Supplementary Information for the article€¦ · Supplementary Information for the article “Robust processivity of myosin-V under off-axis loads” by Yusuke Oguchi, Sergey V

Supplementary Information for the article

“Robust processivity of myosin-V under off-axis loads”

by Yusuke Oguchi, Sergey V. Mikhailenko, Takashi Ohki, Adrian O. Olivares,

Enrique M. De La Cruz & Shin’ichi Ishiwata

Supplementary Results

Supplementary References

Supplementary Tables 1– 3

Supplementary Figures 1– 15

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Nature Chemical Biology: doi: 10.1038/nchembio.322

Supplementary Results

Effect of the off-axis loads on myosin-V stepping. To apply the off-axis load to

myosin-V during processive stepping, the stage was rapidly displaced perpendicularly

to the long axis of actin filament in either of the two directions, such that off-axis load

was applied at either positive or negative angle (Fig. 5). The long axis of actin filament

and the direction of stage displacement were defined as x-axis and y-axis, respectively.

The trap stiffness was 0.019 pN nm–1. The magnitude of stage displacement was

adjusted for each tested bead so that the value of the y-component of the backward load

was 1–2 pN in the stall region, corresponding to approximately –20° or +20° loading

angle (see Supplementary Tables 2 and 3 for the exact values). The stage displacement

was therefore in the range of 100–400 nm, apparently depending on the manner of

myosin-V attachment to the bead. 82 and 60 events were recorded for –20° and +20°

loading angles, respectively. To reliably detect the effect of the off-axis loads on the

processive stepping of myosin-V in the stall region, only the events when the y-axis

load was applied to the myosin-V before the motor had entered the stall region were

counted (namely, 30 and 21 traces of processive movement under –20° and +20°

off-axis load, respectively).

The effect of the off-axis loads on the processive stepping of myosin-V was

characterized by measuring two parameters, the value of the stall force and the number

of steps in the stall region. The stall force was defined here as an on-axis component of

load at which myosin bead dissociates from actin. We found that the stall force was 2.6

± 0.1 pN at both –20° and +20° and 2.3 ± 0.04 pN at 0° (n=164), all data are presented

as mean ± s.e.m.

After a motor had reached the stall region, the back-and-forth movement was

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observed. In addition to characterizing the stepping behavior of myosin-V at stall by

measuring the average dwell time of all steps (Supplementary Fig. 14), we also

analyzed the dwell time separately for the forward and the backward steps

(Supplementary Fig. 15). No noticeable effect of the off-axis loads was detected at

both –20° or +20° loads, as the observed small differences in the dwell time values

correlate with the difference in the corresponding load, namely, the larger load prolongs

the dwell time.

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Supplementary References

1. Oguchi, Y. et al. Load-dependent ADP binding to myosins V and VI: Implications for subunit

coordination and function. Proc. Natl. Acad. Sci. USA. 105, 7714–7719 (2008).

2. Ali, M.Y. et al. Myosin V is a left-handed spiral motor on the right-handed actin helix. Nat. Struct.

Biol. 9, 464–467 (2002).

3. Robblee, J.P., Cao, W., Henn, A., Hannemann, D.E. & De La Cruz, E.M. Thermodynamics of

nucleotide binding to actomyosin V and VI: a positive heat capacity change accompanies strong

ADP binding. Biochemistry 44, 10238–10249 (2005).

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Supplementary Table 1. Characteristic distances for the actin–myosin and myosin–ADP interaction under forward and backward loads at various angles determined by model-based analysis as we previously reported1, for 6IQ and 1IQ constructs. dD and dφ are the characteristic distances for actomyosin bond in the ADP-bound and the nucleotide-free states, respectively. and are the characteristic distances for ADP dissociation and binding rates, respectively. Asterisk denotes values determined in ref. 1.

