Heterogeneous and rate-dependent streptavidin–biotin unbinding revealed by high-speed force spectroscopy and atomistic simulations - Aix-Marseille Université Access content directly
Journal Articles Proceedings of the National Academy of Sciences of the United States of America Year : 2019

Heterogeneous and rate-dependent streptavidin–biotin unbinding revealed by high-speed force spectroscopy and atomistic simulations


Receptor-ligand interactions are essential for biological function and their binding strength is commonly explained in terms of static lock-and-key models based on molecular complementarity. However, detailed information on the full unbinding pathway is often lacking due, in part, to the static nature of atomic structures and ensemble averaging inherent to bulk biophysics approaches. Here we combine molecular dynamics and high-speed force spec-troscopy on the streptavidin-biotin complex to determine the binding strength and unbinding pathways over the widest dynamic range. Experiment and simulation show excellent agreement at overlapping velocities and provided evidence of the unbinding mechanisms. During unbinding, biotin crosses multiple energy barriers and visits various intermediate states far from the binding pocket, while streptavidin undergoes transient induced fits, all varying with loading rate. This multistate process slows down the transition to the unbound state and favors rebinding, thus explaining the long lifetime of the complex. We provide an atomistic, dynamic picture of the unbinding process, replacing a simple two-state picture with one that involves many routes to the lock and rate-dependent induced-fit motions for intermediates, which might be relevant for other receptor-ligand bonds. high-speed atomic force microscopy | high-speed force spectroscopy | receptor/ligand bonds | single molecules | molecular dynamics simulations R eceptor/ligand bonds are at the core of almost every biological process. The early lock-and-key model including possible conformational changes of the binding partners is commonly accepted to describe the affinities and kinetic rates of receptor/ligand complexes and is mainly based on molecular complementarity pictures from static structural data (1, 2). Over the past decades, an impressive amount of knowledge has been accumulated on the structural and energetic determinants of bound states, thus enabling the increasingly successful rational design of nanomolar binders for therapy as well as the quantitative prediction of binding processes and free energies from atomistic simulations (3). While protein folding and unfolding are thought to follow a multiplicity of pathways, the very mechanism of binding or unbinding of receptor/ligand complexes remains less investigated and is generally described by a simple two-state model, or by the lock-and-key analogy. Moreover, little is known on how the (un)binding dynamics is governed by the underlying microscopic processes-despite being key to a quantitative understanding of receptor-ligand complexes. Progress is mostly hampered by the lack of structural and thermodynamic information of the transient ligand/receptor conformations during unbinding, even for extensively studied systems such the complex formed by streptavidin (SA) and the small molecule biotin (b, vitamin H), one of the strongest noncovalent bonds known in nature. SA forms the b-binding pocket with an eight-stranded, anti-parallel beta-barrel capped by loop 3-4 (Fig. 1A). In the native, tetrameric SA form, loop 7-8 from an adjacent monomer provides a closing lid to the pocket (4). b binds by forming an intricate and extensive network of hydrogen bonds with polar residues of SA (4, 5). Its high affinity (K d ∼ 10 −13 M) and long lifetime (τ ∼ 10 d, k off ∼ 10 −6 s −1) (6-9) make the SA/b system extensively used in biotechnology and biophysics. Dynamic forced disruption of the SA-b complex by atomic force microscopy (AFM) and other techniques pioneered single-molecule biomechanics (10-13) and provided estimates of the distance x β to the unbinding transition state and the intrinsic bond lifetime (13-15). Despite its seeming simplicity, AFM (11, 16-19), optical tweezers (20), and bio-membrane force probe (10, 21) experiments of SA/b unbinding have reported dissimilar results, suggesting an impressive complexity and heterogeneity in the unbinding pathway (SI Appendix). Furthermore, the results from single-molecule studies were incompatible with ensemble bond-lifetime measurements (6). Although recent experimental developments accessing the microsecond timescale have shed light into the complexity of single-molecule transition paths of protein and nucleic acid (un) folding (22-25), the amount of transient structural information extracted from single-molecule experiments is rather limited. Significance Protein-ligand interactions are commonly described in terms of a two-state or a lock-and-key mechanism. To provide a more detailed and dynamic description of receptor-ligand bonds and their (un)binding path, we combined high-speed force spec-troscopy and molecular dynamics simulations to probe the prototypical streptavidin-biotin complex. The excellent agreement observed, never used for force-field refinement, provides the most direct test of the "computational microscope." The so-far largest dynamic range of loading rates explored (11 decades) enabled accurate reconstruction of the free-energy landscape. We revealed velocity-dependent unbinding pathways and intermediate states that enhance rebinding, explaining the long lifetime of the bond. We expect similar behavior in most receptor-ligand complexes, implying unbinding pathways governed by transient, timescale-dependent induced fits.
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hal-02106342 , version 1 (22-04-2019)



Felix Rico, Andreas Russek, Laura Gonzalez, Helmut Grubmüller, Simon Scheuring. Heterogeneous and rate-dependent streptavidin–biotin unbinding revealed by high-speed force spectroscopy and atomistic simulations. Proceedings of the National Academy of Sciences of the United States of America, 2019, 116 (14), pp.6594-6601. ⟨10.1073/pnas.1816909116⟩. ⟨hal-02106342⟩
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