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Structure of myosin-1c tail bound to calmodulin provides insights into calcium-mediated conformational coupling

Nature Structural & Molecular Biology volume 22, pages 81–88 (2015)Cite this article

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Abstract

Class I myosins can sense cellular mechanical forces and function as tension-sensitive anchors or transporters. How mechanical load is transduced from the membrane-binding tail to the force-generating head in myosin-1 is unknown. Here we determined the crystal structure of the entire tail of mouse myosin-1c in complex with apocalmodulin, showing that myosin-1c adopts a stable monomer conformation suited for force transduction. The lever-arm helix and the C-terminal extended PH domain of the motor are coupled by a stable post-IQ domain bound to calmodulin in a highly unusual mode. Ca2+ binding to calmodulin induces major conformational changes in both IQ motifs and the post-IQ domain and increases flexibility of the myosin-1c tail. Our study provides a structural blueprint for the neck and tail domains of myosin-1 and expands the target binding modes of the master Ca2+-signal regulator calmodulin.

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Figure 1: Overall structure of the Myo1c tail bound to apo-CaM.
Figure 2: The N lobe of CaM bound to the Myo1c post-IQ domain adopts an uncoupled conformation.
Figure 3: Validation of the interaction between post-IQ Myo1c and CaM.
Figure 4: The new interaction between CaM and the Myo1c post-IQ domain is required for proper function of Myo1c.
Figure 5: Calcium-induced structural changes of the Myo1c tail.
Figure 6: Schematic model for Myo1c-mediated tethering between actin filaments and Myo1c-bound membranes.

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Acknowledgements

We thank the BL17U station at the Shanghai Synchrotron Radiation Facility for X-ray beam time; Y. Zhang for technical help; and members of the Zhang laboratory for comments on the manuscript. This work was supported by grants from the Research Grant Council of Hong Kong (663811, 663812, 664113, HKUST6/CRF/10, SEG_HKUST06, T13-607/12R and AoE/M09/12 to M.Z.) and the National Key Basic Research Program of China (2014CB910204 to M.Z.). We thank C. Petit (Institut Pasteur) for the full-length Myo1c.

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  1. Qing Lu and Jianchao Li: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.,

    Qing Lu, Jianchao Li, Fei Ye & Mingjie Zhang

  2. Center of Systems Biology and Human Health, School of Science and Institute for Advanced Study, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.,

    Fei Ye & Mingjie Zhang

  3. State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.,

    Mingjie Zhang

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  1. Qing Lu
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Contributions

Q.L., J.L. and M.Z. designed experiments and analyzed data. Q.L., J.L. and F.Y. performed experiments. Q.L., J.L. and M.Z. wrote the manuscript. M.Z. coordinated the research.

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Correspondence to Mingjie Zhang.

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Integrated supplementary information

Supplementary Figure 1 Myo1c tail binds to three apo-CaM.

(a) Sedimentation velocity curves fitting results of myo1c fragments with saturated amount of apo-CaM. IQ1-end and IQ2-end do not contain any fusion tags. CaM in complex with IQ3-end contained a Trx fusion tag, which was used to facilitate the purification of the complex. (b) The molecular masses of various Myo1c fragments with saturated amount of apo-CaM derived from sedimentation velocity analysis (the second column). Black star: the “IQ3-end” protein was purified in the Trx-fused form, and the mass of fusion tag was subtracted from “IQ3-end” for easy comparison. The measured molecular mass of “IQ1-end” is ~10 kD larger than the theoretical value, this discrepancy is likely due to the fitting error caused by friction ratio increase for the highly elongated shape of the complex. (c) The molecular masses of three Myo1c fragments, each saturated with apo-CaM, derived from FPLC coupled with static light scattering experiments (indicated on the top of each peak). The theoretical masses for the IQ1-852–(CaM)3, IQ2-852–(CaM)2 and IQ3-852–CaM are 68.6 kD, 49.1 kD and 29.8 kD, respectively.

Supplementary Figure 2 Electron density maps of the myo1c tail and detailed interactions of the myo1c post-IQ region with the N-terminal IQ3 motif and the C-terminal extended PH domain.

