Motor domain phosphorylation increases nucleotide exchange and turns MYO6 into a faster and stronger motor

doi:10.1038/s41467-024-49898-3

. 2024 Aug 7;15(1):6716.

doi: 10.1038/s41467-024-49898-3.

Motor domain phosphorylation increases nucleotide exchange and turns MYO6 into a faster and stronger motor

Janeska J de Jonge^#¹, Andreas Graw^#^{2

3}, Vasileios Kargas^{1

4

5}, Christopher Batters^{1

2

3}, Antonino F Montanarella^{2

3}, Tom O'Loughlin¹, Chloe Johnson¹, Susan D Arden¹, Alan J Warren^{1

4

5}, Michael A Geeves⁶, John Kendrick-Jones⁷, Nathan R Zaccai¹, Markus Kröss^{2

3}, Claudia Veigel^{8

9}, Folma Buss¹⁰

Affiliations

¹ Cambridge Institute for Medical Research, Department of Clinical Biochemistry, University of Cambridge, Cambridge Biomedical Campus, The Keith Peters Building, Hills Road, Cambridge, CB2 0XY, UK.
² Department of Cellular Physiology, Biomedical Centre (BMC), Ludwig-Maximilians-Universität München, Grosshadernerstrasse 9, 82152, Planegg-Martinsried, Germany.
³ Centre for NanoScience (CeNS), Ludwig-Maximilians-Universität München, Schellingstrasse 4, 80799, München, Germany.
⁴ Wellcome MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK.
⁵ Department of Haematology, University of Cambridge, Cambridge, UK.
⁶ School of Biosciences, University of Kent, Canterbury, UK.
⁷ MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, CB2 0QH, UK.
⁸ Department of Cellular Physiology, Biomedical Centre (BMC), Ludwig-Maximilians-Universität München, Grosshadernerstrasse 9, 82152, Planegg-Martinsried, Germany. Claudia.Veigel@med.lmu.de.
⁹ Centre for NanoScience (CeNS), Ludwig-Maximilians-Universität München, Schellingstrasse 4, 80799, München, Germany. Claudia.Veigel@med.lmu.de.
¹⁰ Cambridge Institute for Medical Research, Department of Clinical Biochemistry, University of Cambridge, Cambridge Biomedical Campus, The Keith Peters Building, Hills Road, Cambridge, CB2 0XY, UK. fb207@cam.ac.uk.

^# Contributed equally.

PMID: 39112473
PMCID: PMC11306250
DOI: 10.1038/s41467-024-49898-3

Motor domain phosphorylation increases nucleotide exchange and turns MYO6 into a faster and stronger motor

Janeska J de Jonge et al. Nat Commun. 2024.

. 2024 Aug 7;15(1):6716.

doi: 10.1038/s41467-024-49898-3.

Authors

Affiliations

¹ Cambridge Institute for Medical Research, Department of Clinical Biochemistry, University of Cambridge, Cambridge Biomedical Campus, The Keith Peters Building, Hills Road, Cambridge, CB2 0XY, UK.
² Department of Cellular Physiology, Biomedical Centre (BMC), Ludwig-Maximilians-Universität München, Grosshadernerstrasse 9, 82152, Planegg-Martinsried, Germany.
³ Centre for NanoScience (CeNS), Ludwig-Maximilians-Universität München, Schellingstrasse 4, 80799, München, Germany.
⁴ Wellcome MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK.
⁵ Department of Haematology, University of Cambridge, Cambridge, UK.
⁶ School of Biosciences, University of Kent, Canterbury, UK.
⁷ MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, CB2 0QH, UK.
⁸ Department of Cellular Physiology, Biomedical Centre (BMC), Ludwig-Maximilians-Universität München, Grosshadernerstrasse 9, 82152, Planegg-Martinsried, Germany. Claudia.Veigel@med.lmu.de.
⁹ Centre for NanoScience (CeNS), Ludwig-Maximilians-Universität München, Schellingstrasse 4, 80799, München, Germany. Claudia.Veigel@med.lmu.de.
¹⁰ Cambridge Institute for Medical Research, Department of Clinical Biochemistry, University of Cambridge, Cambridge Biomedical Campus, The Keith Peters Building, Hills Road, Cambridge, CB2 0XY, UK. fb207@cam.ac.uk.

^# Contributed equally.

