Research ArticleRESEARCH METHODS

High-speed AFM reveals accelerated binding of agitoxin-2 to a K+ channel by induced fit

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Science Advances  03 Jul 2019:
Vol. 5, no. 7, eaax0495
DOI: 10.1126/sciadv.aax0495
  • Fig. 1 Schematic illustrations and AFM imaging of AgTx2 binding to the extracellular surface of the KcsA channel.

    (A) X-ray crystallographic structure of the KcsA channel [Protein Data Bank (PDB) ID: 1K4C] (14) and solution structure of AgTx2 obtained by NMR (1AGT) (15). Gray dots are K+ ions. Only two diagonal subunits of the tetrameric channel are shown. The side chain of the K27 residue on AgTx2 is shown as a stick structure. (B) Arrangements of the reconstituted channel with (right) or without (left) AgTx2 on the substrate. (C) Typical AFM images of the channel with (right) or without (left) AgTx2. The KcsA channel is reconstituted in the DMPC bilayer. The AgTx2 is added to an imaging buffer. Height profiles along the white dotted lines in the AFM images are shown below the images. The background illustration behind the height profiles indicates the corresponding structures of the channel and AgTx2. The binding model (model II) (12) was downloaded from B. Roux’s web page (http://thallium.bsd.uchicago.edu/RouxLab/sub/gallery/agtx_shaker.html). Imaged samples were in 10 mM Hepes (pH 7.5) containing 200 mM KCl and 20 nM AgTx2. Scale bars, 2 nm. Z scale, 15 Å.

  • Fig. 2 HS-AFM captured AgTx2 binding to the channel with single-molecule resolution.

    (A) Time-lapse images of AgTx2 binding to and dissociation from the KcsA channels (top; see also movie S1) and time courses of the averaged height h (nm) around the center of the extracellular surface (0.8 nm by 0.8 nm area) of two corresponding K+ channels (bottom). White dotted squares represent regions of interest for visualization of the tetrameric channels. Spontaneous AgTx2 bindings on the channels are indicated by white arrowheads (top). These two channels showed discrete changes in the h (nm), representing bound and unbound states of the channels (bottom). The bathing electrolyte solution contains 10 mM Hepes (pH 7.5) and 100 mM KCl. The scale bar in the first AFM image represents 5 nm. (B) Height histograms at 10, 20, 100, and 1000 nM AgTx2. The height of the extracellular surface of the AgTx2-unbound channel was set to 0 nm. Height transitions used in the histograms were measured in 10 mM Hepes (pH 7.5) containing 200 mM KCl. The number of data frames used for the histograms were 8604, 250,846, 33,701 and 35,453 for 10, 20, 100, and 1000 nM AgTx2, respectively.

  • Fig. 3 Event-oriented analysis of AgTx2 binding.

    (A) Representative data of height transitions and definitions for the event-oriented analysis. The AgTx2-bound and AgTx2-unbound states are indicated by white and black arrowheads, respectively. Red and blue arrowheads indicate binding and dissociation events, respectively. For an arbitrary dissociation event (blue arrowhead), tpersist was defined from the instance of dissociation (blue). The time origin of Δt was the end of tpersist. The time variable definitions are the same as for the binding event (red arrowhead). (B and C) A representative dataset and the event-oriented analysis. The AgTx2-bound and AgTx2-unbound states are indicated by white and black arrowheads, respectively. All dissociation events were detected from the long-lasting height transition data, and strips of data before and after the event were collected and aligned at the dissociation events (Δt = 0) (blue arrowhead). Five examples are shown. For tpersist = 0 s (B), all the strips were ensemble-averaged, providing Pboundt) for tpersist = 0 s. For tpersist = 1 s (C), strips of data persisting in the unbound state longer than tpersist after the dissociation event were used for ensemble averaging. (D) Time course of the binding probability after dissociation. Pboundt) for different tpersist values ranging from immediately after dissociation (tpersist = 0 s) to 1 s from dissociation (tpersist = 1 s) are shown as a function of Δt measured from the end of tpersist. As tpersist increased, the time course of Pboundt) could be expressed by a single-exponential function. (E) Time course of the binding probability after binding. Pboundt) for different tpersist values from immediately after binding (tpersist = 0 s) to 1 s (tpersist = 1 s) after binding are shown. The binding transitions were measured in 10 mM Hepes (pH 7.5) containing 200 mM KCl and 20 nM AgTx2.

