Cooperative hydrogen bond interactions in the streptavidin–biotin system
Abstract
The thermodynamic and structural cooperativity between the Ser45– and D128–biotin hydrogen bonds was measured by calorimetric and X-ray crystallographic studies of the S45A/D128A double mutant of streptavidin. The double mutant exhibits a binding affinity ~2 × 10 times lower than that of wild-type streptavidin at 25°C. The corresponding reduction in binding free energy (ΔΔG) of 10.1 kcal/mol was nearly completely due to binding enthalpy losses at this temperature. The loss of binding affinity is 11-fold greater than that predicted by a linear combination of the single-mutant energetic perturbations (8.7 kcal/mol), indicating that these two mutations interact cooperatively. Crystallographic characterization of the double mutant and comparison with the two single mutant structures suggest that structural rearrangements at the S45 position, when the D128 carboxylate is removed, mask the true energetic contribution of the D128–biotin interaction. Taken together, the thermodynamic and structural analyses support the conclusion that the wild-type hydrogen bond between D128–OD and biotin–N2 is thermodynamically stronger than that between S45–OG and biotin–N1.
The binding of biotin (vitamin H) to streptavidin and avidin has been the subject of considerable fundamental and applied interest. This protein–ligand pair represents one of the strongest noncovalent affinities known. From the structure of the bound complex, it is known that the binding energy derives from multiple types of interactions between the protein and biotin (Hendrickson et al. 1989; Weber et al. 1989). There are large hydrophobic and van der Waals contributions arising from tryptophan contacts to biotin (Chilkoti et al. 1995; Sano and Cantor 1995; Dixon and Kollman 1999). There are also seven specific hydrogen bonding interactions, five of them deep within the pocket and shielded from competition with solvent, including three to a single oxygen on biotin (Klumb et al. 1998; Freitag et al. 1999; Hyre et al. 2000, 2002). Weber et al. (1992) postulated that a ure-ido-oxyanion resonance form of biotin is stabilized in the bound state, leading to the prediction that the hydrogen bonding contributions would be large.
Mutational analysis has demonstrated that the biotin–streptavidin hydrogen bonds indeed display exceptionally large mutational free energy alterations, among the largest mutational free energies ever observed (Klumb et al. 1998; Freitag et al. 1999; Hyre et al. 2000). Biophysical characterization of the S45A and D128A single mutants have shown that loss of hydrogen bonds to the ureido nitrogens decrease binding free energy by ~4.2 kcal/mol apiece at 37°C. Both mutants also displayed nearly identical equilibrium thermodynamic perturbations (Freitag et al. 1999; Hyre et al. 2000). The equilibrium binding enthalpy and entropy, and hence free energy, were within 0.2 kcal/mol of one another, as were the activation-barrier free energies. The only energetic difference was in the balance between enthalpic and entropic contributions to the dissociation barrier. The S45A/D128A double-mutant streptavidin (45/128) was created to determine the level of cooperativity between the interactions of these residues with biotin. Herein, we characterize the double mutant both thermodynamically and structurally, showing that subtle structural alterations in the D128A single mutant likely masks the full energetic contribution of the abrogated hydrogen bond.
Acknowledgments
We gratefully acknowledge the NIH for support of this project ({"type":"entrez-nucleotide","attrs":{"text":"EB000237","term_id":"90538378","term_text":"EB000237"}}EB000237 and GM62617). Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL), a national user facility operated by Stanford University on behalf of the U.S. Department of Energy (DOE) Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the NIH National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences. Other portions were carried out at the Advanced Light Source, which is supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences Division of the DOE under contract no. DE-AC03-76SF00098 at Lawrence Berkeley National Laboratory. Access to the CSC ITC 4200 calorimeter was graciously provided by our UW colleague Dr. Wim Hol, HHMI.
Abbreviations
ITC, isothermal calorimetry
MD, molecular dynamics
SPR, surface plasmon resonance
StAv, streptavidin
45/128 S45A/D128A, double mutant of streptavidin
Notes
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051970306.