1. Physics of Living Systems
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Increasing evidence of mechanical force as a functional regulator in smooth muscle myosin light chain kinase

  1. Fabian Baumann
  2. Magnus Sebastian Bauer
  3. Martin Rees
  4. Alexander Alexandrovich
  5. Mathias Gautel
  6. Diana Angela Pippig
  7. Hermann Eduard Gaub  Is a corresponding author
  1. Ludwig-Maximilians-Universität München, Germany
  2. King's College London BHF Centre of Research Excellence, United Kingdom
Research Article
Cite as: eLife 2017;6:e26473 doi: 10.7554/eLife.26473
5 figures

Figures

Overview of the experimental configuration for applying controlled mechanical stress to smMLCK.

(A) Schematic illustration of the investigated smMLCK construct. It consists of the kinase domain surrounded by several Ig-like domains (Ig) and a fibronectin-like domain (Fn3). Possible substrate interactions are indicated (ATP, Ca2+/CaM and RLC). RLC interaction is prevented by the auto-inhibitory pseudosubstrate sequence that is released upon Ca2+/CaM binding. For covalent attachment onto the surface, the construct harbors a C-terminal ybbR-tag. (B) Representative force-distance curves (red, blue) depicting the characteristic transitions of the kinase through different conformational states (S1, S2, S3) and subsequent unfolding of the adjacent Fn3 and Ig-like domains. Whereas most force-distance curves rupture before or after Fn3 unfolding (as shown in red) due to comparable rupture forces of Fn3 and the employed handle system, the blue curve illustrates a descriptive example with additional unfolding of Fn3 and Ig-like domains depicting the further force-distance pattern given by the construct. Structural interpretation and assignment of the detected force-distance pattern is schematically depicted above the curve. (C) Contour length transformation of 99 unfolding events with respective contour length increments. L12 and L23 are released at the transition of the kinase domain from conformational state S1 to S2 and S2 to S3 respectively. The contour lengths of Fn3 and Ig-like domains are additionally depicted.

https://doi.org/10.7554/eLife.26473.002
Figure 1—source data 1

Contour length plot of 99 unfolding events of MLCK with 0 mM ATP present, aligned as described in the data analysis section.

https://doi.org/10.7554/eLife.26473.003
Figure 2 with 2 supplements
Structural effects of ATP binding on smMLCK’s characteristic sequence of conformational states.

(A) Stabilization of the S1→S2 transition upon ligand binding. For better illustration, a heatmap of 560 aligned curves is depicted. (B) Contour-length transformation of the respective events in the presence of ATP. Lij is associated to the contour length released at the transition from state Si to Sj. (C) Statistical evaluation of S1→S2 stabilization via force histograms fitted with the Bell-Evans model. An increase in the most probable transition force of about 30 pN is observed upon ATP addition. Both data sets were recorded within one experiment with the same cantilever.

https://doi.org/10.7554/eLife.26473.004
Figure 2—source data 1

Contour length plot of 560 unfolding events of MLCK in the presence of 3 mM ATP, aligned as described in the data analysis section.

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Figure 2—source data 2

Force histogram of S1→S2 transition in the presence of 0 mM ATP.

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Figure 2—source data 3

Force histogram of S1→S2 transition in the presence of 3 mM ATP.

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Figure 2—figure supplement 1
Effects of ATP or Ca2+/CaM addition on the peak forces for the respective transitions S1→S2 and S2→S3 and for the Fn3 unfolding force.

All data was collected within one experiment and absolute force values can directly be compared. Whereas S1→S2 is stabilized by ATP as described in the main part of the manuscript, S2→S3 and Fn3 appear not to be significantly changed by substrate interaction. The force histograms of the Fn3 domain are used in other experiments for normalizing forces to the same value.

https://doi.org/10.7554/eLife.26473.008
Figure 2—figure supplement 1—source data 1

Force histogram of S1→S2 transition in the presence of 0 mM ATP.

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Figure 2—figure supplement 1—source data 2

Force histogram of S1→S2 transition in the presence of 3 mM ATP.

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Figure 2—figure supplement 1—source data 3

Force histogram of S1→S2 transition in the presence of 3 mM ATP, 25 µM CaM, 2 mM Ca2+.

https://doi.org/10.7554/eLife.26473.011
Figure 2—figure supplement 1—source data 4

Force histogram of S2→S3 transition in the presence of 0 mM ATP.

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Figure 2—figure supplement 1—source data 5

Force histogram of S2→S3 transition in the presence of 3 mM ATP.

https://doi.org/10.7554/eLife.26473.013
Figure 2—figure supplement 1—source data 6

Force histogram of S2→S3 transition in the presence of 3 mM ATP, 25 µM CaM, 2 mM Ca2+.

