2m5a

Publication Abstract from PubMed

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, KD, of approximately 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 koff </= 5 x 10-6 s-1 and an estimated KD </= 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.

High-affinity binding to staphylococcal protein A by an engineered dimeric Affibody molecule.,Lindborg M, Dubnovitsky A, Olesen K, Bjorkman T, Abrahmsen L, Feldwisch J, Hard T Protein Eng Des Sel. 2013 Aug 7. PMID:23924760^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.