Publications by authors named "Peter J Knight"

46 Publications

Structure of the shutdown state of myosin-2.

Nature 2020 12 2;588(7838):515-520. Epub 2020 Dec 2.

The Astbury Centre for Structural and Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.

Myosin-2 is essential for processes as diverse as cell division and muscle contraction. Dephosphorylation of its regulatory light chain promotes an inactive, 'shutdown' state with the filament-forming tail folded onto the two heads, which prevents filament formation and inactivates the motors. The mechanism by which this happens is unclear. Here we report a cryo-electron microscopy structure of shutdown smooth muscle myosin with a resolution of 6 Å in the head region. A pseudo-atomic model, obtained by flexible fitting of crystal structures into the density and molecular dynamics simulations, describes interaction interfaces at the atomic level. The N-terminal extension of one regulatory light chain interacts with the tail, and the other with the partner head, revealing how the regulatory light chains stabilize the shutdown state in different ways and how their phosphorylation would allow myosin activation. Additional interactions between the three segments of the coiled coil, the motor domains and the light chains stabilize the shutdown molecule. The structure of the lever in each head is competent to generate force upon activation. This shutdown structure is relevant to all isoforms of myosin-2 and provides a framework for understanding their disease-causing mutations.
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http://dx.doi.org/10.1038/s41586-020-2990-5DOI Listing
December 2020

Dr Gerald W. Offer (1938-2019); an appreciation.

J Muscle Res Cell Motil 2019 Dec 23;40(3-4):275-278. Epub 2019 Oct 23.

School of Physiology, Pharmacology & Neurosciences, University of Bristol, Bristol, UK.

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http://dx.doi.org/10.1007/s10974-019-09561-7DOI Listing
December 2019

Dr. Gerald Offer, 1938-2019.

Meat Sci 2019 Jun 4;157:107854. Epub 2019 Jun 4.

University of Leeds, United Kingdom. Electronic address:

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http://dx.doi.org/10.1016/j.meatsci.2019.05.025DOI Listing
June 2019

Determining Stable Single Alpha Helical (SAH) Domain Properties by Circular Dichroism and Atomic Force Microscopy.

Methods Mol Biol 2018 ;1805:185-211

Astbury Centre for Structural Molecular Biology and School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK.

Stable, single α-helical (SAH) domains exist in a number of unconventional myosin isoforms, as well as other proteins. These domains are formed from sequences rich in charged residues (Arg, Lys, and Glu), they can be hundreds of residues long, and in isolation they can tolerate significant changes in pH and salt concentration without loss in helicity. Here we describe methods for the preparation and purification of SAH domains and SAH domain-containing constructs, using the myosin 10 SAH domain as an example. We go on to describe the use of circular dichroism spectroscopy and force spectroscopy with the atomic force microscope for the elucidation of structural and mechanical properties of these unusual helical species.
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http://dx.doi.org/10.1007/978-1-4939-8556-2_10DOI Listing
March 2019

Affimer proteins for F-actin: novel affinity reagents that label F-actin in live and fixed cells.

Sci Rep 2018 04 26;8(1):6572. Epub 2018 Apr 26.

School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.

Imaging the actin cytoskeleton in cells uses a wide range of approaches. Typically, a fluorescent derivative of the small cyclic peptide phalloidin is used to image F-actin in fixed cells. Lifeact and F-tractin are popular for imaging the cytoskeleton in live cells. Here we characterised novel affinity reagents called Affimers that specifically bind to F-actin in vitro to determine if they are suitable alternatives as eGFP-fusion proteins, to label actin in live cells, or for labeling F-actin in fixed cells. In vitro experiments showed that 3 out of the 4 Affimers (Affimers 6, 14 and 24) tested bind tightly to purified F-actin, and appear to have overlapping binding sites. As eGFP-fusion proteins, the same 3 Affimers label F-actin in live cells. FRAP experiments suggest that eGFP-Affimer 6 behaves most similarly to F-tractin and Lifeact. However, it does not colocalise with mCherry-actin in dynamic ruffles, and may preferentially bind stable actin filaments. All 4 Affimers label F-actin in methanol fixed cells, while only Affimer 14 labels F-actin after paraformaldehyde fixation. eGFP-Affimer 6 has potential for use in selectively imaging the stable actin cytoskeleton in live cells, while all 4 Affimers are strong alternatives to phalloidin for labelling F-actin in fixed cells.
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http://dx.doi.org/10.1038/s41598-018-24953-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5920084PMC
April 2018

