2M5A

Protein A binding by an engineered Affibody molecule

Summary for 2M5A

• Entry DOI: 10.2210/pdb2m5a/pdb
• Related: 1H0T
• NMR Information: BMRB: 19043
• Descriptor: Immunoglobulin G-binding protein A, ZpA963 (2 entities in total)
• Functional Keywords: binding protein, protein engineering, protein a, z domain, affibody molecule, protein binding
• Biological source: Staphylococcus aureus; More
• Cellular location: Secreted, cell wall; Peptidoglycan-anchor (Potential): P38507
• Total number of polymer chains: 2
• Total formula weight: 13042.39

Authors

Hard, T. (deposition date: 2013-02-19, release date: 2013-08-21, Last modification date: 2024-05-01)

Primary citation

Lindborg, M.,Dubnovitsky, A.,Olesen, K.,Bjorkman, T.,Abrahmsen, L.,Feldwisch, J.,Hard, T.
High-affinity binding to staphylococcal protein A by an engineered dimeric Affibody molecule.
Protein Eng.Des.Sel., 26:635-644, 2013

PubMed 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.

PubMed: 23924760
DOI: 10.1093/protein/gzt038

Experimental method

SOLUTION NMR