Biotin and Biotin analogs binding to Streptavidin and Avidin

Proteins, 47:194-208 (2002)

Biotin in the binding site of streptavidin



The free energy of binding of a ligand to a macromolecule was formally decomposed into the (effective) energy of interaction, reorganization energy of the ligand and the macromolecule, conformational entropy change of the ligand and the macromolecule, and translational and rotational entropy loss of the ligand. Molecular dynamics simulations with implicit solvation were used to evaluate these contributions in the binding of biotin, biotin analogs, and two peptides to avidin and streptavidin. It was found that the largest contribution opposing binding is the protein reorganization energy, which was calculated to be from 10 to 30 kcal/mol for the ligands considered here. The ligand reorganization energy was also significant for flexible ligands. The translational/rotational entropy was 4.5-6 kcal/mol at 1 M standard state and room temperature. The calculated binding free energies are in the correct range, but the large statistical uncertainty in the protein reorganization energy precludes precise predictions. For some complexes the simulations showed multiple binding modes, different from the one observed in the crystal structure. This is probably due to deficiencies in the force field but may also reflect considerable ligand flexibility.
 
 
 

Unpublished data:
Ab initio calculations of the binding site

Weber et al. (Weber, P.C., Ohlendorf, D.H., Wendoloski, J.J., Salemme, F.R. Structural origins of high-affinity biotin binding to streptavidin. Science 243:85-8, 1989) proposed that stabilization of ionic resonance forms of biotin by the protein environment contributes to the strong binding of biotin to streptavidin. To test this hypothesis, and to test the ability of our classical, nonpolarizable force field to reproduce biotin-protein interactions, we conducted ab initio calculations for two model systems simulating biotin/streptavidin and biotin/avidin complexes. The Gaussian 98 suite of programs 70 was used in all calculations. Two levels of theory were considered: semiempirical with the AM1 Hamiltonian, and ab-initio at the HF/6-31G* level. Standard counterpoise correction (Boys, S.F., Bernardi, F. Calculation of small molecular interactions by differences of separate total energies. Some procedures with reduced errors. Mol. Phys. 19:553-, 1970) was used at the ab-initio level.

The model for the biotin/streptavidin complex (system I) was built as follows. The polar groups of the side-chains from 5 residues (Asn11, Ser15, Tyr31, Ser 33, Asp116), forming hydrogen bonds to the biotin molecule in the crystal structure were identified and truncated to the nearest carbon atom (except for Tyr31, where the whole phenyl ring was included). The charged side-chain of biotin was replaced by a methyl group. The hydrogen atoms were placed geometrically according to the hybridization of the heavy atoms. In the case of OH groups this procedure was ambiguous and several variants were considered, all consistent with the formation of a hydrogen bonding network. As some contacts in the crystal structure were too short, the total interaction was found repulsive at the AM1 level. Four systems different in the placement of H atoms were fully optimized at the AM1 level, but only one of them retained the hydrogen bonds between the biotin and the protein environment observed in the crystal structure. This system was fully optimized at the HF/6-31G* level, and conserved the above hydrogen bonds at each optimization step.

To construct the model for the biotin/avidin complex (system II), we selected 5 side-chain polar groups (Asn10, Ser14, Tyr31, Thr33, Asn116) forming hydrogen bonds to biotin in the crystal structure. They were truncated analogously to system I, so that the only difference in stoichiometry was a neutral NH2 group replacing an O- atom of COO-. Hydrogen atoms were placed to reproduce the topology of hydrogen bonding network in system I. The geometry was completely optimized at HF/6-31G* level. While initial optimization steps led mostly to relaxation in hydrogen bonding distances, after 20 steps the two Asn residues formed a new hydrogen bond and one of them consequently broke the hydrogen bond to biotin. One possible reason for this deviation from the crystal structure is the absence of the geometrical constraints exerted by the rest of the protein upon the binding site. For this reason we report the interaction energy (defined as the total energy of the complex less the sum of total energy for the biotin and the rest of the model system in the geometry of the complex) obtained in partial optimization (16 steps) along with complete optimization results in Table 3. Complete optimization of each system converged in about 160 steps, and took over 9 days of CPU time on a Compaq DS20 Alpha machine. As one can see from the Table 3, the intermolecular interaction in system I after the counterpoise correction is 7 kcal/mol stronger than in system II for the fully optimized structures and 10.6 kcal/mol for the partially optimized structures.

To compare the DE values to the MM results we used the standard CHARMM 19 force field (fully charged ionic residues, e=1, and no solvation term) and calculated the interaction energy between the biotin bicyclic ring and the 5 residues included in the QM calculations. We did this calculation on the PDB structures minimized for 300 ABNR steps. The values obtained are -37 kcal/mol for streptavidin and either -30 or -35 kcal/mol for avidin depending on which monomer is used (there are two distinct subunits in the crystal structure). These values are quite close to the QM results, despite differences in system preparation and the lack of protein connectivity constraints in the QM optimizations. The interaction between streptavidin's Asp 116 and biotin is -10.7 kcal/mol, whereas the interaction of avidin's Asn 116 and biotin is -6.7 kcal/mol. Thus, the DDE between avidin and streptavidin tends to be a little underestimated by the CHARMM19 force field (2-7 kcal/mol vs 7-10.7 kcal/mol from quantum mechanics). The polarization of biotin by the charged Asp 116 may contribute a couple of kcal/mol extra to the binding free energy in streptavidin. It is noted that in EEF1 the Asp residue, like all other ionizable residues, is neutralized and its solvation free energy is reduced accordingly. In addition, e=r is used in the calculations. The effective energy between Asp 116 and biotin in EEF1 is -4.3 kcal/mol (van der Waals -2.3, electrostatic -4, desolvation +2). If we use a charged Asp 116 and assign to its carboxylate the appropriate solvation free energy (~ -80 kcal/mol) and use e=1, the effective energy is -4 (van der Waals -2.3, electrostatic -8.4, desolvation +6.7). Thus, neutralizing the Asp and reducing its reference solvation free energy produces compensating changes so that the effective interaction remains about the same.