Ser45 plays an important role in managing both the equilibrium and transition state energetics of the streptavidin—biotin system

Abstract

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.

Motifs for molecular recognition exploiting hydrophobic enclosure in protein–ligand binding

“…When mutated to Ala, residues Asp-128 and Ser-45 exhibit 1.4 kcal of binding cooperativity, driven by a 3.0 kcal/mol entropic term. This finding is consistent with a destabilization of the water ring solvating the active site due to the loss of the hydrogen binding groups maintaining it (8). Circular deletion of the mobile loop formed by residues 47-51 that hydrophobically enclose the ligand from above, most notably by Val-47, loses 8 kcal/mol lower than the unmutated protein; this is much more than continuum methods predict but in agreement with our estimate (9).…”

Design and Construction of Highly Stable, Protease-resistant Chimeric Avidins

“…The activation thermodynamic parameters for AVR4 and avidin were determined by analysis of the dependence of the dissociation rate upon temperature using the global fit of all data as described in Ref. 22.…”

Crystallographic Analysis of a Full-length Streptavidin with Its C-terminal Polypeptide Bound in the Biotin Binding Site

“…The tight binding between streptavidin and biotin is also the subject of biophysical, structural and computational investigations probing the nature of the protein-ligand interactions. [2][3][4][5] Streptavidin, after expression in Streptomyces avidinii as a polypeptide of 159 residues, is rapidly cleaved by proteases to yield several truncated versions of the protein. 6 The most stable and wellstudied form of the protein contains residues 13-139 and is called core-streptavidin.…”

“…Streptavidin/ligand interactions have been probed both by mutating the protein 5,9,10 and by varying the structure of the ligand. [11][12][13][14] These studies have been driven by theoretical as well as biotechnical considerations, and a number of crystal structures of streptavidin and its ligands are available in the Protein Data Bank.…”