Tracking molecular recognition at the atomic level with a new protein scaffold based on the OB-fold.
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.
Department of Biological Sciences, University of Waikato, Hamilton, New Zealand.
John D
JD
Steemson
Department of Biological Sciences, University of Waikato, Hamilton, New Zealand.
Matthias
M
Baake
Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand.
Jasna
J
Rakonjac
Department of Biological Sciences, University of Waikato, Hamilton, New Zealand.
Vickery L
VL
Arcus
OBodies Limited, Hamilton, New Zealand.
Mark T
MT
Liddament
D000595:Amino Acid Sequence; D000818:Animals; D000926:Anticodon / Q000378:metabolism; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001226:Aspartate-tRNA Ligase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002645:Chickens; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D055550:Protein Stability; D041543:Pyrobaculum / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D000926:Anticodon; D019843:Archaeal Proteins; C421864:hen egg lysozyme; D009113:Muramidase; D001226:Aspartate-tRNA Ligase