A1603P and K1617del, Mutations in β-Cardiac Myosin Heavy Chain that Cause Laing Early-Onset Distal Myopathy, Affect Secondary Structure and Filament Formation In Vitro and In Vivo.

J Mol Biol 2018 05 14;430(10):1459-1478. Epub 2018 Apr 14.

School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK; Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK. Electronic address:

Over 20 mutations in β-cardiac myosin heavy chain (β-MHC), expressed in cardiac and slow muscle fibers, cause Laing early-onset distal myopathy (MPD-1), a skeletal muscle myopathy. Most of these mutations are in the coiled-coil tail and commonly involve a mutation to a proline or a single-residue deletion, both of which are predicted to strongly affect the secondary structure of the coiled coil. To test this, we characterized the effects of two MPD-1 causing mutations: A1603P and K1617del in vitro and in cells. Both mutations affected secondary structure, decreasing the helical content of 15 heptad and light meromyosin constructs. Both mutations also severely disrupted the ability of glutathione S-transferase-light meromyosin fusion proteins to form minifilaments in vitro, as demonstrated by negative stain electron microscopy. Mutant eGFP-tagged β-MHC accumulated abnormally into the M-line of sarcomeres in cultured skeletal muscle myotubes. Incorporation of eGFP-tagged β-MHC into sarcomeres in adult rat cardiomyocytes was reduced. Molecular dynamics simulations using a composite structure of part of the coiled coil demonstrated that both mutations affected the structure, with the mutation to proline (A1603P) having a smaller effect compared to K1617del. Taken together, it seems likely that the MPD-1 mutations destabilize the coiled coil, resulting in aberrant myosin packing in thick filaments in muscle sarcomeres, providing a potential mechanism for the disease.
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http://dx.doi.org/10.1016/j.jmb.2018.04.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5958240PMC
May 2018

Characterization of long and stable de novo single alpha-helix domains provides novel insight into their stability.

Sci Rep 2017 03 13;7:44341. Epub 2017 Mar 13.

Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK.

Naturally-occurring single α-helices (SAHs), are rich in Arg (R), Glu (E) and Lys (K) residues, and stabilized by multiple salt bridges. Understanding how salt bridges promote their stability is challenging as SAHs are long and their sequences highly variable. Thus, we designed and tested simple de novo 98-residue polypeptides containing 7-residue repeats (AEEEXXX, where X is K or R) expected to promote salt-bridge formation between Glu and Lys/Arg. Lys-rich sequences (EK3 (AEEEKKK) and EK2R1 (AEEEKRK)) both form SAHs, of which EK2R1 is more helical and thermo-stable suggesting Arg increases stability. Substituting Lys with Arg (or vice versa) in the naturally-occurring myosin-6 SAH similarly increased (or decreased) its stability. However, Arg-rich de novo sequences (ER3 (AEEERRR) and EK1R2 (AEEEKRR)) aggregated. Combining a PDB analysis with molecular modelling provides a rational explanation, demonstrating that Glu and Arg form salt bridges more commonly, utilize a wider range of rotamer conformations, and are more dynamic than Glu-Lys. This promiscuous nature of Arg helps explain the increased propensity of de novo Arg-rich SAHs to aggregate. Importantly, the specific K:R ratio is likely to be important in determining helical stability in de novo and naturally-occurring polypeptides, giving new insight into how single α-helices are stabilized.
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http://dx.doi.org/10.1038/srep44341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347031PMC
March 2017

Direct observation shows superposition and large scale flexibility within cytoplasmic dynein motors moving along microtubules.