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Figure 2—figure supplement 1—source data 7

Force histogram of Fn3 unfolding in the presence of 0 mM ATP.

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Figure 2—figure supplement 1—source data 8

Force histogram of Fn3 unfolding in the presence of 3 mM ATP.

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Figure 2—figure supplement 1—source data 9

Force histogram of Fn3 unfolding in the presence of 3 mM ATP, 25 µM CaM, 2 mM Ca2+.

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Figure 2—figure supplement 2
Stabilization of the S1→S2 transition upon ADP or AMP-PNP binding.
https://doi.org/10.7554/eLife.26473.018
Figure 2—figure supplement 2—source data 1

Force histogram of S1→S2 transition in the presence of 3 mM AMP-PNP, 30 µM CaM, 3 mM Ca2+, 280 µM RLC.

https://doi.org/10.7554/eLife.26473.019
Figure 2—figure supplement 2—source data 2

Force histogram of Fn3 unfolding in the presence of 3 mM AMP-PNP, 30 µM CaM, 3 mM Ca2+, 280 µM RLC is used for normalizing forces to the same value.

Actual histogram of Fn3 forces is not shown here and is just used for normalization.

https://doi.org/10.7554/eLife.26473.020
Figure 2—figure supplement 2—source data 3

Force histogram data of S1→S2 transition in the presence of 4 mM ADP.

https://doi.org/10.7554/eLife.26473.021
Figure 2—figure supplement 2—source data 4

Force histogram of the Fn3 unfolding in the presence of 4 mM ADP is used for normalizing forces to the same value.

Actual histogram of Fn3 forces is not shown here and is just used for normalization.

https://doi.org/10.7554/eLife.26473.022
Figure 3 with 1 supplement
Structural effects of Ca2+/CaM binding on smMLCK’s characteristic sequence of conformational states.

(A) Attenuated S0→S1 transition in the characteristic force-distance pattern of the smMLCK construct due to conformational changes upon Ca2+/CaM binding. This effect is emphasized by a heatmap comparison of several hundred overlaid force-distance curves. Both data sets were collected within one measurement. (B) Structural model interpretation. The S0→S1 transition is assigned to a force-induced rearrangement in the kinase domain that correlates with the conformational changes induced by Ca2+/CaM binding – the release of the inhibitory pseudosubstrate.

https://doi.org/10.7554/eLife.26473.023
Figure 3—figure supplement 1
Missing effects by addition of Ca2+ without CaM.

The atypical stretching behavior indicating a transition from state S0 to S1 is still observable in the presence of Ca2+: only in combination with CaM is the barrier not detected in the unfolding pattern.

https://doi.org/10.7554/eLife.26473.024
Figure 4 with 1 supplement
Structural effects of RLC peptide binding on smMLCK’s characteristic sequence of conformational states.

(A) Qualitative observation of an increased mechanical stability in the large kinase lobe illustrated by the higher forces in the S2→S3 transition. The effect is emphasized by heatmaps of aligned force-distance curves obtained under different substrate conditions. The stabilizing effect is detected independently of the presence of ATP. (B) Quantitative evaluation of the increased S2→S3 transition force. The force histograms were approximated with a kernel-density function for extracting the most probable rupture force. It reveals a significant shift of about 30 pN due to the stable interaction of the RLC peptide with the catalytic core. Since this binding is stated to be prevented by an auto-inhibition process according to the conventional view of smMLCK activation, the experimental observation hints at an additional path of kinase regulation modulated by force.

https://doi.org/10.7554/eLife.26473.025
Figure 4—source data 1

Force histogram of S2→S3 transition in the presence of 3 mM ATP.

https://doi.org/10.7554/eLife.26473.026
Figure 4—source data 2

Force histogram of S2→S3 transition in the presence of 3 mM ATP and 280 µM RLC.

https://doi.org/10.7554/eLife.26473.027
Figure 4—source data 3

Force histogram of S2→S3 transition in the presence of 280 µM RLC.

https://doi.org/10.7554/eLife.26473.028
Figure 4—figure supplement 1
Ca2+/CaM-dependent RLC phosphorylation of the investigated smMLCK construct.

Western blot probing RLC phospho-serine 19 following smMLCK in vitro kinase assay time-course shows only phosphorylation in presence of Ca2+/CaM.

https://doi.org/10.7554/eLife.26473.029
Structural interpretation of the stabilized S2 state upon RLC interaction.

Mechnical stress forces the construct into a conformational state S1 equivalent to the state reached by Ca2+/CaM binding. By release of the pseudosubstrate sequence the conformational state is capable of RLC binding which is detected by a significant stabilization of the S2 state. Both initially different activation pathways eventually result in the same sequence of conformational states, with the only difference being the presence or absence of bound Ca2+/CaM, depicted in light grey in the S2 state.

https://doi.org/10.7554/eLife.26473.030

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