11 We dissected the coupled folding and binding thermodynamics, and suggested that the origin of moderate affinity (dissociation constant K d ∼ 1 μM) of Z SPA-1 is the entropic penalty associated with conformational stabilization upon binding, rather than defects in the binding interface or the poor folding stability of Z SPA-1. On the other hand, it was possible to deduce the structural basis for the selection of Z SPA-1 as a Z-domain binder. Overall, these studies illustrate how seemingly simple structural topologies may conceal a great deal of complexity that arises when coupled folding/binding and induced fit operate simultaneously in molecular recognition. In previous work, we examined structural and thermodynamic properties of the complex between the Z SPA-1 affibody and its target, the Z domain.10., 11., 12., 13. 5 Binders to a large number of protein targets, with dissociation constants in the range micromolar to approximately nanomolar have been reported.6., 7., 8., 9. Thirteen residues in the binding interface are randomized to create a combinatorial library from which specific binders can be selected using phage display. 4 The Fc binding surface of the Z domain is used as template for creation of affibody binding proteins. 3 The Z domain shows, like the other five homologous SPA domains, antibody Fc binding activity, but it lacks the Fab affinity found in the parental domain. We are using a class of engineered binding proteins named affibody proteins as models in studies of the mechanisms of molecular recognition and protein–protein binding interactions.Īffibody binders are based on the three-helix bundle scaffold of the Z domain, which was originally engineered from the B domain of staphylococcal protein A (SPA). Beside their practical applicability, engineered proteins provide attractive model systems for studying fundamental aspects of structure, stability and function. The high levels of binding affinity and specificity of antibodies have been exploited extensively in medicine and biotechnology, and the large range of applications has motivated development of novel artificial binding proteins to be used as cost-effective alternatives or which allow for more flexibility with regard to conditions such as pH, temperature, redox potential etc.1., 2. A thermodynamic characterization of Z Taq and anti-Z Taq is presented in an accompanying paper. 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. 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. 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. The interaction surface is large (∼1670 Å 2) and unusually non-polar (70 %) compared to other protein–protein complexes. The complex is formed with helices 1 and 2 of anti-Z Taq in perpendicular contact with helices 1 and 2 of Z Taq. 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 ☌ in 50 mM NaCl with 20 mM potassium phosphate buffer at pH 6.4). 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. 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. 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).
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