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)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.
Affibody AB, Gunnar Asplunds Allé 24, SE-171 63 Solna, Sweden.
Malin
M
Lindborg
Anatoly
A
Dubnovitsky
Kenneth
K
Olesen
Tomas
T
Björkman
Lars
L
Abrahmsén
Joachim
J
Feldwisch
Torleif
T
Härd
D000595:Amino Acid Sequence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding; D015202:Protein Engineering; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000378:metabolism*
D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A
molecular recognition; phage display; protein engineering; protein structure; protein–protein interactions