  • Fig. 4 The four-state binding model includes high- and low-affinity channels.

    (A) The four-state model with high and low affinity of AgTx2 binding. The optimized rate constants are shown. (B) The binding probability Pbound evaluated by experimental and theoretical calculations using the four-state model. The experimental Pbound values were calculated from the height histograms in Fig. 2B. (C) Binding transitions obtained by HS-AFM imaging and simulation. The rate constants as shown in (A) were used for the simulation at 10, 20, 100, and 1000 nM AgTx2 (top to bottom). White and black arrowheads indicate the AgTx2-bound and AgTx2-unbound states of the channel, respectively. Red and blue points indicate the high- and low-affinity states of channel, respectively. The sampling rate for the simulation of the binding transitions was 10 Hz. (D) The percentage of high-affinity channels (CH and CHTx) in the whole system calculated by kinetic model. (E) The percentage of the induced-fit pathway over the total (induced fit and conformational selection) pathway for binding. The preference was calculated by using the kinetic model.

  • Fig. 5 Binding dynamics of the noninactivating channel.

    (A) Side view of the KcsA channel and enlarged view of the selectivity filter region with the conductive state (PDB ID: 1K4C) and the collapsed state (1K4D). (B) Height transitions of the E71A and WT channels upon AgTx2 binding. White and black arrowheads indicate the AgTx2-bound and AgTx2-unbound states of the channels, respectively. The noninactivating mutant (E71A) showed markedly high affinity to AgTx2 compared with WT. The binding probabilities of AgTx2 to the E71A mutant obtained by HS-AFM imaging and simulation with only the high-affinity channel are plotted in Fig. 4B. (C and D) Time course of Pbound immediately after or for a persistence of up to 1 or 0.3 s from the dissociation (C) and the binding (D) events. Δt is the time elapsed from the end of tpersist. The height transitions used in this analysis were measured in 10 mM Hepes (pH 7.5) containing 200 mM KCl and 20 nM AgTx2.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/7/eaax0495/DC1

    Fig. S1. Binding transitions obtained by HS-AFM.

    Fig. S2. Dwell time distributions and the fitted time course of Pbound.

    Fig. S3. Simulated binding transitions.

    Fig. S4. The AgTx2 concentration dependence on kobs.

    Fig. S5. Boltzmann’s H function.

    Fig. S6. Dynamic disorder of binding transitions and correlation of individual Pbound values and interchannel distance.

    Fig. S7. Dwell time distributions obtained by dwell time analysis, event-oriented analysis, and correlation function.

    Fig. S8. Removing fluctuations in the baseline.

    Movie S1. HS-AFM movie of AgTx2 binding on the KcsA channels.

    Movie S2. HS-AFM movie showing dynamic disorder of binding transitions.

    Reference (61)

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Binding transitions obtained by HS-AFM.
    • Fig. S2. Dwell time distributions and the fitted time course of Pbound.
    • Fig. S3. Simulated binding transitions.
    • Fig. S4. The AgTx2 concentration dependence on kobs.
    • Fig. S5. Boltzmann’s H function.
    • Fig. S6. Dynamic disorder of binding transitions and correlation of individual Pbound values and interchannel distance.
    • Fig. S7. Dwell time distributions obtained by dwell time analysis, event-oriented analysis, and correlation function.
    • Fig. S8. Removing fluctuations in the baseline.
    • Reference (61)

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). HS-AFM movie of AgTx2 binding on the KcsA channels.
    • Movie S2 (.mp4 format). HS-AFM movie showing dynamic disorder of binding transitions.

    Files in this Data Supplement:

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