Tracking molecular recognition at the atomic level with a new protein scaffold based on the OB-fold

PLoS One. 2014 Jan 20;9(1):e86050. doi: 10.1371/journal.pone.0086050. eCollection 2014.


The OB-fold is a small, versatile single-domain protein binding module that occurs in all forms of life, where it binds protein, carbohydrate, nucleic acid and small-molecule ligands. We have exploited this natural plasticity to engineer a new class of non-immunoglobulin alternatives to antibodies with unique structural and biophysical characteristics. We present here the engineering of the OB-fold anticodon recognition domain from aspartyl tRNA synthetase taken from the thermophile Pyrobaculum aerophilum. For this single-domain scaffold we have coined the term OBody. Starting from a naïve combinatorial library, we engineered an OBody with 3 nM affinity for hen egg-white lysozyme, by optimising the affinity of a naïve OBody 11,700-fold over several affinity maturation steps, using phage display. At each maturation step a crystal structure of the engineered OBody in complex with hen egg-white lysozyme was determined, showing binding elements in atomic detail. These structures have given us an unprecedented insight into the directed evolution of affinity for a single antigen on the molecular scale. The engineered OBodies retain the high thermal stability of the parental OB-fold despite mutation of up to 22% of their residues. They can be expressed in soluble form and also purified from bacteria at high yields. They also lack disulfide bonds. These data demonstrate the potential of OBodies as a new scaffold for the engineering of specific binding reagents and provide a platform for further development of future OBody-based applications.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Anticodon / metabolism
  • Archaeal Proteins / chemistry*
  • Archaeal Proteins / genetics
  • Archaeal Proteins / metabolism*
  • Aspartate-tRNA Ligase / chemistry*
  • Aspartate-tRNA Ligase / genetics
  • Aspartate-tRNA Ligase / metabolism*
  • Binding Sites
  • Chickens
  • Crystallography, X-Ray
  • Models, Molecular
  • Molecular Sequence Data
  • Muramidase / metabolism
  • Protein Conformation
  • Protein Engineering*
  • Protein Stability
  • Pyrobaculum / chemistry*
  • Pyrobaculum / genetics
  • Pyrobaculum / metabolism*


  • Anticodon
  • Archaeal Proteins
  • hen egg lysozyme
  • Muramidase
  • Aspartate-tRNA Ligase

Associated data

  • PDB/4GLA
  • PDB/4GLV
  • PDB/4GN3
  • PDB/4GN4
  • PDB/4GN5

Grant support

This project was funded by a grant from the Marsden Fund of New Zealand with additional funds from the Maurice Wilkins Centre for Molecular Biodiscovery, New Enterprise Research Fund, AgResearch Limited, The University of Waikato and OBodies Ltd. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.