PMID: 39112473
PMCID: PMC11306250
DOI: 10.1038/s41467-024-49898-3

Abstract

Myosin motors perform many fundamental functions in eukaryotic cells by providing force generation, transport or tethering capacity. Motor activity control within the cell involves on/off switches, however, few examples are known of how myosins regulate speed or processivity and fine-tune their activity to a specific cellular task. Here, we describe a phosphorylation event for myosins of class VI (MYO6) in the motor domain, which accelerates its ATPase activity leading to a 4-fold increase in motor speed determined by actin-gliding assays, single molecule mechanics and stopped flow kinetics. We demonstrate that the serine/threonine kinase DYRK2 phosphorylates MYO6 at S267 in vitro. Single-molecule optical-tweezers studies at low load reveal that S267-phosphorylation results in faster nucleotide-exchange kinetics without change in the working stroke of the motor. The selective increase in stiffness of the acto-MYO6 complex when proceeding load-dependently into the nucleotide-free rigor state demonstrates that S267-phosphorylation turns MYO6 into a stronger motor. Finally, molecular dynamic simulations of the nucleotide-free motor reveal an alternative interaction network within insert-1 upon phosphorylation, suggesting a molecular mechanism, which regulates insert-1 positioning, turning the S267-phosphorylated MYO6 into a faster motor.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Fig. 1. DYRK2 phosphorylates MYO6 at S267 in the motor domain in vitro.**
a SDS-PAGE of GFP-tagged MYO6 immunoprecipitated from RPE cell lysate using GFP nanobodies (right) and endogenous MYO6 immunoprecipitated from A431 cell lysate using MYO6 antibodies (left). A representative image is shown of the experiment performed more than 10 times. The arrows indicate the position of GFP-MYO6 or endogenous MYO6. b MYO6 sequence peptide coverage by mass spectrometry. Identified peptides shown in shades of grey. c Sequence of the identified phosphopeptide LHLSSPDNFR for the S267 and VSLTTRVMLTTAGGTKGTVIK for the T405 and site probabilities for S266 and S267 or T405. d Predicted *AlphaFold* (v2.0) structure of MYO6: motor domain (grey), neck domain (green/turquoise), neck domain extension (gold), SAH domain (pink) and C-terminal cargo-binding domain (blue). The inset A shows the position of S267 (red) and insert-1 (yellow) and inset B highlights the position of T405 (red) in the surface loop (purple) at the actin binding site. e Conservation of MYO6 at S267 across species. f Pie charts illustrating the percentages of S267 or T405 phosphorylation in MYO6 immunoprecipitated from A431 cells grown in serum-free media (starved) or after stimulation with EGF for 15 min or phorbol 12-myristate 13-acetate (PMA) for 20 min. g Table summarising the percentages of S267 or T405 phosphorylation for MYO6 immunoprecipitated from A431 cells grown in serum-free media (starved) or after stimulation with EGF for 15 min or phorbol 12-myristate 13-acetate (PMA) for 20 min.

**Fig. 2. DYRK2 specifically phosphorylates the MYO6 S267 peptide and the MYO6 full-length protein.**
a Activities (HotSpot^TM kinase assay) of 31 kinases with respect to the MYO6 peptide, compared with their ideal substrates. b A representative SDS-PAGE image of recombinant DYRK2 purified from *E. coli*. The purification has been performed several times. c Schematic overview of the ADP-glow assay used to measure DYRK2 activity. d ADP-glow^TM kinase assays show that recombinant DYRK2 has ATPase activity with respect to both DYRK-specific substrate and wild-type MYO6 peptide containing the SSP and the ASP sequence, but not to peptides with the mutant SAP or AAP sequence. e DYRK2 shows ATPase activity towards the MYO6 motor domain in vitro. Before the ADP-glow assay full length MYO6 was heat-treated at 37 °C or 60 °C to inactivate endogenous ATPase activity that would interfere with the kinase assay. f MYO6 was immunoprecipitated from A431 cells grown in serum-free media (starved) and incubated with purified DYRK2 for 1 h before determining the level of S267 phosphorylation by mass spectrometry. g DYRK2 does not phosphorylate MYO6 at T405 using the wildtype MYO6 T405 peptide in the ADP-glow assay in vitro.

