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

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.

Dubnovitsky, AnatolySwedish University of Agricultural Sciences,Sveriges lantbruksuniversitet,Institutionen för molekylärbiologi,Department of Molecular Biology,Karolinska Institute(Swepub:slu)51034 (författare)
Olesen, Kenneth,1966Gothenburg University,Göteborgs universitet,Institutionen för biomedicin,Institute of Biomedicine(Swepub:gu)xoleke (författare)
Bjorkman, T. (författare)
Abrahmsen, L. (författare)
Feldwisch, J. (författare)
Härd, Torleif,1959Swedish University of Agricultural Sciences,Sveriges lantbruksuniversitet,Institutionen för molekylärbiologi,Department of Molecular Biology(Swepub:slu)50056 (författare)
Sveriges lantbruksuniversitetInstitutionen för molekylärbiologi (creator_code:org_t)
Sveriges lantbruksuniversitet

Ingår i:Protein Engineering Design & Selection: Oxford University Press (OUP)26:10, s. 635-6441741-01261741-0134

Av författaren/redakt...: Lindborg, M.; Dubnovitsky, Ana ...; Olesen, Kenneth, ...; Bjorkman, T.; Abrahmsen, L.; Feldwisch, J.; visa fler...; Härd, Torleif, 1 ...; visa färre...

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