Nat Commun 2015 Sep 14;6:8179. Epub 2015 Sep 14.

School of Molecular and Cellular Biology and Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.

Cytoplasmic dynein is a dimeric AAA(+) motor protein that performs critical roles in eukaryotic cells by moving along microtubules using ATP. Here using cryo-electron microscopy we directly observe the structure of Dictyostelium discoideum dynein dimers on microtubules at near-physiological ATP concentrations. They display remarkable flexibility at a hinge close to the microtubule binding domain (the stalkhead) producing a wide range of head positions. About half the molecules have the two heads separated from one another, with both leading and trailing motors attached to the microtubule. The other half have the two heads and stalks closely superposed in a front-to-back arrangement of the AAA(+) rings, suggesting specific contact between the heads. All stalks point towards the microtubule minus end. Mean stalk angles depend on the separation between their stalkheads, which allows estimation of inter-head tension. These findings provide a structural framework for understanding dynein's directionality and unusual stepping behaviour.
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http://dx.doi.org/10.1038/ncomms9179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4579568PMC
September 2015

The Inner Centromere Protein (INCENP) Coil Is a Single α-Helix (SAH) Domain That Binds Directly to Microtubules and Is Important for Chromosome Passenger Complex (CPC) Localization and Function in Mitosis.

J Biol Chem 2015 Aug 14;290(35):21460-72. Epub 2015 Jul 14.

From The Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Max Born Crescent, Edinburgh EH9 3BF, Scotland, United Kingdom and

The chromosome passenger complex (CPC) is a master regulator of mitosis. Inner centromere protein (INCENP) acts as a scaffold regulating CPC localization and activity. During early mitosis, the N-terminal region of INCENP forms a three-helix bundle with Survivin and Borealin, directing the CPC to the inner centromere where it plays essential roles in chromosome alignment and the spindle assembly checkpoint. The C-terminal IN box region of INCENP is responsible for binding and activating Aurora B kinase. The central region of INCENP has been proposed to comprise a coiled coil domain acting as a spacer between the N- and C-terminal domains that is involved in microtubule binding and regulation of the spindle checkpoint. Here we show that the central region (213 residues) of chicken INCENP is not a coiled coil but a ∼ 32-nm-long single α-helix (SAH) domain. The N-terminal half of this domain directly binds to microtubules in vitro. By analogy with previous studies of myosin 10, our data suggest that the INCENP SAH might stretch up to ∼ 80 nm under physiological forces. Thus, the INCENP SAH could act as a flexible "dog leash," allowing Aurora B to phosphorylate dynamic substrates localized in the outer kinetochore while at the same time being stably anchored to the heterochromatin of the inner centromere. Furthermore, by achieving this flexibility via an SAH domain, the CPC avoids a need for dimerization (required for coiled coil formation), which would greatly complicate regulation of the proximity-induced trans-phosphorylation that is critical for Aurora B activation.
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http://dx.doi.org/10.1074/jbc.M115.645317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4571873PMC
August 2015

Myosin tails and single α-helical domains.

Biochem Soc Trans 2015 Feb;43(1):58-63

*Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, U.K.

The human genome contains 39 myosin genes, divided up into 12 different classes. The structure, cellular function and biochemical properties of many of these isoforms remain poorly characterized and there is still some controversy as to whether some myosin isoforms are monomers or dimers. Myosin isoforms 6 and 10 contain a stable single α-helical (SAH) domain, situated just after the canonical lever. The SAH domain is stiff enough to be able to lengthen the lever allowing the myosin to take a larger step. In addition, atomic force microscopy and atomistic simulations show that SAH domains unfold at relatively low forces and have a high propensity to refold. These properties are likely to be important for protein function, enabling motors to carry cargo in dense actin networks, and other proteins to remain attached to binding partners in the crowded cell.
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http://dx.doi.org/10.1042/BST20140302DOI Listing
February 2015

Stable single α-helices are constant force springs in proteins.