**Fig. 3. Increased actin-gliding velocity of MYO6^S267E in vitro is achieved by faster nucleotide-exchange rates without change in the W/S of the motor.**
a Scheme of the in vitro actin-gliding assay. Monomeric MYO6, immobilised on a nitrocellulose-coated glass surface, translocates rhodamine-phalloidin labelled F-actin in the presence of ATP. b, c Actin-gliding velocity (2 mM ATP, 22 °C) increased 4-fold from 45 nm s⁻¹ (MYO6^WT) and 56 nm s⁻¹ (MYO6^S267A) to 172 nm s⁻¹ for MYO6^S267E. 3 independent experiments from 3 protein purifications were performed; Statistical analysis was performed using an unpaired two-sided t-test. ***P < 0.01. MYO6^WT n = 567, average 0.045 µm/s SD ± 0.008283, MYO6^S267A n = 557, average 0.0557 µm/s SD ± 0.010488, MYO6^S267E n = 569, average 0.1725 µm/s SD ± 0.0345.; d Scheme of the inverted in vitro motility assay. Dimerised MYO6 translocates on fascin-stabilised actin bundles. e Fraction of processive runs of MYO6^WT (48.6%), MYO6^S267A (36.2%) and MYO6^S267E (24%). f Median run-length of MYO6^WT, MYO6^S267A and MYO6^S267E was determined in the inverted motility assay using 3 different flow cells for each protein. Statistical analysis was performed using the Kruskal-Wallis test by ranks, a non-parametric method for testing whether samples originate from the same distribution. It is used for comparing two or more independent samples of equal or different sample sizes. ***P < 0.01. MYO6^WT n = 7926, average 0.22 µm SEM ± 0.0047, MYO6^S267A n = 3981, average 0.148 µm SEM ± 0.0058 and MYO6^S267E n = 2367, average 0.083 µm SEM ± 0.0061. g Actin-gliding velocity (2 mM ATP, 22 °C) for the double mutants MYO6^S267A/E plus MYO6^T406A/E were determined by the mutation at S267 and independent of the mutation at T405 (S267A+T406A 50 ± 14 nm s⁻¹; S267A+T405E 41 ± 17 nm s⁻¹; S267E+T405A 134 ± 25 nm s⁻¹; S267E+T405E 133 ± 35 nm s⁻¹ mean ± SD, N = 51 filaments for each double mutant, ***P < 0.01, unpaired two-sided t-test; 3 experiments from 3 different protein preparations, Fig. 4). h Scheme of single-molecule mechanical experiments using optical tweezers. i Raw data traces of single MYO6^S267A and MYO6^S267E molecules interacting with F-actin (single trap-stiffness κ_trap ~ 0.02 pN nm⁻¹; 100 µM ATP). MYO6 binding events to actin were detected by changes in the variance of thermal motion. Grey bars indicate actin-attached dwell times. j To determine the working stroke (W/S) for the MYO6^S267A and MYO6^S267E the displacement distribution of actin-binding events was analysed at 100 µM ATP. k Characterisation of the apparent duty ratio and apparent on-rate of actin binding for the MYO6 A-and E-mutants. Source data are provided as a Source Data file.

**Fig. 4. Kinetics from single molecule mechanical experiments and gliding filament assays on the monomeric MYO6 phospho-mutants.**
Kinetics from single molecule mechanical experiments and gliding-filament assay on the monomeric MYO6 phospho-mutants. a Scheme of the chemo-mechanical ATPase cycle of a single motor head. b Rate constants for single molecule MYO6 interactions with F-actin measured using optical tweezers at 22 °C, single trap stiffness ktrap 0.02 pN nm⁻¹. The cumulative dwell time distributions were fitted with a double exponential function, with f(t) = A₁ exp(−k₁ t) + A₂ exp(−k₂ t). StdErr = standard error, N = number of binding events in each condition, R² = regression coefficient for the fit function. The ensemble average displacement data and stiffness data were fitted by single exponentials. The load dependence of the rates k₁ and k₂ was obtained by fitting, using k = k_o exp(−Fd /k_BT), with ko rate at zero load, F force, d distance parameter and k_BT thermal energy. All single molecule mechanical data were obtained from at least 10 different motor heads in all different conditions. For comparison, values measured here and previously for MYO6^WT in single molecule mechanical experiments (SMM) and in bulk solution (stopped-flow, SF) were included in the ‘Literature’ section of the table; (a) Lister et al., (b) Altman et al., (c) DeLaCruz et al., (d) Polypenko et al.. Gliding filament assay for monomeric double mutants S267A/E plus T405A/E; mean velocity and standard deviation SD.

