4GN4

OBody AM2EP06 bound to hen egg-white lysozyme

Summary for 4GN4

• Entry DOI: 10.2210/pdb4gn4/pdb
• Related: 4GLA 4GLV 4GN3 4GN5
• Descriptor: OBody AM2EP06, Lysozyme C, GLYCEROL, ... (4 entities in total)
• Functional Keywords: beta barrel, ob-fold, protein-protein complex, novel scaffold, muraminidase, enzyme inhibition, engineered binding protein, inhibitor, de novo protein-hydrolase complex, de novo protein/hydrolase
• Biological source: Pyrobaculum aerophilum; More
• Cellular location: Secreted: P00698
• Total number of polymer chains: 2
• Total formula weight: 26637.12

Authors

Steemson, J.D.,Liddament, M.T. (deposition date: 2012-08-16, release date: 2013-08-21, Last modification date: 2024-10-16)

Primary citation

Steemson, J.D.,Baake, M.,Rakonjac, J.,Arcus, V.L.,Liddament, M.T.
Tracking Molecular Recognition at the Atomic Level with a New Protein Scaffold Based on the OB-Fold.
Plos One, 9:e86050-e86050, 2014

PubMed Abstract: 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.

PubMed: 24465865
DOI: 10.1371/journal.pone.0086050

Experimental method

X-RAY DIFFRACTION (1.861 Å)