SUPPLEMENTARY INFORMATION

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1 Results Construct purification and coupling. Two A1-GP1bα ReaLiSM constructs, with and without cysteine residues near the N and C-termini (Fig. S2a), were expressed and purified by Ni affinity chromatography (Fig. S2b) and Superdex 200 gel filtration (Fig. S2c). ReaLiSM constructs both with and without cysteines were coupled to DNA (Fig. S2d). Only the construct with cysteines yielded handle-protein-handle product, whereas handle-handle byproduct was seen with both constructs, demonstrating specific DNA coupling to the introduced cysteines (Fig. S2d). Furthermore, only the construct with cysteines showed a band shifted to higher molecular weight with antibody to GP1bα (Fig. S2d). Note that the gels are stained for nucleic acid with SYBR green, and therefore only nucleic acid or nucleic acid-protein complexes are visualized. At slower rates of laser trap movement, multiple extension and contraction events were sometimes observed in a single cycle (Fig. S3). 1

2 Figure S1. Conceptual summary. Schematic model of flexed and extended states of the A1-GP1bα flex-bond, showing direction of tensile force (horizontal arrows), and estimated constants for conversion between the two states of the receptor-ligand bond (k 21 and k 12 ) and off-rates in absence of force (k 1off 0 and k 2off 0 and mechanical properties of the bonds showing how much force exponentiates off-rates (σ1 and σ2). A1 is shown in cyan, GP1ba in magenta, and a linker polypeptide that fuses them together as a grey curved line. 2

3 Figure S2. ReaLiSM construct design, purification, and coupling to DNA handles. a. Schematic design of covalently tethered VWF A1-GPIbα constructs. Constructs with N and C-terminal cysteines for coupling to DNA handles (protein construct) or without the added cysteines (control protein construct) were expressed and purified. Each construct was expressed and purified similarly. b. Protein purification using Ni-NTA affinity chromatography. All three fractions of the elution (column volumes 1-3) were concentrated and applied to the S200 size exclusion column. c. Purification on Superdex S200. Lanes 1-4 represent successive even-numbered fractions from the gel filtration column. The fraction shown in lane 3 was used for coupling to DNA handles. Panels b and c show nonreducing SDS 10% PAGE and Coomassie Blue staining; markers are in lane M. d. Protein coupling to DNA handles. Coupled material, with or without a mab to GPIbα (mab 6D1, from Dr. B. Coller, Rockefeller U., New York, NY) to assay a shift in migration, was subjected to electrophoresis in 4-20% native gels and stained with SYBR green nucleic acid gel stain. 3

4 Figure S3. Force-extension trace for one cycle of force increase (black) and decrease (red) for a representative cycle in which multiple unbinding and binding events were seen at 5 nm/s. Receptor-ligand unbinding and binding events are highlighted in yellow. Bead position (Z bead ) is calculated from trap position (Z trap ) as Z bead = Z trap - F/k, with an average stiffness k from several different beads. 4

5 Figure S4. The effect of the linker in A1-GP1bα ReaLiSM constructs. All results in the main text are with the 43-residue linker, except where a 26-residue linker is noted. (a) Fit of receptor-ligand extension (unbinding) data to the WLC model. Data is as shown in main text Fig. 1f, with the addition of data on the 26-residue linker (green symbols) and fit to WLC model (red line). This fit yielded a persistence length of 0.6 nm and contour length of 10.6 ± 0.6 nm. The expected contour length of 11.8 nm is calculated as the linker length of 26 residues times an extension of 3.8 Å/residue (9.9 nm), plus the N to C- terminal distances in the A1 (1.9 nm) and GPIbα (7.0 nm) crystal structures, minus the distance between the N-terminus of A1 and C-terminus of GPIbα in the complex structure (7.0 nm). (b and c) Distribution of unbinding forces with a 43-residue linker (b) and 26-residue linker (c). Error bars show Poisson noise as h k (h k is bin height). Loading rate averages and SD are over all rupture events at pulling rate. Curves show the predicted rupture force distributions using the constants estimated in Fig. 2g. 5

6 Figure S5. a. Extension over representative 60 s periods at pn. b. Segmentation of the extension data into bound and unbound intervals. 6

7 Figure S6. Comparison of off rates determined using different systems. Data from this publication on the A1-linker-GPIbα fusion protein- DNA handle ReaLiSM construct are shown in black open or filled symbols. Black lines show fits of this data to the Bell model. The circle with gray fill is from 1, and shows bulk phase measurements of soluble A1 domain dissociation from GPIbα on beads or on platelets (k off of and s - 1 ). Fitting of our data on k 1off at different forces to the Bell equation (dashed line) extrapolates to a k 1off 0 of s -1, close to the bulk phase results. Blue symbols show data from Fig. 2a of 2, on dissociation of A1 domains physically adsorbed to polystyrene Petri dishes from GPIbα (glycocalicin) physically adsorbed to atomic force microscope cantilevers. No fits to force spectroscopy dynamics equations were reported in 2 ; the blue dashed and solid lines are visual fits of their data to straight lines. The fit of the catch bond regime (dashed line) extrapolates to a k off 0 of 10 s -1, more than three orders of magnitude away from the ReaLiSM and bulk phase results. 7

8 Table S1. Estimates of flex-bond kinetics at three different forces. All four rates were estimated by fitting bond survival data (Fig. 3f to h) to equations S2-S4. Estimates with reasonable confidence intervals are obtained for k 1 off. However, because the other three parameters are correlated with one another, their confidence intervals are large. Force k 1 off, s -1 k 2 off, s -1 k 12, s -1 k 21, s pn pn pn Table S2. Values of k 12, k 21, and k 1 off estimated from force-clamp experiments using equations S2 to S4 when k 2 off was fixed at values estimated in Fig. 2g. Uncertainties are shown as 95% confidence intervals in parentheses. Force k 1 off, s -1 k 2 off, s -1 k 12, s -1 k 21, s pn 2.2 (1.1, 3.3) (0.32, 0.82) 0.12 (0, 0.28) pn 2.2 (1.9, 2.6) (0.35, 0.42) 0.34 (0.31, 0.38) pn 3.6 (2.4, 4.8) (0.79, 1.5) 0.75 (0.59, 0.91) 8

9 References 1 Miura, S. et al. Interaction of von Willebrand factor domain A1 with platelet glycoprotein Ibα (1 289). Slow intrinsic binding kinetics mediate rapid platelet adhesion. J. Biol. Chem. 275, (2000). 2 Yago, T. et al. Platelet glycoprotein Ibalpha forms catch bonds with human WT vwf but not with type 2B von Willebrand disease vwf. J Clin Invest 118, (2008). 9