The streptavidin/biotin complex involves one of the strongest non-covalent interactions observed in biology, and is an ideal model system for the study of high-affinity protein/ligand interactions. Analysis of high- resolution crystal structures for the streptavidin/biotin complex reveals numerous favorable protein-ligand contacts characteristic of a tightly bound complex. However, our extensive structural and thermodynamic characterization of numerous streptavidin mutants strongly suggests that direct protein-ligand contacts cannot fully explain the extremely tight biotin binding. Our previous studies also suggest that biotin follows a well defined reaction coordinate during ligand binding and dissociation reactions, and that certain mutations can alter the activation energy barrier for the binding/dissociation reactions, without any significant effect on the equilibrium structure. These results lead us to propose that streptavidin equilibrium dynamics, or structural fluctuations, help determine the equilibrium binding energy and activation energy barrier. We will test this hypothesis by performing molecular dynamics simulations and detailed crystallographic analyses of anisotropic temperature factors for wild-type and selected mutant streptavidin/biotin complexes, and then look for correlations between structural fluctuations and calorimetric measurements in wild-type versus mutant complexes. Our previous results also suggest that specific water molecules play a key energetic role in the biotin binding/dissociation reactions. We will use molecular dynamics simulations to test this proposal by further characterizing water interactions in the streptavidin binding site. We will use these simulation results to suggest mutations that can alter the equilibrium water behavior in the complex, and thus modulate the ligand binding free energy and/or activation energy. These studies will enhance our understanding of high-affinity protein/ligand binding interactions, and will help us elucidate important concepts that should be useful in structure-based ligand design projects. Such information will be helpful in rational drug design applications for therapeutically important protein targets.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Special Emphasis Panel (ZRG1-BCMB-Q (90))
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Preusch, Peter C
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Vanderbilt University Medical Center
Schools of Medicine
United States
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Baugh, Loren; Le Trong, Isolde; Stayton, Patrick S et al. (2016) A Streptavidin Binding Site Mutation Yields an Unexpected Result: An Ionized Asp128 Residue Is Not Essential for Strong Biotin Binding. Biochemistry 55:5201-3
Le Trong, Isolde; Chu, Vano; Xing, Yi et al. (2013) Structural consequences of cutting a binding loop: two circularly permuted variants of streptavidin. Acta Crystallogr D Biol Crystallogr 69:968-77
Baugh, Loren; Le Trong, Isolde; Cerutti, David S et al. (2012) Second-contact shell mutation diminishes streptavidin-biotin binding affinity through transmitted effects on equilibrium dynamics. Biochemistry 51:597-607
Le Trong, Isolde; Wang, Zhizhi; Hyre, David E et al. (2011) Streptavidin and its biotin complex at atomic resolution. Acta Crystallogr D Biol Crystallogr 67:813-21
Baugh, Loren; Le Trong, Isolde; Cerutti, David S et al. (2010) A distal point mutation in the streptavidin-biotin complex preserves structure but diminishes binding affinity: experimental evidence of electronic polarization effects? Biochemistry 49:4568-70
Cerutti, David S; Le Trong, Isolde; Stenkamp, Ronald E et al. (2009) Dynamics of the streptavidin-biotin complex in solution and in its crystal lattice: distinct behavior revealed by molecular simulations. J Phys Chem B 113:6971-85
Cerutti, David S; Duke, Robert E; Darden, Thomas A et al. (2009) Staggered Mesh Ewald: An extension of the Smooth Particle-Mesh Ewald method adding great versatility. J Chem Theory Comput 5:2322
Cerutti, David S; Le Trong, Isolde; Stenkamp, Ronald E et al. (2008) Simulations of a protein crystal: explicit treatment of crystallization conditions links theory and experiment in the streptavidin-biotin complex. Biochemistry 47:12065-77
Cerutti, David S; Duke, Robert; Freddolino, Peter L et al. (2008) Vulnerability in Popular Molecular Dynamics Packages Concerning Langevin and Andersen Dynamics. J Chem Theory Comput 4:1669-1680