Structural mimicry of a native protein by a minimized binding domain

Department of Protein Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080

Edited by Peter G. Wolynes, University of Illinois, Urbana, IL, and approved June 26, 1997 (received for review June 2, 1997)

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.

Footnotes

↵ * To whom reprint requests should be addressed.
This paper was submitted directly (Track II) to the Proceedings Office.
Abbreviations: 2D, two dimensional; COSY, correlated spectroscopy; NOESY, nuclear Overhauser effect spectroscopy.
Data deposition: The atomic coordinates for the ensembles and minimized mean structures have been deposited in the Protein Data Bank, Biochemistry Department, Brookhaven National Laboratory, Upton, NY 11973 (references 1Zda and 1Zdb for Z38 and 1Zdc and 1Zdd for Z34c).