DOMAIN, STRUCTURAL BASIS, COMPLEX, STABILIZATION, RECOGNITION, LIBRARIES, LAGLIO F, 1995, JOURNAL OF BIOMOLECULAR NMR, V6, P277, UDIER FW, 1990, METHODS IN ENZYMOLOGY, V185, P60, ATES OF AMERICA, V107, P15039
Subject categories Clinical Medicine


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)(2)htt 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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