J Biol Chem 2014 Oct 13;289(40):27825-35. Epub 2014 Aug 13.

From the Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom

Single α-helix (SAH) domains are rich in charged residues (Arg, Lys, and Glu) and stable in solution over a wide range of pH and salt concentrations. They are found in many different proteins where they bridge two functional domains. To test the idea that their high stability might enable these proteins to resist unfolding along their length, the properties and unfolding behavior of the predicted SAH domain from myosin-10 were characterized. The expressed and purified SAH domain was highly helical, melted non-cooperatively, and was monomeric as shown by circular dichroism and mass spectrometry as expected for a SAH domain. Single molecule force spectroscopy experiments showed that the SAH domain unfolded at very low forces (<30 pN) without a characteristic unfolding peak. Molecular dynamics simulations showed that the SAH domain unfolds progressively as the length is increased and refolds progressively as the length is reduced. This enables the SAH domain to act as a constant force spring in the mechanically dynamic environment of the cell.
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http://dx.doi.org/10.1074/jbc.M114.585679DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4183817PMC
October 2014

Flexibility within the heads of muscle myosin-2 molecules.

J Mol Biol 2014 Feb 9;426(4):894-907. Epub 2013 Dec 9.

School of Molecular and Cellular Biology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK. Electronic address:

We show that negative-stain electron microscopy and image processing of nucleotide-free (apo) striated muscle myosin-2 subfragment-1 (S1), possessing one light chain or both light chains, is capable of resolving significant amounts of structural detail. The overall appearance of the motor and the lever is similar in rabbit, scallop and chicken S1. Projection matching of class averages of the different S1 types to projection views of two different crystal structures of apo S1 shows that all types most commonly closely resemble the appearance of the scallop S1 structure rather than the methylated chicken S1 structure. Methylation of chicken S1 has no effect on the structure of the molecule at this resolution: it too resembles the scallop S1 crystal structure. The lever is found to vary in its angle of attachment to the motor domain, with a hinge point located in the so-called pliant region between the converter and the essential light chain. The chicken S1 crystal structure lies near one end of the range of flexion observed. The Gaussian spread of angles of flexion suggests that flexibility is driven thermally, from which a torsional spring constant of ~23 pN·nm/rad² is estimated on average for all S1 types, similar to myosin-5. This translates to apparent cantilever-type stiffness at the tip of the lever of 0.37 pN/nm. Because this stiffness is lower than recent estimates from myosin-2 heads attached to actin, we suggest that binding to actin leads to an allosteric stiffening of the motor-lever junction.
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http://dx.doi.org/10.1016/j.jmb.2013.11.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3919154PMC
February 2014

MYH9-related disease: a novel prognostic model to predict the clinical evolution of the disease based on genotype-phenotype correlations.

Hum Mutat 2014 Feb 12;35(2):236-47. Epub 2013 Dec 12.

Department of Internal Medicine, IRCCS Policlinico San Matteo Foundation and University of Pavia, Pavia, Italy.

