Atomic resolution crystallographic studies of streptavidin and its biotin complex have been carried out at 1.03 and 0.95 Å, respectively. The wild‐type protein crystallized with a tetramer in the asymmetric unit, while the crystals of the biotin complex contained two subunits in the asymmetric unit. Comparison of the six subunits shows the various ways in which the protein accommodates ligand binding and different crystal‐packing environments. Conformational variation is found in each of the polypeptide loops connecting the eight strands in the β‐sandwich subunit, but the largest differences are found in the flexible binding loop (residues 45–52). In three of the unliganded subunits the loop is in an `open' conformation, while in the two subunits binding biotin, as well as in one of the unliganded subunits, this loop `closes' over the biotin–binding site. The `closed' loop contributes to the protein's high affinity for biotin. Analysis of the anisotropic displacement parameters included in the crystallographic models is consistent with the variation found in the loop structures and the view that the dynamic nature of the protein structure contributes to the ability of the protein to bind biotin so tightly.

The contribution of the Ser45 hydrogen bond to biotin binding activation and equilibrium thermodynamics was investigated by biophysical and X-ray crystallographic studies. The S45A mutant exhibits a 1,700-fold greater dissociation rate and 907-fold lower equilibrium affinity for biotin relative to wild-type streptavidin at 37 8C, indicating a crucial role in binding energetics. The crystal structure of the biotin-bound mutant reveals only small changes from the wild-type bound structure, and the remaining hydrogen bonds to biotin retain approximately the same lengths. No additional water molecules are observed to replace the missing hydroxyl, in contrast to the previously studied D128A mutant. The equilibrium DG8, DH8, DS8, DC8 P , and activation DG ‡ of S45A at 37 8C are Ϫ13.7 6 0.1 kcal0mol, Ϫ21.1 6 0.5 kcal0mol, Ϫ23.7 6 1.8 cal0mol K, Ϫ223 6 12 cal0mol K, and 20.0 6 2.5 kcal0mol, respectively. Eyring analysis of the large temperature dependence of the S45A off-rate resolves the DH ‡ and DS ‡ of dissociation, 25.8 6 1.2 kcal0mol and 18.7 6 4.3 cal0mol K. The large increases of DH ‡ and DS ‡ in the mutant, relative to wild-type, indicate that Ser45 could form a hydrogen bond with biotin in the wild-type dissociation transition state, enthalpically stabilizing it, and constraining the transition state entropically. The postulated existence of a Ser45-mediated hydrogen bond in the wild-type streptavidin transition state is consistent with potential of mean force simulations of the dissociation pathway and with molecular dynamics simulations of biotin pullout, where Ser45 is seen to form a hydrogen bond with the ureido oxygen as biotin slips past this residue after breaking the native hydrogen bonds.

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 7 times lower than that of wild-type streptavidin at 258C. The corresponding reduction in binding free energy (DDG) 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.

It is currently unclear whether small molecules dissociate from a protein binding site along a defined pathway or through a collection of dissociation pathways. We report herein a joint crystallographic, computational, and biophysical study that suggests the Asp-128 3 Ala (

No abstract

The chicken avidin gene family consists of avidin and seven separate avidin-related genes (AVRs) 1-7. Avidin protein is a widely used biochemical tool, whereas the other family members have only recently been produced as recombinant proteins and characterized. In our previous study, AVR4 was found to be the most stable biotin binding protein thus far characterized (T m ‫؍‬ 106.4°C). In this study, we studied further the biotin-binding properties of AVR4. A decrease in the energy barrier between the biotin-bound and unbound state of AVR4 was observed when compared with that of avidin. The high resolution structure of AVR4 facilitated comparison of the structural details of avidin and AVR4. In the present study, we used the information obtained from these comparative studies to transfer the stability and functional properties of AVR4 to avidin. A chimeric avidin protein, ChiAVD, containing a 21-amino acid segment of AVR4 was found to be significantly more stable (T m ‫؍‬ 96.5°C) than native avidin (T m ‫؍‬ 83.5°C), and its biotin-binding properties resembled those of AVR4. Optimization of a crucial subunit interface of avidin by an AVR4-inspired point mutation, I117Y, significantly increased the thermostability of the avidin mutant (T m ‫؍‬ 97.5°C) without compromising its high biotin-binding properties. By combining these two modifications, a hyperthermostable ChiAVD(I117Y) was constructed (T m ‫؍‬ 111.1°C). This study provides an example of rational protein engineering in which another member of the protein family has been utilized as a source in the optimization of selected properties.

Pretargeted radioimmunotherapy specifically targets radiation to tumors using antibody-streptavidin conjugates followed by radiolabeled biotin. A potential barrier to this cancer therapy is the presence of endogenous biotin in serum, which can block the biotin-binding sites of the antibody-streptavidin conjugate before the administration of radiolabeled biotin. Serum-derived biotin can also be problematic in clinical diagnostic applications. Due to the extremely slow dissociation of the biotin-streptavidin complex, this endogenous biotin can irreversibly block the biotin-binding sites of streptavidin and reduce therapeutic efficacy, as well as reduce sensitivity in diagnostic assays. We tested a streptavidin mutant (SAv-Y43A), which has a 67-fold lower affinity for biotin than wild type streptavidin, and three bivalent bis-biotin constructs as replacements for wild-type streptavidin and biotin used in pretargeting and clinical diagnostics. Biotin dimers were engineered with certain parameters including water solubility, biotinidase resistance, and linker lengths long enough to span the distance between two biotin-binding sites of streptavidin. The bivalent biotins were compared to biotin in exchange, retention, and off-rate assays. The faster off-rate of SAv-Y43A allowed efficient exchange of prebound biotin by the biotin dimers. In fluorescent competition experiments, the biotin dimer ligands displayed high avidity binding and essentially irreversible retention with SAv-Y43A. The off-rate of a biotinidase-stabilized biotin dimer from SAv-Y43A was 4.36 x 10(-)(6) s(-)(1), over 640 times slower compared to biotin. These findings strongly suggest that employing a mutant streptavidin in concert with a bivalent biotin can mitigate the deleterious impact of endogenous biotin, by allowing exchange of bound biotin and retention of the biotin dimer carriers.

The streptavidin-biotin system has provided a unique opportunity to investigate the molecular details of ligand dissociation pathways. An underlying mechanistic question is whether ligand dissociation proceeds with a relatively ordered process of bond breaking and ligand escape. Here we report a joint computational and crystallographic study of the earliest events in biotin dissociation. In molecular dynamics potential of mean force simulations, a water molecule from a defined access channel intercalated into the hydrogen bond between Asp 128 and biotin, bridging them and stabilizing an intermediate state. In forced biotin dissociation simulations, this event led to subsequent bond breaking steps and ligand escape. In equilibrium simulations, the water molecule was sometimes observed to move back to the access channel with re-formation of the biotin hydrogen bond. Analysis of streptavidin crystal structures revealed a close overlap of crystallographically defined and simulated waters in the water access channel. These results suggest that biotin dissociation is initiated by stochastic coupling of water entry with lengthening of a specific biotin hydrogen-bonding interaction.