title: Characterization of monobody scaffold interactions with ligand via force spectroscopy and steered molecular dynamics.
abstract: Monobodies are antibody alternatives derived from fibronectin that are thermodynamically stable, small in size, and can be produced in bacterial systems. Monobodies have been engineered to bind a wide variety of target proteins with high affinity and specificity. Using alanine-scanning mutagenesis simulations, we identified two scaffold residues that are critical to the binding interaction between the monobody YS1 and its ligand, maltose-binding protein (MBP). Steered molecular dynamics (SMD) simulations predicted that the E47A and R33A mutations in the YS1 scaffold substantially destabilize the YS1-MBP interface by reducing the bond rupture force and the lifetime of single hydrogen bonds. SMD simulations further indicated that the R33A mutation weakens the hydrogen binding between all scaffold residues and MBP and not just between R33 and MBP. We validated the simulation data and characterized the effects of mutations on YS1-MBP binding by using single-molecule force spectroscopy and surface plasmon resonance. We propose that interfacial stability resulting from R33 of YS1 stacking with R344 of MBP synergistically stabilizes both its own bond and the interacting scaffold residues of YS1. Our integrated approach improves our understanding of the monobody scaffold interactions with a target, thus providing guidance for the improved engineering of monobodies.
authors: 1] Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland [2] Johns Hopkins Institute for NanoBioTechnology, The Johns Hopkins University, Baltimore, Maryland [3] Johns Hopkins Physical Sciences-Oncology Center, The Johns Hopkins University, Baltimore, Maryland.
authors: Luthur Siu-Lun
authors: LS
authors: Cheung
authors: Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland.
authors: Daniel J
authors: DJ
authors: Shea
authors: Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland.
authors: Nathan
authors: N
authors: Nicholes
authors: Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland.
authors: Amol
authors: A
authors: Date
authors: 1] Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland [2] Johns Hopkins Institute for NanoBioTechnology, The Johns Hopkins University, Baltimore, Maryland.
authors: Marc
authors: M
authors: Ostermeier
authors: 1] Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland [2] Johns Hopkins Institute for NanoBioTechnology, The Johns Hopkins University, Baltimore, Maryland [3] Johns Hopkins Physical Sciences-Oncology Center, The Johns Hopkins University, Baltimore, Maryland [4] Center of Cancer Nanotechnology Excellence, The Johns Hopkins University, Baltimore, Maryland.
authors: Konstantinos
authors: K
authors: Konstantopoulos
mesh_terms: D001665:Binding Sites; D005353:Fibronectins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D058619:Maltose-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D055550:Protein Stability; D020349:Surface Plasmon Resonance
chemical_list: D005353:Fibronectins; D008024:Ligands; D058619:Maltose-Binding Proteins
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