−ADPd +

ADPd

d D, nm d φ, nm d -ADP, nm d +

ADP, nm d D, nm d φ, nm d -ADP, nm d +

ADP, nm-45° 9.4 ± 0.3 8.0 ± 0.5 -1.8 ± 0.4 5.0 ± 0.2 9.4 ± 0.2 6.7 ± 2.8 4.1 ± 0.2 7.5 ± 0.9-20° 9.3 ± 0.5 7.1 ± 0.8 -0.1 ± 0.3 5.0 ± 0.5 7.2 ± 0.6 7.0 ± 0.7 6.2 ± 1.8 4.2 ± 2.70°* 9.8 ± 1.2 7.1 ± 2.2 -1.3 ± 0.2 4.2 ± 0.8 5.8 ± 2.2 6.5 ± 1.3 6.4 ± 1.1 5.7 ± 0.9

+20° 9.7 ± 0.5 8.3 ± 0.6 -1.3 ± 0.6 5.0 ± 0.4 6.4 ± 0.4 9.3 ± 0.3 2.6 ± 1.1 1.8 ± 0.3+45° 9.8 ± 0.2 8.0 ± 0.2 -1.3 ± 0.4 5.4 ± 0.5 8.9 ± 0.5 9.0 ± 0.7 －－

d D, nm d φ, nm d -ADP, nm d +

ADP, nm d D, nm d φ, nm d -ADP, nm d +

ADP, nm-45° 9.4 ± 0.4 8.1 ± 0.4 -0.7 ± 0.5 5.0 ± 0.5 9.4 ± 0.5 6.7 ± 0.2 0.6 ± 0.4 4.0 ± 1.0-20° 9.4 ± 0.7 7.0 ± 0.2 -0.4 ± 0.2 4.4 ± 0.3 7.1 ± 0.3 8.0 ± 0.4 5.0 ± 0.3 1.0 ± 0.10° 9.0 ± 1.0 6.5 ± 1.1 -0.5 ± 0.4 5.2 ± 0.8 7.3 ± 0.6 6.9 ± 0.1 -0.2 ± 0.3 4.9 ± 0.5

+20° 9.5 ± 1.6 6.9 ± 0.4 -1.5 ± 0.3 5.4 ± 0.3 9.3 ± 0.4 6.8 ± 0.2 -0.4 ± 0.4 5.1 ± 0.4+45° 9.5 ± 0.5 7.1 ± 0.2 -1.6 ± 0.2 5.5 ± 0.3 6.6 ± 0.3 7.0 ± 0.4 5.0 ± 1.5 2.9 ± 1.3

Backward load

Forward load Backward loadCharacteristic distances for 6IQ

Characteristic distances for 1IQForward load

Angle

Angle

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Supplementary Table 2. Summary of the forces at stall under the off-axis loads. The

loading angle, the values of the total load (Fx+y) and its on-axis (Fx) and off-axis (Fy)

components at the moment of an actomyosin bond rupture are averaged over all traces.

All data are presented as mean ± s.e.m.

On-axis load-20° +20° 0°

N 30 21 164angle (°) -26.9 ± 2.38 +26.0 ± 2.43 -2.22 ± 0.82

Fx (=stall force) (pN) 2.63 ± 0.09 2.56 ± 0.14 2.35 ± 0.04Fy (pN) 1.40 ± 0.14 1.28 ± 0.14 0.37 ± 0.02

Fx+y (pN) 3.10 ± 0.12 2.92 ± 0.16 2.39 ± 0.04

Off-axis load

Supplementary Table 3. Parameters summarizing the back-and-forth stepping in the stall region under the off-axis loads. The average number of back-and-forth steps in the stall region was determined by fitting the distributions to the single exponentials. The average loading angle, the total load (Fx+y) and its on-axis (Fx) and off-axis (Fy) components were determined by averaging the according values during all dwells observed in the stall region for each record and further averaged for all traces. All data are presented as mean ± s.e.m.

