A hot-spot motif characterizes the interface between a designed ankyrin-repeat protein and its target ligand.
Nonantibody scaffolds such as designed ankyrin repeat proteins (DARPins) can be rapidly engineered to detect diverse target proteins with high specificity and offer an attractive alternative to antibodies. Using molecular simulations, we predicted that the binding interface between DARPin off7 and its ligand (maltose binding protein; MBP) is characterized by a hot-spot motif in which binding energy is largely concentrated on a few amino acids. To experimentally test this prediction, we fused MBP to a transmembrane domain to properly orient the protein into a polymer-cushioned lipid bilayer, and characterized its interaction with off7 using force spectroscopy. Using this, to our knowledge, novel technique along with surface plasmon resonance, we validated the simulation predictions and characterized the effects of select mutations on the kinetics of the off7-MBP interaction. Our integrated approach offers scientific insights on how the engineered protein interacts with the target molecule.
Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland, USA.
Luthur Siu-Lun
LS
Cheung
Manu
M
Kanwar
Marc
M
Ostermeier
Konstantinos
K
Konstantopoulos
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017089:Ankyrin Repeat; D007700:Kinetics; D008024:Ligands; D008051:Lipid Bilayers / Q000378:metabolism; D058619:Maltose-Binding Proteins / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D015202:Protein Engineering; D013057:Spectrum Analysis; D020349:Surface Plasmon Resonance; D013816:Thermodynamics
D008024:Ligands; D008051:Lipid Bilayers; D058619:Maltose-Binding Proteins