An affibody in complex with a target protein: Structure and coupled folding

Abstract

Combinatorial protein engineering provides powerful means for functional selection of novel binding proteins. One class of engineered binding proteins, denoted affibodies, is based on the three-helix scaffold of the Z domain derived from staphylococcal protein A. The Z SPA-1 affibody has been selected from a phagedisplayed library as a binder to protein A. Z SPA-1 also binds with micromolar affinity to its own ancestor, the Z domain. We have characterized the Z SPA-1 affibody in its uncomplexed state and determined the solution structure of a Z:Z SPA-1 protein-protein complex. Uncomplexed Z SPA-1 behaves as an aggregation-prone molten globule, but folding occurs on binding, and the original (Z) three-helix bundle scaffold is fully formed in the complex. The structural basis for selection and strong binding is a large interaction interface with tight steric and polar/nonpolar complementarity that directly involves 10 of 13 mutated amino acid residues on Z SPA-1. We also note similarities in how the surface of the Z domain responds by induced fit to binding of Z SPA-1 and Ig Fc, respectively, suggesting that the Z SPA-1 affibody is capable of mimicking the morphology of the natural binding partner for the Z domain.protein engineering ͉ protein-protein interactions ͉ molecular recognition ͉ NMR spectroscopy ͉ induced fit T here is an interest in generating novel classes of binding proteins that can be used as an alternative to immunoglobulins in various biochemical assays and biotechnological applications. To this end, carefully chosen protein domains can be used as framework structures for combinatorial protein engineering. Affibodies constitute a class of engineered binding proteins for which the three-helix bundle Z domain is used as a scaffold. The 58-aa residue Z domain is derived from one of five homologous domains (the B domain) in Staphylococcus aureus protein A (SPA). SPA binds strongly to the Fc region of immunoglobulins, and Z was originally developed as a stabilized gene fusion partner for affinity purification of recombinant proteins by using IgG-containing resins (1). The structure of a complex between the B domain of SPA and an Fc fragment shows that the binding surface consists of residues that are exposed on helices 1 and 2, whereas helix 3 is not directly involved in binding (2). Affibodies are selected from combinatorial libraries in which typically 13 residues at the Fc-binding surface of helices 1 and 2 are randomized. Specific binders to target proteins are then identified by biopanning the phage-displayed library against desired targets (3). Several Z-based affibodies with specific proteinbinding properties have in this way been developed and used as affinity tools in a number of applications (4-7).Structural studies of engineered protein-binding domains and their complexes are of interest for methods development in biotechnology as well as for basic studies of protein-protein interactions and the mechanisms of biomolecular recognition. Here we describe the (solution) structural and biophysic...

Coupled folding–binding versus docking: A lattice model study

“…8 This and similar lattice-based models have also been used to gain insights into topics such as protein evolution, 9,10,11 prion-like conformational propagation 12,13 and protein aggregation. 14,15 Our study is inspired by recent experiments by Wahlberg et al 16 on the in vitro evolved protein Z SPA−1 . This sequence was engineered 17 from the Z domain of staphylococcal protein A, a well characterized three-helix-bundle protein.…”

“…In the Z:Z SPA−1 complex, Z SPA−1 adopts a structure similar to the solution structure of the Z domain. 16,19 However, in solution, Z SPA−1 turns out to be structurally disordered. 16 The engineered Z SPA−1 thus exhibits coupled folding-binding.…”

Crystal Structure of the Measles Virus Phosphoprotein Domain Responsible for the Induced Folding of the C-terminal Domain of the Nucleoprotein

“…The Z-domain has been shown to be able to induce the folding of another protein, namely an affibody targeted against the Z-domain (52). In the absence of the Z-domain, the affibody is a molten globule which has little ordered structure, although upon addition of the Z-domain, it folds into an ordered structure (52).…”

“…In the absence of the Z-domain, the affibody is a molten globule which has little ordered structure, although upon addition of the Z-domain, it folds into an ordered structure (52). The interface by which the Z-domain interacts with its binding partners, either the immunoglobulin Fc or the anti-Z affibody, is formed by helices ␣1 and ␣2.…”

Latest folding game results: Protein A barely frustrates computationalists

“…Biotechnologists (36), trying to make convenient fish hooks for protein purification, have created another homologue, known as Z-SPA-1, which folds upon binding to the Z domain. There is significant rearrangement at the binding interface, referred to as ''induced fit,'' largely involving side-chain reorientation (37).…”