MYH9-related disease (MYH9-RD) is a rare autosomal-dominant disorder caused by mutations in the gene for nonmuscle myosin heavy chain IIA (NMMHC-IIA). MYH9-RD is characterized by a considerable variability in clinical evolution: patients present at birth with only thrombocytopenia, but some of them subsequently develop sensorineural deafness, cataract, and/or nephropathy often leading to end-stage renal disease (ESRD). We searched for genotype-phenotype correlations in the largest series of consecutive MYH9-RD patients collected so far (255 cases from 121 families). Association of genotypes with noncongenital features was assessed by a generalized linear regression model. The analysis defined disease evolution associated to seven different MYH9 genotypes that are responsible for 85% of MYH9-RD cases. Mutations hitting residue R702 demonstrated a complete penetrance for early-onset ESRD and deafness. The p.D1424H substitution associated with high risk of developing all the noncongenital manifestations of disease. Mutations hitting a distinct hydrophobic seam in the NMMHC-IIA head domain or substitutions at R1165 associated with high risk of deafness but low risk of nephropathy or cataract. Patients with p.E1841K, p.D1424N, and C-terminal deletions had low risk of noncongenital defects. These findings are essential to patients' clinical management and genetic counseling and are discussed in view of molecular pathogenesis of MYH9-RD.
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http://dx.doi.org/10.1002/humu.22476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233870PMC
February 2014

Functions and mechanics of dynein motor proteins.

Nat Rev Mol Cell Biol 2013 Nov 25;14(11):713-26. Epub 2013 Sep 25.

1] Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK. [2] Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.

Fuelled by ATP hydrolysis, dyneins generate force and movement on microtubules in a wealth of biological processes, including ciliary beating, cell division and intracellular transport. The large mass and complexity of dynein motors have made elucidating their mechanisms a sizable task. Yet, through a combination of approaches, including X-ray crystallography, cryo-electron microscopy, single-molecule assays and biochemical experiments, important progress has been made towards understanding how these giant motor proteins work. From these studies, a model for the mechanochemical cycle of dynein is emerging, in which nucleotide-driven flexing motions within the AAA+ ring of dynein alter the affinity of its microtubule-binding stalk and reshape its mechanical element to generate movement.
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http://dx.doi.org/10.1038/nrm3667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3972880PMC
November 2013

Cardiomyopathy mutations in the tail of β-cardiac myosin modify the coiled-coil structure and affect integration into thick filaments in muscle sarcomeres in adult cardiomyocytes.

J Biol Chem 2013 Nov 18;288(44):31952-62. Epub 2013 Sep 18.

From the School of Molecular and Cellular Biology and.

It is unclear why mutations in the filament-forming tail of myosin heavy chain (MHC) cause hypertrophic or dilated cardiomyopathy as these mutations should not directly affect contraction. To investigate this, we first investigated the impact of five hypertrophic cardiomyopathy-causing (N1327K, E1356K, R1382W, E1555K, and R1768K) and one dilated cardiomyopathy-causing (R1500W) tail mutations on their ability to incorporate into muscle sarcomeres in vivo. We used adenoviral delivery to express full-length wild type or mutant enhanced GFP-MHC in isolated adult cardiomyocytes. Three mutations (N1327K, E1356K, and E1555K) reduced enhanced GFP-MHC incorporation into muscle sarcomeres, whereas the remainder had no effect. No mutations significantly affected contraction. Fluorescence recovery after photobleaching showed that fluorescence recovery for the mutation that incorporated least well (N1327K) was significantly faster than that of WT with half-times of 25.1 ± 1.8 and 32.2 ± 2.5 min (mean ± S.E.), respectively. Next, we determined the effects of each mutation on the helical properties of wild type and seven mutant peptides (7, 11, or 15 heptads long) from the myosin tail by circular dichroism. R1382W and E1768K slightly increased the α-helical nature of peptides. The remaining mutations reduced α-helical content, with N1327K showing the greatest reduction. Only peptides containing residues 1301-1329 were highly α-helical suggesting that this region helps in initiation of coiled coil. These results suggest that small effects of mutations on helicity translate into a reduced ability to incorporate into sarcomeres, which may elicit compensatory hypertrophy.
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http://dx.doi.org/10.1074/jbc.M113.513291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814791PMC
November 2013

Mammalian myosin-18A, a highly divergent myosin.

J Biol Chem 2013 Mar 4;288(13):9532-48. Epub 2013 Feb 4.

