Structural Basis for Molecular Recognition in an Affibody:Affibody Complex

Abstract

Affibody molecules constitute a class of engineered binding proteins based on the 58-residue three-helix bundle Z domain derived from staphylococcal protein A (SPA). Affibody proteins are selected as binders to target proteins by phage display of combinatorial libraries in which typically 13 side-chains on the surface of helices 1 and 2 in the Z domain have been randomized. The Z Taq:anti-Z Taq affibody–affibody complex, consisting of Z Taq, originally selected as a binder to Taq DNA polymerase, and anti-Z Taq, selected as binder to Z Taq, is formed with a dissociation constant K d ∼100 nM. We have determined high-precision solution structures of free Z Taq and anti-Z Taq, and the Z Taq:anti-Z Taq complex under identical experimental conditions (25 °C in 50 mM NaCl with 20 mM potassium phosphate buffer at pH 6.4). The complex is formed with helices 1 and 2 of anti-Z Taq in perpendicular contact with helices 1 and 2 of Z Taq. The interaction surface is large (∼1670 Å 2) and unusually non-polar (70 %) compared to other protein–protein complexes. It involves all varied residues on anti-Z Taq, most corresponding ( Taq DNA polymerase binding) side-chains on Z Taq, and several additional side-chain and backbone contacts. Other notable features include a substantial rearrangement (induced fit) of aromatic side-chains in Z Taq upon binding, a close contact between glycine residues in the two subunits that might involve aliphatic glycine Hα to backbone carbonyl hydrogen bonds, and four hydrogen bonds made by the two guanidinium N ηH 2 groups of an arginine side-chain. Comparisons of the present structure with other data for affibody proteins and the Z domain suggest that intrinsic binding properties of the originating SPA surface might be inherited by the affibody binders. A thermodynamic characterization of Z Taq and anti-Z Taq is presented in an accompanying paper.