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

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

Single-molecule and biochemical characterization of the human myosin isoforms IIb, VI and IXa

Myosins are actin-dependent molecular motors which convert chemical energy from ATP hydrolysis into mechanical work. They form a large superfamily but all of them share a common domain structure consisting of three parts: a head, neck and tail region. The catalytic head region is evolutionary conserved and includes a nucleotide-binding site together with an actin-binding site. The head is followed by the neck domain, which binds additional accessory proteins (light chains, e.g. calmodulin) and acts as a lever arm. Finally, the tail region is the most variable domain and regulates cellular functions and dynamics of the motor. The myosin superfamily interacts with actin filaments to generate a variety of cellular functions including muscle contraction, exo- and endocytosis, cytokinesis and even signal transduction. Twelve different myosin classes have evolved in humans to adapt to their specific cellular function. In this work I selected isoforms from three myosin classes to investigate their biochemical and single-molecule properties. The isoforms IIb, VI and IXa are structurally, biochemically and kinetically tailored to their specific cellular tasks and not much is known about their regulation (e.g. calcium, phosphorylation) to adapt to changing cellular environments or external stimuli. The aim of the study is to elucidate the molecular roles of the myosin isoforms IIb, VI and IXa and how their structural and regulatory characteristic lead to their specific cellular functions. Besides diverse biochemical methods and functional in vitro assays an optical tweezers transducer was used to study the single-molecule behaviour of the different myosin isoforms. These optical tweezers experiments are able to resolve the nanometre displacements and piconewton forces generated by single motor molecules and allow insights into the basic molecular mechanism of the chemo-mechanical energy transduction cycle. Myosin IXa is a intriguing molecular motor and is localized in acto-myosin networks at cell-cell adhesions. Recent studies identified myosin IXa as a key requirement for collective cell migration, which is an important mechanism during epithelial morphogenesis, tissue regeneration and cancer metastasis. On a structural level, myosin IXa contains several unique features: The tail domain links this motor to signalling functions as it contains a Rho-GTPase activating domain (RhoGAP) which can alter the dynamics of the actin cytoskeleton. Additionally, the head domain comprises a unique, ∼200 aa insertion in the loop 2 region involved in actin binding. This work clearly demonstrates that this insertion allows myosin IXa to bundle actin filaments into actomyosin networks. Quantitative TIRF microscopy revealed that myosin IXa assembles actin into bundles of up to seven or even more filaments. A collaboration project using transmission electron microscopy combined with single particle image processing showed that the bundles consisted of a highly ordered 2D actomyosin network, with three distinct conformations. The novelty found in this work is that the bundles were also present at micromolar ATP concentrations and bundle dissociation occurred in a stepwise manner, suggesting a dynamic assembly and disassembly of the actomyosin networks in a cellular context. Furthermore, the presence of a calmodulin-binding site in the unique loop 2 insertion indicates that the motor and bundling activity of myosin IXa is regulated by calcium/calmodulin, which was also in the focus of this work. At the cellular level, the actin lattices might introduce specific tracks for other myosin motors and represent platforms, which allows specific RhoGAP activity from the tail of myosin IXa to be focused to distinct intracellular locations. In contrast to myosin IXa, the non-muscle myosin isoform IIb (NMIIb) is ubiquitously expressed and has key functions in fundamental cellular processes such as cytokinesis, cell migration and control of cell morphology. This double-headed myosin contains a coiledcoil motif in the tail domain, enabling it to form large filaments. In this work I compared the single-molecule properties of single and double-headed human NMIIb to gain insights into the cooperative nature of the myosin heads. The results indicate that the single myosin heads act in an independent, non-cooperative manner. Furthermore, I show that single and double headed NMIIb produces two types of power strokes: A ∼5 nm working stroke known also from other myosin II isoforms, and unusual close-to-zero attachment events. The results suggest that the latter events might represent transient, weak binding states of myosin to actin. These weak binding interactions could have important cellular relevance to bind and tether the NMIIb myosin filament to actin without the consumption of further ATP, especially as this NMII isoform is known to be involved in tension maintenance and structural anchoring. Myosin VI is the only known member of the myosin superfamily moving towards the minus end of actin filaments. This molecular motor is involved in diverse cellular functions such as vesicle transport, endocytosis and intracellular membrane trafficking. In this work I investigate the role of myosin head phosphorylation on the mechanical functions of myosin VI. In vitro motility assays from a collaboration partner at the University of Cambridge (UK) have shown that phosphorylation of serine at position 267 had a huge impact on the actin gliding velocity. Using an optical tweezers transducer, I show that the differences observed in the ensemble assay can be explained by tuning the ADP release and ATP binding rate in the actomyosin cycle without a change in the power stroke size. This dramatic effects on the in vitro properties of myosin VI might have pronounced cellular consequences on myosin VI activity in membrane dynamics and cytoskeletal organisation

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

Acknowledgements: We thank Dr. Robin Antrobus and Dr Harriet Pearson for help with the proteomics and Dr Zuzana Kadlekova for many helpful discussions. This work was supported in the Buss laboratory by a CIMR-funded studentship to JJ, a Wellcome Trust PhD studentship to TOL, the Isaac Newton Trust Cambridge, a Henry Wellcome Fellowship to CAJ, and a program grant from the Medical Research Council (MR/S007776/1). Work in the AJW laboratory is supported by the UK MRC (MR/T012412/1), Blood Cancer UK (21002) and the Rosetrees Trust (PGL22/100032). CIMR is supported by a Wellcome Trust equipment grant (108415/Z/15/Z). We like to thank Prof. Martin Zacharias (TUM) for helpful discussions and Dr. John M. Davis for his editorial assistance. Work in the Veigel laboratory by AG, MK, CB, AFM and CV was funded by DFG SFB-863, SFB-1032, Friedrich-Baur-Stiftung and the LMU-Munich.Funder: Isaac Newton Trust Cambridge, Henry Wellcome Fellowship,Funder: CIMR-funded PhD studentshipFunder: Friedrich-Baur-Stiftung and the LMU-MunichFunder: Blood Cancer UK (21002)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