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 spectroscopy 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.

Structural basis for cooperativity of CRM1 export complex formation.

In eukaryotes, the nucleocytoplasmic transport of macromolecules is mainly mediated by soluble nuclear transport receptors of the karyopherin-ß superfamily termed importins and exportins. The highly versatile exportin chromosome region maintenance 1 (CRM1) is essential for nuclear depletion of numerous structurally and functionally unrelated protein and ribonucleoprotein cargoes. CRM1 has been shown to adopt a toroidal structure in several functional transport complexes and was thought to maintain this conformation throughout the entire nucleocytoplasmic transport cycle. We solved crystal structures of free CRM1 from the thermophilic eukaryote Chaetomium thermophilum. Surprisingly, unbound CRM1 exhibits an overall extended and pitched superhelical conformation. The two regulatory regions, namely the acidic loop and the C-terminal α-helix, are dramatically repositioned in free CRM1 in comparison with the ternary CRM1-Ran-Snurportin1 export complex. Single-particle EM analysis demonstrates that, in a noncrystalline environment, free CRM1 exists in equilibrium between extended, superhelical and compact, ring-like conformations. Molecular dynamics simulations show that the C-terminal helix plays an important role in regulating the transition from an extended to a compact conformation and reveal how the binding site for nuclear export signals of cargoes is modulated by different CRM1 conformations. Combining these results, we propose a model for the cooperativity of CRM1 export complex assembly involving the long-range allosteric communication between the distant binding sites of GTP-bound Ran and cargo.