Structural mimicry of a native protein by a minimized binding domain

Abstract

The affinity between molecules depends both on the nature and presentation of the contacts. Here, we observe coupling of functional and structural elements when a protein binding domain is evolved to a smaller functional mimic. Previously, a 38-residue form of the 59-residue B-domain of protein A, termed Z38, was selected by phage display. Z38 contains 13 mutations and binds IgG only 10-fold weaker than the native B-domain. We present the solution structure of Z38 and show that it adopts a tertiary structure remarkably similar to that observed for the first two helices of B-domain in the B-domain/Fc complex [Deisenhofer, J. (1981) Biochemistry 20, 2361-2370], although it is significantly less stable. Based on this structure, we have improved on Z38 by designing a 34-residue disulfide-bonded variant (Z34C) that has dramatically enhanced stability and binds IgG with 9-fold higher affinity. The improved stability of Z34C led to NMR spectra with much greater chemical shift dispersion, resulting in a more precisely determined structure. Z34C, like Z38, has a structure virtually identical to the equivalent region from native protein A domains. The well-defined hydrophobic core of Z34C reveals key structural features that have evolved in this small, functional domain. Thus, the stabilized two-helix peptide, about half the size and having one-third of the remaining residues altered, accurately mimics both the structure and function of the native domain.

Figure 1

(A) CD spectra at 8°C (pH 7.5) and (B) temperature dependence of the ellipticity at 222 nm (θ₂₂₂) for Z-domain (▴), Z38 (▾), and Z34C (•).

Figure 2

Overlay of the fingerprint region of COSY spectra for Z38 (black) and Z34C (red). Spectra were acquired under identical conditions at 8°C (pH 5.1). Selected crosspeaks are labeled for Z34C; arrows show the location of the same crosspeak in Z38. Complete ¹H resonance assignments are available from the authors upon request.

Figure 3

Amide region of the 2D NOESY spectra of (A) Z38 and (B) Z34C acquired with a 150-ms mixing time. NOEs involving neighboring amide protons from residues in helix-2 are indicated.

Figure 4

NMR structure ensembles for (A) Z38 and (B) Z34C. In each case, 24 models are shown aligned using the backbone atoms of residues 10–36. Shown also are the coordinates for residues 7–38 from the crystal structure of the B-domain/Fc complex (red) (6). The disordered N-terminal residues 1–5 from Z38 are colored gray. Side chains from the hydrophobic core of Z34C are shown (Phe-6, Cys-10, Phe-14, Ala-17, Leu-18, Leu-23, Ile-32, Ile-35, and Cys-39). (C) The minimized mean structure of Z34C showing all side chains colored by amino acid type: hydrophobic (yellow), positively charged (blue), negatively charged (red), and polar (green); only those that are labeled are well defined in the NMR ensemble with χ1 angular order parameters >0.9. Note that only the χ1 = −60° orientation for Phe-14 is shown, but this side chain also exists in the χ1 = 180° conformation in solution. (D) Same as C, but rotated 90° with the Fc binding surface on top.