On-axis load-20° +20° 0°

N 30 21 164angle (°) -26.3 ± 1.40 +29.5 ± 1.70 -1.94 ± 0.41

number of steps 2.3 ± 0.3 0.9 ± 0.6 2.8 ± 0.1Fx (pN) 2.43 ± 0.05 2.32 ± 0.07 2.13 ± 0.02Fy (pN) 1.29 ± 0.08 1.37 ± 0.10 0.32 ± 0.01

Fx+y (pN) 2.83 ± 0.07 2.78 ± 0.09 2.16 ± 0.02

Off-axis load

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Supplementary Figure 1. Schematic illustration of the off-axis load. Myosin-V follows the left-handed helix during the unconstrained movement around actin filament in vitro, recording 34.6-nm steps, which correspond to landing on the 11th and 13th actin subunits2. The direction of the intramolecular load thus deviates from the filament’s axis.

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Supplementary Figure 2. Schematic representation of the experimental setup. The angle between the direction of stage displacement and the axis of actin filament was –45º, –20º, 0º, +20º, or +45º, and load was applied towards the barbed (B-) or the pointed (P-) end (the forward or the backward loading, respectively).

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Supplementary Figure 3. Effect of the lever length on the actin-binding affinity in the nucleotide-free (a) and the ADP-bound state (b) plotted as the difference in the unbinding forces between 6IQ and 1IQ constructs at each loading angle.

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Supplementary Figure 4. Unbinding force distributions for 6IQ construct at various loading angles in the range of [ADP] (0–1 mM). The slender curves show fits to double Gaussians (see Text for details).

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Supplementary Figure 5. Unbinding force distributions for 1IQ construct at various loading angles in the range of [ADP] (0–1 mM). The slender curves are fits to double Gaussians (see Text for details).

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Supplementary Figure 6. Dependence of the average unbinding forces on [ADP]. Average unbinding forces gradually decrease with an increase in [ADP], which correlates with weaker unbinding force in the ADP-bound state compared to the nucleotide-free state (with the exception of the backward load at +20º, where the relation is opposite).

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Supplementary Figure 7. Dependence of Kd1 (a, c) and Kd2 (b, d) on loading angle under forward and backward loads for 6IQ (a, b) and 1IQ (c, d) constructs. The lines, which divide the panel into different colors, denote values of Kd1 and Kd2 under no load from ref. 3. The intermediate state in 6IQ construct (e) and 1IQ construct (f) is stabilized by backward loads in the –20º to –45º range and the +10º to –45º range, respectively.

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Supplementary Figure 8. Unbinding force distributions modeled with the obtained characteristic distances for 6IQ construct.

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Supplementary Figure 9. Unbinding force distributions modeled with the obtained characteristic distances for 1IQ construct.

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Supplementary Figure 10. Load-dependence of ADP dissociation rates. The ADP dissociation rates modulated by forward and backward loads at various angles in 6IQ construct (a) and 1IQ (b) construct, respectively. In all panels, colored areas represent the parameter error.

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Supplementary Figure 11. Load-dependence of ADP binding rates. The ADP binding rates modulated by forward and backward loads at various angles in 6IQ construct (a) and 1IQ construct (b), respectively. In all panels, colored areas represent the parameter error.

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Supplementary Figure 12. The typical traces of the stepping motion under external backward loads at 0°. The top and middle panels show traces of the bead displacement in the x and y directions, respectively. The bottom panel shows the y-displacement of the stage.

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Supplementary Figure 13. Distributions of the number of steps in the stall region under –20°, 0°, and +20° directional load are fitted with single exponentials, yielding the average number of steps for each loading angle. All data are presented as mean ± s.e.m.

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Supplementary Figure 14. Dwell time distributions fitted with single exponentials (a) and the obtained average dwell times (b) in the stall region under –20°, 0°, and +20° load. The values of load shown in (a) are calculated by averaging loads at stall during all dwells (Supplementary Table 3). All data are presented as mean ± s.e.m.

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Supplementary Figure 15. Dwell time distributions obtained separately for the backward (a) and forward (c) steps fitted with single exponentials to obtain the average dwell times for the backward (b) and forward (d) steps in the stall region under directional loads. The values of load shown in (a) and (c) are calculated by averaging loads during dwell prior to the backward and forward steps. All data are presented as mean ± s.e.m.

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Supplementary Information for the article€¦ · Supplementary Information for the article “Robust processivity of myosin-V under off-axis loads” by Yusuke Oguchi, Sergey V