**Fig. 5. Single-molecule mechanics and solution kinetics reveal faster ADP-release and ATP-binding rates for the phosphomimetic MYO6^S267E.**
a Scheme of the actomyosin ATPase cycle. b Cumulative plots of the actin-attached dwell times for single MYO6^S267A (yellow, red) and MYO6^S267E (light and dark blue) molecules, measured with 10 µM and 100 µM ATP. Distributions can be described by two exponential components, k₁ (ATP-independent) and k₂ (ATP-dependent). Mean rates ± StdErr, see Fig. 4; data from > 3 experiments and > 3 different protein preparations. c Ensemble averaging of the displacement events for MYO6^S267E, with rates k₁ (light grey) and k₂ (dark grey) shown in (b), see also Fig. 4. d Scheme of stopped flow experiments. e Time course of ADP displacement from a pyrene-actin.MYO6.ADP complex by ATP excess in the absence of calcium (2 mM ATP, 100 nM MYO6, 200 nM pyrene-actin, 100 µM ADP). Single exponential fits yield ADP dissociation rates (k₁) of 3.7 s^{−1 (}MYO6^WT, blue), 3.0 s⁻¹ (MYO6^S267A, yellow) and 16.3 s⁻¹ (MYO6^S267E, blue). f ATP-induced dissociation of pyrene-actin.MYO6 (k_obs) in the absence of calcium. k₂ values were 20.8 mM⁻¹ s⁻¹ (MYO6^WT, grey), 24.2 mM⁻¹ s⁻¹ (MYO6S^267A, yellow) and 46.4 mM⁻¹ s⁻¹ (MYO6^S267E, blue). Source data are provided as a Source Data file.

**Fig. 6. Intracellular effects of phosphomimic MYO6^S267E.**
a Scheme highlighting the design of MYO6+, the plus-end directed mutant, in which the insert-2 (reverse gear), lever-arm extension and IQ motif are replaced with six IQ motifs of MYO5. b Filopodia numbers per cell expressing either GFP-tagged MYO6+^WT, MYO6+^S267A or MYO6+^S267E are shown for 3 experiments. Statistical significance was determined using one-way ANOVA and post-hoc testing. ***P < 0.01. MYO6+^WT n = 2786, average 65 filopodia/cell SD ± 26.834, MYO6+^S267A n = 1824, average 52 filopodia/cell SD ± 9.3, MYO6+^S267E n = 6163, average 121 filopodia/cell SD ± 18.62. c Filopodia length was measured in 30–50 cells per construct in 3 experiments expressing either GFP-tagged MYO6+^WT, MYO6+^S267A or MYO6+^S267E. The lengths of filopodia were not significantly different between cells expressing MYO6+^S267A n = 50, average 4.72 ± 1.82 SD, MYO6+^S267E n = 55, average 5.5 ± 4.43 SD and MYO6+^WT n = 56, average 5.05 ± 3.02 SD. The lack of statistical significance was determined using a two-sided t-test. d GFP and actin localisation in HeLa cells transfected with GFP-MYO6+^S267A or GFP-MYO6+^S267E. The accumulation of either construct in filopodia tips was observed in several independent experiments. A representative image is shown. Scale bar, 10 μm and 5 μm for enlarged images. e HeLa cells co-expressing mCherry-MYO6+^WT (red) and either GFP-MYO6+^WT, GFP-MYO6+^S267A or GFP-MYO6+^S267E (green) shown in high-resolution images of single filopodia. This experiment has been performed at least 3 times, representative images are shown. Schemes highlight the relative distribution of mCherry-MYO6+^WT and GFP-MYO6+^WT, GFP-MYO6+^S267A or GFP-MYO6+^S267E. Scale bar 5 μm. f Degree of co-localization between mCherry-MYO6+^WT and GFP-tagged MYO6+^WT, GFP-MYO6+^S267A or GFP-MYO6+^S267E was determined in 3 independent experiments from confocal images using *Pearson’s* correlation coefficient with automatic *Costes* threshold. Statistical significance was determined using a two-sided t-test ***P < 0.01. mCherry-MYO6+^WT and GFP-MYO6+^WT n = 152, average 0.6569 SD ± 0.1054, mCherry-MYO6+^WT and GFP-MYO6+^S267A n = 150, average 0.7077 SD ± 0.0961, mCherry-MYO6+^WT and GFP-MYO6+ ^S267E n = 145, average 0.2098 SD ± 0.1123. Source data are provided as a Source Data file.

**Fig. 7. Single molecule stiffness of acto-MYO6^S267E/WT through the cycle.**
a Schematic of single molecule stiffness measurements. b Schematic and experimental time courses of the ensemble-averaged stiffness of the acto.MYO6 complex measured for MYO6^WT and MYO6^S267E, see also Fig. 4. c Load-dependent rates k₁ and k₂ describe the change in stiffness of the acto.MYO6^S267E complex at 100 µM ATP at different loads. Light and dark grey circles, k₁ and k₂ from ≈30 measurements at 100 µM ATP and different loads (mean ± StdErr), see also Fig. 4. Solid lines are fitted using k = k_o exp(−Fd/kT), with k_o rate at zero load, F force, d distance parameter and kT thermal energy; d₁ = 1.8 nm, d₂ = 2.8 nm. Extrapolated load-dependent rate $k_{2}^{*}$ at 1 mM ATP (dotted line) and $k_{2}^{* *}$ at 2 mM ATP (dashed line). Source data are provided as a Source Data file.

