Structural basis for exquisite specificity of affinity clamps, synthetic binding proteins generated through directed domain-interface evolution

Abstract

We have established a new protein-engineering strategy termed "directed domain-interface evolution" that generates a binding site by linking two protein domains and then optimizing the interface between them. Using this strategy, we have generated synthetic two-domain "affinity clamps" using PDZ and fibronectin type III (FN3) domains as the building blocks. While these affinity clamps all had significantly higher affinity toward a target peptide than the underlying PDZ domain, two distinct types of affinity clamps were found in terms of target specificity. One type conserved the specificity of the parent PDZ domain, and the other increased the specificity dramatically. Here, we characterized their specificity profiles using peptide phage-display libraries and scanning mutagenesis, which suggested a significantly enlarged recognition site of the high-specificity affinity clamps. The crystal structure of a high-specificity affinity clamp showed extensive contacts with a portion of the peptide ligand that is not recognized by the parent PDZ domain, thus rationalizing the improvement of the specificity of the affinity clamp. A comparison with another affinity clamp structure showed that, although both had extensive contacts between PDZ and FN3 domains, they exhibited a large offset in the relative position of the two domains. Our results indicate that linked domains could rapidly fuse and evolve as a single functional module, and that the inherent plasticity of domain interfaces allows for the generation of diverse active-site topography. These attributes of directed domain-interface evolution provide facile means to generate synthetic proteins with a broad range of functions.

Fig. 1

Directed domain-interface engineering produces new protein functions. (a) Scheme of domain-interface engineering. Two domains are connected and a new recognition site is produced at the interface of the newly connected domains. (b) Sequences and binding parameters of affinity clamps. The data were taken from Huang et al.

Fig. 2

Specificity profiles of affinity clamps. (a) Sequences of the C-terminal peptides of ARVCF and δ-catenin. (b) and (c) Weblogo ; representations of specificity profiles for ePDZ-b family clamps. The sequence conservation at each position is represented by the overall height of the stack. The height of symbols within the stack indicates the relative frequency of each amino. The grey horizontal bars indicate positions that were kept constant in the libraries. (b) shows the results with the hepta-peptide library and (c) with the penta-peptide library. The library design is shown above the profiles. (d) Effects of Ala substitution at the indicated positions of the ARVCF peptide as measured by SPR. The values for the free energy changes (ΔΔ G) are shown on the left axis and those for the K_d ratio (K_d ^mutant) are shown on the right axis.

Fig. 3

X-ray crystal structures of ePDZ-b1 and ePDZ-a in complex with the ARVCF peptide. (a) and (b) Ribbon (left panel) and surface (right panel) representations of the overall structures of ePDZ-b1 (a) and ePDZ-a (b). The PDZ and FN3 portions and the peptide are shown in gray, cyan and yellow, respectively. In ePDZ-b1, the linker of is shown in orange. The weighted 2F_obs-F_calc electron density map of the linker segment contoured at 1.5 σ is shown in the enlarged box. In ePDZ-a, the missing residues for the linker segment are indicated with dashed lines. The concave surface around the linker area is highlighted in red circle. (c) Superposition of the two structures on the PDZ portion. ePDZ-b1 is colored as in (a); ePDZ-a is colored in light brown for PDZ and dark blue for FN3. The peptide was omitted for clarity. (d) Interactions of the FN3 loops with the PDZ/peptide complex in ePDZ-b1 (left panel) and ePDZ-a (right panel). The three FN3 loops (BC, DE and FG loops) are shown as sticks in blue, cyan and red, respectively. The surface of the PDZ portion is shown in gray, and the peptide as yellow spheres. In the bottom panel, the surfaces of the PDZ and peptide portions in contact with the BC, DE and FG loops are shown in blue, cyan and red, respectively, and those in contact with both BC and FG loops are in magenta. The peptide surfaces without FN3 contact are shown in yellow, and the black lines enclose the bound peptide. The N- and C-termini of the peptide are also labeled.

Fig. 4

Interactions of ARVCF peptide with affinity clamps. (a) Surface burial of peptide residues by affinity clamps and Erbin PDZ. (b) Comparison of the peptide binding to the underlying PDZ domain. The ePDZ-a and ePDZ-b1 structures are superposed on the PDZ portion. The PDZ portion is shown in surface representation and the peptide is shown in stick. PDZ of ePDZ-b1 is colored in gray and peptide in yellow; PDZ in ePDZ-a is colored in light brown and peptide in green. (c) Comparison of N-terminal three residues interacting with ePDZ-b1 (left panel) and ePDZ-a (right panel). The peptide and affinity clamps are colored as in Fig. 3. The orientation of the two structures are flipped by 180° to obtain a clear view of the N-terminal residues. Residues involved in H-bonds (dashed black lines) on the affinity clamps are shown in sticks with atomic coloring for nitrogen and oxygen. (d) A close view of H-bond pattern of the C-terminal five residues.