The Crystal Structure of Monovalent Streptavidin

Zhang, Min; Biswas, Sanjib; Deng, Wenbin; Yu, Hongjun

doi:10.1038/srep35915

Cited by 11 publications

(9 citation statements)

References 22 publications

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“…The structure of a monovalent Streptomyces avidinii SA (mSA) had been solved by means of x-ray crystallography at 1.65 Å resolution and was available at the PDB (PDB: 5TO2) (26). Because this crystallographic structure does not contain a biotin bound to the binding pocket, the structure of the tetravalent S. avidinii SA bound to biotin (PDB: 1MK5) (27), solved at 1.4 Å resolution, was used to place the biotin on to its binding site at chain D of the mSA.…”

Section: System Setupmentioning

confidence: 99%

Streptavidin/biotin: Tethering geometry defines unbinding mechanics

et al. 2020

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Macromolecules tend to respond to applied forces in many different ways. Chemistry at high shear forces can be intriguing, with relatively soft bonds becoming very stiff in specific force-loading geometries. Largely used in bionanotechnology, an important case is the streptavidin (SA)/biotin interaction. Although SA’s four subunits have the same affinity, we find that the forces required to break the SA/biotin bond depend strongly on the attachment geometry. With AFM-based single-molecule force spectroscopy (SMFS), we measured unbinding forces of biotin from different SA subunits to range from 100 to more than 400 pN. Using a wide-sampling approach, we carried out hundreds of all-atom steered molecular dynamics (SMD) simulations for the entire system, including molecular linkers. Our strategy revealed the molecular mechanism that causes a fourfold difference in mechanical stability: Certain force-loading geometries induce conformational changes in SA’s binding pocket lowering the energy barrier, which biotin has to overcome to escape the pocket.

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Section: System Setupmentioning

confidence: 99%

Streptavidin/biotin: Tethering geometry defines unbinding mechanics

et al. 2020

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“… (a) Crystal structure of mSA (pdb identification code 5TO2 [ 15 ], overlaid with 1MK5 [ 16 ] to show the position of biotin). The functional subunit (green) with biotin (red) bound is stabilized by the three non-functional subunits (grey).…”

Section: Introductionmentioning

confidence: 99%

Monodisperse measurement of the biotin-streptavidin interaction strength in a well-defined pulling geometry

et al. 2017

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The widely used interaction of the homotetramer streptavidin with the small molecule biotin has been intensively studied by force spectroscopy and has become a model system for receptor ligand interaction. However, streptavidin’s tetravalency results in diverse force propagation pathways through the different binding interfaces. This multiplicity gives rise to polydisperse force spectroscopy data. Here, we present an engineered monovalent streptavidin tetramer with a single cysteine in its functional subunit that allows for site-specific immobilization of the molecule, orthogonal to biotin binding. Functionality of streptavidin and its binding properties for biotin remain unaffected. We thus created a stable and reliable molecular anchor with a unique high-affinity binding site for biotinylated molecules or nanoparticles, which we expect to be useful for many single-molecule applications. To characterize the mechanical properties of the bond between biotin and our monovalent streptavidin, we performed force spectroscopy experiments using an atomic force microscope. We were able to conduct measurements at the single-molecule level with 1:1-stoichiometry and a well-defined geometry, in which force exclusively propagates through a single subunit of the streptavidin tetramer. For different force loading rates, we obtained narrow force distributions of the bond rupture forces ranging from 200 pN at 1,500 pN/s to 230 pN at 110,000 pN/s. The data are in very good agreement with the standard Bell-Evans model with a single potential barrier at Δx0 = 0.38 nm and a zero-force off-rate koff,0 in the 10−6 s-1 range.

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“…(A) Crystal structure of monovalent streptavidin (PDB 5TO2, biotin from PDB 1MK5). Biotin is bound in the functional subunit (light orange).…”

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confidence: 99%

“…Recently, the crystal structure of mSA was solved (Figure S4). 12 Crystallographic data suggest that in the nonfunctional subunit, the L3/4-loop is fixed in an open state−similar to the open state of wild-type apo-SA.…”

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confidence: 99%

Direction Matters: Monovalent Streptavidin/Biotin Complex under Load

et al. 2018

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Novel site-specific attachment strategies combined with improvements of computational resources enable new insights into the mechanics of the monovalent biotin-streptavidin complex under load and forced us to rethink the diversity of rupture forces reported in the literature. We discovered that the mechanical stability of this complex depends strongly on the geometry in which force is applied. By atomic force microscopy-based single molecule force spectroscopy we found unbinding of biotin to occur beyond 400 pN at force loading rates of 10 nN/s when monovalent streptavidin was tethered at its C-terminus. This value is about twice as high than that for Nterminal attachment. Steered molecular dynamics simulations provided a detailed picture of the mechanics of the unbinding process in the corresponding force loading geometries. Using machine learning techniques, we connected findings from hundreds of simulations to the experimental results, identifying different force propagation pathways. Interestingly, we observed that depending on force loading geometry, partial unfolding of N-terminal region of monovalent streptavidin occurs before biotin is released from the binding pocket.

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The Crystal Structure of Monovalent Streptavidin

Cited by 11 publications

References 22 publications

Streptavidin/biotin: Tethering geometry defines unbinding mechanics

Streptavidin/biotin: Tethering geometry defines unbinding mechanics

Monodisperse measurement of the biotin-streptavidin interaction strength in a well-defined pulling geometry

Direction Matters: Monovalent Streptavidin/Biotin Complex under Load

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