title: Second-contact shell mutation diminishes streptavidin-biotin binding affinity through transmitted effects on equilibrium dynamics.
abstract: We report a point mutation in the second contact shell of the high-affinity streptavidin-biotin complex that appears to reduce binding affinity through transmitted effects on equilibrium dynamics. The Y54F streptavidin mutation causes a 75-fold loss of binding affinity with 73-fold faster dissociation, a large loss of binding enthalpy (ΔΔH = 3.4 kcal/mol at 37 °C), and a small gain in binding entropy (TΔΔS = 0.7 kcal/mol). The removed Y54 hydroxyl is replaced by a water molecule in the bound structure, but there are no observable changes in structure in the first contact shell and no additional changes surrounding the mutation. Molecular dynamics simulations reveal a large increase in the atomic fluctuation amplitudes for W79, a key biotin contact residue, compared to the fluctuation amplitudes in the wild-type. The increased W79 atomic fluctuation amplitudes are caused by loss of water-mediated hydrogen bonds between the Y54 hydroxyl group and peptide backbone atoms in and near W79. We propose that the increased atomic fluctuation amplitudes diminish the integrity of the W79-biotin interaction and represents a loosening of the "tryptophan collar" that is critical to the slow dissociation and high affinity of streptavidin-biotin binding. These results illustrate how changes in protein dynamics distal to the ligand binding pocket can have a profound impact on ligand binding, even when equilibrium structure is unperturbed.
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, Washington 98195, United States.
authors.forename: Loren
authors.initials: L
authors.lastname: Baugh
authors.forename: Isolde
authors.initials: I
authors.lastname: Le Trong
authors.forename: David S
authors.initials: DS
authors.lastname: Cerutti
authors.forename: Nital
authors.initials: N
authors.lastname: Mehta
authors.forename: Susanne
authors.initials: S
authors.lastname: Gülich
authors.forename: Patrick S
authors.initials: PS
authors.lastname: Stayton
authors.forename: Ronald E
authors.initials: RE
authors.lastname: Stenkamp
authors.forename: Terry P
authors.initials: TP
authors.lastname: Lybrand
mesh_terms: D001665:Binding Sites; D001710:Biotin / Q000378:metabolism*; D018360:Crystallography, X-Ray; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D019809:Streptavidin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D050505:Mutant Proteins; D001710:Biotin; D019809:Streptavidin