Laboratory of Molecular Physiology, NHLBI, National Institutes of Health, Bethesda, MD 20892-8015, USA.

The Mus musculus myosin-18A gene is expressed as two alternatively spliced isoforms, α and β, with reported roles in Golgi localization, in maintenance of cytoskeleton, and as receptors for immunological surfactant proteins. Both myosin-18A isoforms feature a myosin motor domain, a single predicted IQ motif, and a long coiled-coil reminiscent of myosin-2. The myosin-18Aα isoform, additionally, has an N-terminal PDZ domain. Recombinant heavy meromyosin- and subfragment-1 (S1)-like constructs for both myosin-18Aα and -18β species were purified from the baculovirus/Sf9 cell expression system. These constructs bound both essential and regulatory light chains, indicating an additional noncanonical light chain binding site in the neck. Myosin-18Aα-S1 and -18Aβ-S1 molecules bound actin weakly with Kd values of 4.9 and 54 μm, respectively. The actin binding data could be modeled by assuming an equilibrium between two myosin conformations, a competent and an incompetent form to bind actin. Actin binding was unchanged by presence of nucleotide. Both myosin-18A isoforms bound N-methylanthraniloyl-nucleotides, but the rate of ATP hydrolysis was very slow (<0.002 s(-1)) and not significantly enhanced by actin. Phosphorylation of the regulatory light chain had no effect on ATP hydrolysis, and neither did the addition of tropomyosin or of GOLPH3, a myosin-18A binding partner. Electron microscopy of myosin-18A-S1 showed that the lever is strongly angled with respect to the long axis of the motor domain, suggesting a pre-power stroke conformation regardless of the presence of ATP. These data lead us to conclude that myosin-18A does not operate as a traditional molecular motor in cells.
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http://dx.doi.org/10.1074/jbc.M112.441238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3611021PMC
March 2013

ATP-driven remodeling of the linker domain in the dynein motor.

Structure 2012 Oct 2;20(10):1670-80. Epub 2012 Aug 2.

Astbury Centre for Structural Molecular Biology, Institute of Molecular & Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.

Dynein ATPases are the largest known cytoskeletal motors and perform critical functions in cells: carrying cargo along microtubules in the cytoplasm and powering flagellar beating. Dyneins are members of the AAA+ superfamily of ring-shaped enzymes, but how they harness this architecture to produce movement is poorly understood. Here, we have used cryo-EM to determine 3D maps of native flagellar dynein-c and a cytoplasmic dynein motor domain in different nucleotide states. The structures show key sites of conformational change within the AAA+ ring and a large rearrangement of the "linker" domain, involving a hinge near its middle. Analysis of a mutant in which the linker "undocks" from the ring indicates that linker remodeling requires energy that is supplied by interactions with the AAA+ modules. Fitting the dynein-c structures into flagellar tomograms suggests how this mechanism could drive sliding between microtubules, and also has implications for cytoplasmic cargo transport.
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http://dx.doi.org/10.1016/j.str.2012.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3469822PMC
October 2012

Role of the tail in the regulated state of myosin 2.

J Mol Biol 2011 May 23;408(5):863-78. Epub 2011 Mar 23.

Institute of Molecular and Cellular Biology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.

Myosin 2 from vertebrate smooth muscle or non-muscle sources is in equilibrium between compact, inactive monomers and thick filaments under physiological conditions. In the inactive monomer, the two heads pack compactly together, and the long tail is folded into three closely packed segments that are associated chiefly with one of the heads. The molecular basis of the folding of the tail remains unexplained. By using electron microscopy, we show that compact monomers of smooth muscle myosin 2 have the same structure in both the native state and following specific, intramolecular photo-cross-linking between Cys109 of the regulatory light chain (RLC) and segment 3 of the tail. Nonspecific cross-linking between lysine residues of the folded monomer by glutaraldehyde also does not perturb the compact conformation and stabilizes it against unfolding at high ionic strength. Sequence comparisons across phyla and myosin 2 isoforms suggest that the folding of the tail is stabilized by ionic interactions between the positively charged N-terminal sequence of the RLC and a negatively charged region near the start of tail segment 3 and that phosphorylation of the RLC could perturb these interactions. Our results support the view that interactions between the heads and the distal tail perform a critical role in regulating activity of myosin 2 molecules through stabilizing the compact monomer conformation.
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http://dx.doi.org/10.1016/j.jmb.2011.03.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3776433PMC
May 2011

