Research ArticleBIOPHYSICS

Streptavidin/biotin: Tethering geometry defines unbinding mechanics

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Science Advances  25 Mar 2020:
Vol. 6, no. 13, eaay5999
DOI: 10.1126/sciadv.aay5999
  • Fig. 1 Force spectroscopy of the SA/biotin complex with different valences.

    (A) Crystal structure of SA. SA comprises four subunits, each consisting of a β barrel into which a biotin molecule can be bound. At the C terminus of subunit D, a unique cysteine is used as anchor point for site-specific covalent immobilization by maleimide–polyethylene glycol (PEG) linkers onto a functionalized glass surface. (B) Combining nonfunctional (light gray cylinders) and functional subunits (colored cylinders) allows preparation of SA of different valences. These different SA variants are immobilized at different areas on the surface: 0SA (gray), 1SA (red), 3SA (green), and 4SA (blue) are all examined with the same cantilever. Biotinylated ddFLN4 (purple) with an N-terminal Fgβ peptide (orange) is added to the solution. While biotin (magenta) binds to SA on the surface, the Fgβ peptide can bind to the SdrG domain (brown) immobilized on the cantilever. Retracting the cantilever, ddFLN4 unfolds, and biotin is pulled out of the binding pocket, while the force is recorded. A typical force extension trace is shown in the inset. (C) After sorting the force curves for specific interactions, i.e., for those showing the specific unfolding pattern of ddFLN4, unbinding force histograms are plotted and fitted with Bell-Evans distributions: 1SA (red) is fitted with a single Bell-Evans distribution. To fit 3SA (green), a double Bell-Evans distribution is needed. 4SA (blue lines) is fitted with a triple Bell-Evans. Furthermore, a combination of distributions of 1SA and 3SA can be fitted (red and green dotted lines).

  • Fig. 2 Course of a SMFS measurement.

    (A) For all interactions between cantilever tip and surface (higher than 50 pN), the rupture forces are shown. Interactions recorded on the 0SA spots are shown in black, 1SA spot in red, 3SA spot in green, and 4SA spot in blue. Most rupture forces are smaller than 500 pN. The rare events above 2000 pN correspond to the unbinding of the Fgβ from SdrG. (B) Zoom-in on the start of the measurement. The biotinylated Fgβ-ddFLN4-biotin construct was added after 1200 approach-retraction cycles; before that, only a few nonspecific interactions occur. At first, in every 250 curves, a different surface area is probed and then every 1000 curves. (C) Zoom-in on forces lower than 500 pN. The unbinding from the different SA subunits manifests itself in the clustering of unbinding events around 100, 220, and 450 pN. (D) Specific interactions only. For all interactions, for which the distinct two-step unfolding pattern of ddFLN4 is observed directly before the complex ruptures, the unbinding force is plotted.

  • Fig. 3 Direction dependent unbinding and lid opening.

    (A) Schematics of the force-loading geometries. To simplify MD simulations, biotin bound in subunit D (shown with surface representation) was anchored by the end of its molecular linker, while one of four subunits (A to D) was pulled by its C terminus. Colored lines indicate the four resulting force-loading directions (polymeric biotin linker is not shown). (B and C) The structure of SA stretched via its subunit C and the end of the polymeric linker of biotin bound in subunit D are shown before (B) and after (C) lid opening just before bond rupture. (D to F) Surface representation of SA shows how the stretching of biotin and its linker during subunit C pulling—from initial conformation at time 0 ns (D), to time 32 ns (E), to time 54 ns (F)—induces conformational changes in the binding pocket’s lid (colored by amino acid sequence).

  • Fig. 4 Results of SMD simulations.

    Pulling C termini of SA subunits while holding molecular linker of biotin bound to pocket in subunit D. (A) Exemplary force extension traces for the four geometries. (B) Resulting rupture force histograms fitted with Bell-Evans distributions. (C) Exemplary plots of the distance metric for the L3/4 loop opening (distance between α carbons of Gly48 and Leu124 residues) over time. The red dashed lines denote the moment at which biotin leaves the pocket. (D) Histograms of the distance metric for the L3/4 loop opening for the first 10 ns of the simulation (unloaded condition, gray) and for 10 ns just before the point of rupture (loaded condition, red).

  • Fig. 5 Force propagation pathways through the SA tetramer.

    (A to D) The force propagation pathway is shown for the different subunits close to the point of rupture. Force propagation pathways were obtained from cross-correlation–based network analysis calculated for all 100 replicas in a force-loaded condition. α carbon atoms serve as nodes that are connected by tubes of different diameters corresponding to how likely it is to have force transferred between them. SA is rotated to align the directions of force application horizontally. (E) Overlay of the force propagation pathway of subunits B and D. Within subunit D, the two are similar. For subunit D, a strong correlation is found between the molecular linker of biotin and the fourth β strand of subunit D, revealing a stabilization of the SA/biotin interaction pocket.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/13/eaay5999/DC1

    Fig. S1. SDS-PAGE of different SA variants.

    Fig. S2. SMFS measurements with direct covalent attachment of the biotinylated ddFLN4 domain to the cantilever tip.

    Fig. S3. Exemplary force extension traces.

    Fig. S4. Dynamic force spectrum.

    Fig. S5. Structure of biotin with the adjacent linker and illustration of the simulation box.

    Fig. S6. SMD force histograms.

    Fig. S7. Structure of the SA/biotin complex during L3/4 loop opening.

    Fig. S8. Angle metric for L3/4 loop opening.

    Fig. S9. Distance metric for L3/4 loop opening.

    Table S1. Fit parameters for the Bell-Evans distributions shown in the main text.

    Note S1. Fit parameters of Bell-Evans distributions.

    Note S2. Sequences of protein constructs.

    Movie S1. SA’s crystal structure with highlighted amine groups.

    Movie S2. Exemplary SMD: Holding biotin, pulling on the C terminus of SA subunit A.

    Movie S3. Exemplary SMD: Holding biotin, pulling on the C terminus of SA subunit B.

    Movie S4. Exemplary SMD: Holding biotin, pulling on the C terminus of SA subunit C.

    Movie S5. Exemplary SMD: Holding biotin, pulling on the C-terminus of SA subunit D.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. SDS-PAGE of different SA variants.
    • Fig. S2. SMFS measurements with direct covalent attachment of the biotinylated ddFLN4 domain to the cantilever tip.
    • Fig. S3. Exemplary force extension traces.
    • Fig. S4. Dynamic force spectrum.
    • Fig. S5. Structure of biotin with the adjacent linker and illustration of the simulation box.
    • Fig. S6. SMD force histograms.
    • Fig. S7. Structure of the SA/biotin complex during L3/4 loop opening.
    • Fig. S8. Angle metric for L3/4 loop opening.
    • Fig. S9. Distance metric for L3/4 loop opening.
    • Table S1. Fit parameters for the Bell-Evans distributions shown in the main text.
    • Note S1. Fit parameters of Bell-Evans distributions.
    • Note S2. Sequences of protein constructs.

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). SA’s crystal structure with highlighted amine groups.
    • Movie S2 (.mov format). Exemplary SMD: Holding biotin, pulling on the C terminus of SA subunit A.
    • Movie S3 (.mov format). Exemplary SMD: Holding biotin, pulling on the C terminus of SA subunit B.
    • Movie S4 (.mov format). Exemplary SMD: Holding biotin, pulling on the C terminus of SA subunit C.
    • Movie S5 (.mov format). Exemplary SMD: Holding biotin, pulling on the C-terminus of SA subunit D.

    Files in this Data Supplement:

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