(a) Overview of the experimental determined Au positions (magenta, σ=4) and the phased electron density (blue, σ=1). (b) 2Fo-Fc electron density map (σ=1) of the whole tail. (c) Stereo views of 2Fo-Fc electron density map (Left, σ=1) and ribbon diagram (Right) of the post-IQ region in complex with N-lobe of CaM3. (d) 2Fo-Fc electron density map (Left, σ=1) and ribbon diagram (Right) of the extended PH domain. (e) The interface between the lever arm and post-IQ is mainly composed of hydrophobic interactions between IQ3 and α2. For clarity, CaM is not shown. (f) The interaction between post-IQ and the extended PH domain is mediated by hydrogen bonds and hydrophobic interactions between α4 of post-IQ and α5/α7 of the extended PH domain.

Supplementary Figure 3 Sequence alignment of the neck-tail domain of myo1c from vertebrates and different members of the class I human myosins.

Identical and highly similar residues are colored. The secondary structure elements are labeled using the same color coding scheme shown in Fig. 1b.

Supplementary Figure 4 NMR-based validation of the post-IQ–CaM interaction.

(a) 15N-HSQC spectra of 15N-labeled IQ3-852 in complex with 15N-labeled WT-CaM or with 15N-labeled F16C-CaM. The overlap of most of the peaks of the two spectra indicates that the F16C mutation of CaM does not alter the overall structure of the complex between CaM and IQ3-852. (b) 15N-HSQC spectra of 15N-labeled apo-CaM titrated with the IQ3 peptide. Color coding of the peaks corresponds to different apo-CaM/IQ3 ratios indicated in the figure. For clarity, only a few representative peaks are labeled. Peaks from C lobe that change are highlighted with black boxes.

Supplementary Figure 5 Flexibility change of Myo1c–CaM complex upon addition of Ca2+

(a&b) Time-dependent, partial trypsin digestions of Myo1c IQ1-end (a) and IQ2-end (b) in the absence or presence of Ca2+. Myo1c-CaM complexes were incubated in a digestion buffer with or without calcium (1 mM EDTA/CaCl2, 50 mM Tris PH 7.8, 100 mM NaCl, and 1 mM DTT) containing 1% trypsin (i.e. at a trypsin:Myo1c mass ratio of 1:100). Samples were withdrawn at 0, 1, 5, 15, 30 min after mixing with trypsin for SDS-PAGE analysis using Coomassie Blue staining. (c) Thrombin digestions of Myo1c IQ1-end (left) and IQ2-end (right) in the absence and presence of Ca2+. Myo1c-CaM complexes were cleaved with thrombin at 10 units/mg of complex for 10 h at 37 °C under the same buffer conditions as those in panels a&b. (d) CD spectra of Myo1c IQ2-852 in complex with calmodulin with or without Ca2+. To make sure that the relatively small ellipticity changes are reliable, an aliquot of apo-CaM-bound Myo1c IQ2-852 at a concentration of 4 μM complex (in the buffer of 1 mM EDTA, 50 mM Tris PH 7.8, 100 mM NaCl, and 1 mM DTT) was split into two equal portions. A few μl of concentrated CaCl2 was added to one sample to make the final Ca2+ to 2 mM, and the same volume of H2O was added to the other sample to make sure that the protein concentrations of the two samples for the CD experiments are identical. Upon addition of Ca2+, the ellipticity of IQ2-853 decreases (red curve). It has been well established that binding of Ca2+ to CaM stabilizes helical structures of the protein and thus leads to increase of the ellipticity. The decrease of the helical content of the IQ2-852 and CaM complex upon addition of Ca2+ is most likely resulted from the loss or destabilization of the α-helices from the Myo1c tail.

Supplementary Figure 6 Binding of IQ2-852 and IQ2-829 to Ca2+-CaM.

FPLC coupled with static light scattering analysis of the bindings of IQ2-852 (red line) and IQ2-829 (blue line) to saturated amount of Ca2+-CaM. All samples can be fitted with two CaM molecules bound to the Myo1c fragments, showing that α4 of post-IQ does not involve in binding to Ca2+-CaM.

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Lu, Q., Li, J., Ye, F. et al. Structure of myosin-1c tail bound to calmodulin provides insights into calcium-mediated conformational coupling. Nat Struct Mol Biol 22, 81–88 (2015). https://doi.org/10.1038/nsmb.2923

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