Myosin complexed with ADP and blebbistatin reversibly adopts a conformation resembling the start point of the working stroke.

Proc Natl Acad Sci U S A 2010 Apr 29;107(15):6799-804. Epub 2010 Mar 29.

Department of Biochemistry, Eötvös University, H-1117 Budapest, Pázmány Peter Setany 1/C, Hungary.

The powerstroke of the myosin motor is the basis of cell division and bodily movement, but has eluded empirical description due to the short lifetime and low abundance of intermediates during force generation. To gain insight into this process, we used well-established single-tryptophan and pyrene fluorescent sensors and electron microscopy to characterize the structural and kinetic properties of myosin complexed with ADP and blebbistatin, a widely used inhibitor. We found that blebbistatin does not weaken the tight actin binding of myosin.ADP, but unexpectedly it induces lever priming, a process for which the gamma-phosphate of ATP (or its analog) had been thought necessary. The results indicate that a significant fraction of the myosin.ADP.blebbistatin complex populates a previously inaccessible conformation of myosin resembling the start of the powerstroke.
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http://dx.doi.org/10.1073/pnas.0907585107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2872372PMC
April 2010

Influence of lever structure on myosin 5a walking.

Proc Natl Acad Sci U S A 2010 Feb 25;107(6):2509-14. Epub 2010 Jan 25.

Astbury Centre for Structural Molecular Biology and Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.

Using electron microscopy and image processing, we have observed myosin 5a modified with lever arms of different lengths (four, six, and eight calmodulin-binding IQ domains) and orientations walking along actin filaments. Step lengths were dependent on lever length: 8IQ > 6IQ > 4IQ, which is consistent with myosin 5a having evolved to walk straight along actin. Lead heads were mostly in the prepowerstroke state, tethered there by the trail head. However, improved image processing showed that in 5-10% of molecules the lead motor was in the postpowerstroke state. This is a unique attached state of myosin, where the motor domain has completed its powerstroke at the expense of severe lever distortion, but with little cargo movement. Postpowerstroke lead heads were seen in both wild-type and modified lever molecules, mostly where there was least strain. These data allow the strain dependence of the equilibrium between pre- and postpowerstroke conformations to be measured. Slow rates of ADP dissociation observed from lead heads of these molecules can be explained by the unfavorable equilibrium between the pre- and postpowerstroke conformations preceding ADP loss.
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http://dx.doi.org/10.1073/pnas.0906907107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2823865PMC
February 2010

The SAH domain extends the functional length of the myosin lever.

Proc Natl Acad Sci U S A 2009 Dec 15;106(52):22193-8. Epub 2009 Dec 15.

Astbury Centre for Structural Molecular Biology and Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.

