Structural Basis for Molecular Recognition in an Affibody:Affibody Complex

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 ZTaq:anti-ZTaq affibody–affibody complex, consisting of ZTaq, originally selected as a binder to Taq DNA polymerase, and anti-ZTaq, selected as binder to ZTaq, is formed with a dissociation constant Kd ∼100 nM. We have determined high-precision solution structures of free ZTaq and anti-ZTaq, and the ZTaq:anti-ZTaq 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-ZTaq in perpendicular contact with helices 1 and 2 of ZTaq. The interaction surface is large (∼1670 Å2) and unusually non-polar (70 %) compared to other protein–protein complexes. It involves all varied residues on anti-ZTaq, most corresponding (Taq DNA polymerase binding) side-chains on ZTaq, and several additional side-chain and backbone contacts. Other notable features include a substantial rearrangement (induced fit) of aromatic side-chains in ZTaq 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 NηH2 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 ZTaq and anti-ZTaq is presented in an accompanying paper.