A structural snapshot of an intermediate on the streptavidin–biotin dissociation pathway

Freitag, Stefanie; Chu, V.; Penzotti, Julie E.; Klumb, L.A.; To, Richard; Hyre, David E.; Trong, I. Le; Lybrand, Terry P.; Stenkamp, Ronald E.; Stayton, Patrick S.

doi:10.1073/pnas.96.15.8384

Cited by 63 publications

(95 citation statements)

References 37 publications

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“…A 1,000-fold decrease in binding affinity confirmed this in the D128A mutant Freitag et al, 1999!. A number of thermodynamic and structural features suggest that D128A may mimic an intermediate on the biotin dissociation pathway. We report here the structural and biophysical analysis of the Ser45Ala~S45A!…”

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confidence: 51%

“…Removal of this loop decreases K a by as much as six orders of magnitude~Chu et al, 1998!. Finally, a hydrogen-bonding network that extends from the biotin-binding pocket plays an important energetic role~Klumb et Freitag et al, 1999!. Large interaction energies between these residues and an oxyanion resonance form of the ureido moiety of biotin were postulated from structural data~Weber et al., 1989, 1992!.…”

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confidence: 99%

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

et al. 2000

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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.

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

et al. 2000

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“…41,42 For our studies of ligand-receptor chemomechanics, we first consider the biotin-streptavidin complex to test the effect of initial bound configuration and ECM stiffness on unbinding. Biotinstreptavidin is a ligand-receptor system that has been very well characterized both experimentally and computationally, 22,38,[43][44][45][46][47][48][49][50][51] making this complex an ideal model system for fundamental ligandreceptor studies. Here, we use the biotin-streptavidin results to inform our design of a study for the integrin-RGD complex that is more relevant to cell-substrata adhesion.…”

confidence: 99%

Modeling and simulation of chemomechanics at the cell-matrix interface

Krishnan

Oommen

Walton

et al. 2008

Cell Adhesion & Migration

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Chemomechanical characteristics of the extracellular materials with which cells interact can have a profound impact on cell adhesion and migration. To understand and modulate such complex multiscale processes, a detailed understanding of the feedback between a cell and the adjacent microenvironment is crucial. Here, we use computational modeling and simulation to examine the cell-matrix interaction at both the molecular and continuum lengthscales. Using steered molecular dynamics, we consider how extracellular matrix (ECM) stiffness and extracellular pH influence the interaction between cell surface adhesion receptors and extracellular matrix ligands, and we predict potential consequences for focal adhesion formation and dissolution. Using continuum level finite element simulations and analytical methods to model cell-induced ECM deformation as a function of ECM stiffness and thickness, we consider the implications toward design of synthetic substrata for cell biology experiments that intend to decouple chemical and mechanical cues.

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“…Each streptavidin monomer binds one biotin molecule with femtomolar affinity, one of the strongest non-covalent interactions observed in nature [8]. Over the past 15 years several groups have developed peptide binders to the tetrameric streptavidin protein [9][10][11][12][13] and a variety of protein engineering approaches have been applied to tailor affinity for specific applications [14][15][16]. This provides a simple and clean system for protein purification as a streptavidin binding peptide can be selectively eluted from a streptavidin affinity column using appropriate concentrations of biotin.…”

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Optimisation of a multivalent Strep tag for protein detection

Stadler

Ferrigno

Davis

2010

Biophysical Chemistry

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A structural snapshot of an intermediate on the streptavidin–biotin dissociation pathway

Abstract: 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 (

Cited by 63 publications

References 37 publications

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

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

Modeling and simulation of chemomechanics at the cell-matrix interface

Optimisation of a multivalent Strep tag for protein detection

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