title: Redesigning protein cavities as a strategy for increasing affinity in protein-protein interaction: interferon- γ receptor 1 as a model.
abstract: Combining computational and experimental tools, we present a new strategy for designing high affinity variants of a binding protein. The affinity is increased by mutating residues not at the interface, but at positions lining internal cavities of one of the interacting molecules. Filling the cavities lowers flexibility of the binding protein, possibly reducing entropic penalty of binding. The approach was tested using the interferon-γ receptor 1 (IFNγR1) complex with IFNγ as a model. Mutations were selected from 52 amino acid positions lining the IFNγR1 internal cavities by using a protocol based on FoldX prediction of free energy changes. The final four mutations filling the IFNγR1 cavities and potentially improving the affinity to IFNγ were expressed, purified, and refolded, and their affinity towards IFNγ was measured by SPR. While individual cavity mutations yielded receptor constructs exhibiting only slight increase of affinity compared to WT, combinations of these mutations with previously characterized variant N96W led to a significant sevenfold increase. The affinity increase in the high affinity receptor variant N96W+V35L is linked to the restriction of its molecular fluctuations in the unbound state. The results demonstrate that mutating cavity residues is a viable strategy for designing protein variants with increased affinity.
authors.affiliation: Laboratory of Biomolecular Recognition, Institute of Biotechnology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague, Czech Republic.
authors.forename: Jiří
authors.identifier: 0000-0002-1969-9304
authors.initials: J
authors.lastname: Černý
authors.affiliation: Laboratory of Biomolecular Recognition, Institute of Biotechnology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague, Czech Republic.
authors.forename: Lada
authors.initials: L
authors.lastname: Biedermannová
authors.affiliation: Laboratory of Biomolecular Recognition, Institute of Biotechnology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague, Czech Republic.
authors.forename: Pavel
authors.initials: P
authors.lastname: Mikulecký
authors.affiliation: Laboratory of Biomolecular Recognition, Institute of Biotechnology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague, Czech Republic.
authors.forename: Jiří
authors.initials: J
authors.lastname: Zahradník
authors.affiliation: Laboratory of Biomolecular Recognition, Institute of Biotechnology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague, Czech Republic.
authors.forename: Tatsiana
authors.initials: T
authors.lastname: Charnavets
authors.affiliation: Laboratory of Biomolecular Recognition, Institute of Biotechnology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague, Czech Republic.
authors.forename: Peter
authors.initials: P
authors.lastname: Šebo
authors.affiliation: Laboratory of Biomolecular Recognition, Institute of Biotechnology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague, Czech Republic.
authors.forename: Bohdan
authors.identifier: 0000-0001-7855-3690
authors.initials: B
authors.lastname: Schneider
mesh_terms: D019943:Amino Acid Substitution; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D020125:Mutation, Missense; D011485:Protein Binding; D017510:Protein Folding; D017471:Receptors, Interferon / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000096743:Interferon gamma Receptor
chemical_list: D007371:Interferon-gamma; D017471:Receptors, Interferon; D000096743:Interferon gamma Receptor; C573415:IFNG protein, human