Second-Contact Shell Mutation Diminishes Streptavidin–Biotin Binding Affinity through Transmitted Effects on Equilibrium Dynamics

• Published in: Biochemistry (Easton) 2012-01, Vol.51 (2), p.597-607
• Main Authors: Baugh, Loren, Le Trong, Isolde, Cerutti, David S, Mehta, Nital, Gülich, Susanne, Stayton, Patrick S, Stenkamp, Ronald E, Lybrand, Terry P
• Format: Article
• Language: English
• Subjects: Binding Sites; Biotin - metabolism; Crystallography, X-Ray; Kinetics; Molecular Dynamics Simulation; Mutant Proteins - chemistry; Mutant Proteins - genetics; Mutant Proteins - metabolism; Point Mutation; Protein Binding; Protein Conformation; Streptavidin - chemistry; Streptavidin - genetics; Streptavidin - metabolism; Thermodynamics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi201221j

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.