**Fig. 8. The S267E mutation disrupts the stability of insert-1.**
a MYO6 atomic model used in MD simulations. Insert-1 is highlighted in yellow. Atomic models of the insert-1 domain in WT (left inset) and S267E (right inset) mutant with key residues highlighted after energy minimization. b Snapshots of the insert-1 domain (orange) and 310 loop (magenta) after 1 μs for the WT (replica 2) (left) and S267E (replica 1) (right) trajectories. c Comparison of insert-1 and L310 loop conformations at 0 and 1 μs in WT (replica 2) (left) and S267E (replica 1) (right) mutant trajectories obtained by superposition of residues 260–280 around the insert-1 domain and L310 loop. d, e Probability distribution plots for d the distance between the sidechain oxygens of the indicated pairs of residues and e the RMSD (root mean square deviation) of the insert-1 domain (residues 278–303) and L310 loop (residues 308–312) for the WT and S267E mutant simulations. All distance plots include data from the final 200 ns of all three replicas.

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References

1. Geeves, M. A. The ATPase mechanism of myosin and actomyosin. Biopolymers105, 483–491 (2016). 10.1002/bip.22853 - DOI - PubMed
1. Foth, B. J., Goedecke, M. C. & Soldati, D. New insights into myosin evolution and classification. Proc. Natl. Acad. Sci. USA103, 3681–3686 (2006). 10.1073/pnas.0506307103 - DOI - PMC - PubMed
1. Bloemink, M. J. & Geeves, M. A. Shaking the myosin family tree: biochemical kinetics defines four types of myosin motor. Semin. Cell Dev. Biol.22, 961–967 (2011). 10.1016/j.semcdb.2011.09.015 - DOI - PMC - PubMed
1. Howard, J. Molecular motors: structural adaptations to cellular functions. Nature389, 561–567 (1997). 10.1038/39247 - DOI - PubMed
1. Menetrey, J. et al. The structure of the myosin VI motor reveals the mechanism of directionality reversal. Nature435, 779–785 (2005). 10.1038/nature03592 - DOI - PMC - PubMed

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[1] Geeves, M. A. The ATPase mechanism of myosin and actomyosin. Biopolymers105, 483–491 (2016). 10.1002/bip.22853 - DOI - PubMed

[2] Geeves, M. A. The ATPase mechanism of myosin and actomyosin. Biopolymers105, 483–491 (2016). 10.1002/bip.22853 - DOI - PubMed

[3] Foth, B. J., Goedecke, M. C. & Soldati, D. New insights into myosin evolution and classification. Proc. Natl. Acad. Sci. USA103, 3681–3686 (2006). 10.1073/pnas.0506307103 - DOI - PMC - PubMed

[4] Foth, B. J., Goedecke, M. C. & Soldati, D. New insights into myosin evolution and classification. Proc. Natl. Acad. Sci. USA103, 3681–3686 (2006). 10.1073/pnas.0506307103 - DOI - PMC - PubMed

[5] Bloemink, M. J. & Geeves, M. A. Shaking the myosin family tree: biochemical kinetics defines four types of myosin motor. Semin. Cell Dev. Biol.22, 961–967 (2011). 10.1016/j.semcdb.2011.09.015 - DOI - PMC - PubMed

[6] Bloemink, M. J. & Geeves, M. A. Shaking the myosin family tree: biochemical kinetics defines four types of myosin motor. Semin. Cell Dev. Biol.22, 961–967 (2011). 10.1016/j.semcdb.2011.09.015 - DOI - PMC - PubMed

[7] Howard, J. Molecular motors: structural adaptations to cellular functions. Nature389, 561–567 (1997). 10.1038/39247 - DOI - PubMed

[8] Howard, J. Molecular motors: structural adaptations to cellular functions. Nature389, 561–567 (1997). 10.1038/39247 - DOI - PubMed

[9] Menetrey, J. et al. The structure of the myosin VI motor reveals the mechanism of directionality reversal. Nature435, 779–785 (2005). 10.1038/nature03592 - DOI - PMC - PubMed

[10] Menetrey, J. et al. The structure of the myosin VI motor reveals the mechanism of directionality reversal. Nature435, 779–785 (2005). 10.1038/nature03592 - DOI - PMC - PubMed

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Motor domain phosphorylation increases nucleotide exchange and turns MYO6 into a faster and stronger motor

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Motor domain phosphorylation increases nucleotide exchange and turns MYO6 into a faster and stronger motor

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