A De Novo Protein Binding Pair by Computational Design and Directed Evolution

Mol Cell. 2011 Apr 22;42(2):250-60. doi: 10.1016/j.molcel.2011.03.010. Epub 2011 Mar 31.

Abstract

The de novo design of protein-protein interfaces is a stringent test of our understanding of the principles underlying protein-protein interactions and would enable unique approaches to biological and medical challenges. Here we describe a motif-based method to computationally design protein-protein complexes with native-like interface composition and interaction density. Using this method we designed a pair of proteins, Prb and Pdar, that heterodimerize with a Kd of 130 nM, 1000-fold tighter than any previously designed de novo protein-protein complex. Directed evolution identified two point mutations that improve affinity to 180 pM. Crystal structures of an affinity-matured complex reveal binding is entirely through the designed interface residues. Surprisingly, in the in vitro evolved complex one of the partners is rotated 180° relative to the original design model, yet still maintains the central computationally designed hotspot interaction and preserves the character of many peripheral interactions. This work demonstrates that high-affinity protein interfaces can be created by designing complementary interaction surfaces on two noninteracting partners and underscores remaining challenges.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Binding Sites
  • Chemistry Techniques, Analytical
  • Computer-Aided Design*
  • Models, Molecular
  • Molecular Weight
  • Mutation
  • Protein Binding
  • Protein Conformation
  • Protein Interaction Domains and Motifs*
  • Protein Interaction Mapping*
  • Protein Multimerization
  • Proteins / chemistry*
  • Proteins / genetics
  • Proteins / metabolism
  • Surface Properties

Substances

  • Proteins