Stable, single alpha-helix (SAH) domains are widely distributed in the proteome, including in myosins, but their functions are unknown. To test whether SAH domains can act as levers, we replaced four of the six calmodulin-binding IQ motifs in the levers of mouse myosin 5a (Myo5) with the putative SAH domain of Dictyostelium myosin MyoM of similar length. The SAH domain was inserted between the IQ motifs and the coiled coil in a Myo5 HMM construct in which the levers were truncated from six to two IQ motifs (Myo5-2IQ). Electron microscopy of this chimera (Myo5-2IQ-SAH) showed the SAH domain was straight and 17 nm long as predicted, restoring the truncated lever to the length of wild-type (Myo5-6IQ). The powerstroke (of 21.5 nm) measured in the optical trap was slightly less than that for Myo5-6IQ but much greater than for Myo5-2IQ. Myo5-2IQ-SAH moved processively along actin at physiological ATP concentrations with similar stride and run lengths to Myo5-6IQ in in-vitro single molecule assays. In comparison, Myo5-2IQ is not processive under these conditions. Solution biochemical experiments indicated that the rear head did not mechanically gate the rate of ADP release from the lead head, unlike Myo5-6IQ. These data show that the SAH domain can form part of a functional lever in myosins, although its mechanical stiffness might be lower. More generally, we conclude that SAH domains can act as stiff structural extensions in aqueous solution and this structural role may be important in other proteins.
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http://dx.doi.org/10.1073/pnas.0909851106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2794034PMC
December 2009

A FERM domain autoregulates Drosophila myosin 7a activity.

Proc Natl Acad Sci U S A 2009 Mar 2;106(11):4189-94. Epub 2009 Mar 2.

Laboratory of Molecular Physiology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Full-length Drosophila myosin 7a (myosin 7a-FL) has a complex tail containing a short predicted coiled coil followed by a MyTH4-FERM domain, an SH3 domain, and a C-terminal MyTH4-FERM domain. Myosin 7a-FL expressed in Sf9 cells is monomeric despite the predicted coiled coil. We showed previously that Subfragment-1 (S1) from this myosin has MgATPase of V(max) approximately 1 s(-1) and K(ATPase) approximately 1 microM actin. We find that myosin 7a-FL has V(max) similar to S1 but K(ATPase) approximately 30 microM. Thus, at low actin concentrations (5 microM), the MgATPase of S1 is fully activated, whereas that of myosin 7a-FL is low, suggesting that the tail regulates activity. Electron microscopy of myosin 7a-FL with ATP shows the tail is tightly bent back against the motor domain. Myosin 7a-FL extends at either high ionic strength or without ATP, revealing the motor domain, lever, and tail. A series of C-terminal truncations show that deletion of 99 aa (the MyTH7 subdomain of the C-terminal FERM domain) is sufficient to abolish bending, and the K(ATPase) is then similar to S1. This region is highly conserved in myosin 7a. We found that a double mutation in it, R2140A-K2143A, abolishes bending and reduces K(ATPase) to S1 levels. In addition, the expressed C-terminal FERM domain binds actin with K(d) approximately 30 microM regardless of ATP, similar to the K(ATPase) value for myosin 7a-FL. We propose that at low cellular actin concentrations, myosin 7a-FL is bent and inactive, but at high actin concentrations, it is unfolded and active because the C-terminal FERM domain binds to actin.
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http://dx.doi.org/10.1073/pnas.0808682106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2649957PMC
March 2009

AAA+ Ring and linker swing mechanism in the dynein motor.

Cell 2009 Feb;136(3):485-95

Astbury Centre for Structural Molecular Biology and Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK.

Dynein ATPases power diverse microtubule-based motilities. Each dynein motor domain comprises a ring-like head containing six AAA+ modules and N- and C-terminal regions, together with a stalk that binds microtubules. How these subdomains are arranged and generate force remains poorly understood. Here, using electron microscopy and image processing of tagged and truncated Dictyostelium cytoplasmic dynein constructs, we show that the heart of the motor is a hexameric ring of AAA+ modules, with the stalk emerging opposite the primary ATPase site (AAA1). The C-terminal region is not an integral part of the ring but spans between AAA6 and near the stalk base. The N-terminal region includes a lever-like linker whose N terminus swings by approximately 17 nm during the ATPase cycle between AAA2 and the stalk base. Together with evidence of stalk tilting, which may communicate changes in microtubule binding affinity, these findings suggest a model for dynein's structure and mechanism.
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http://dx.doi.org/10.1016/j.cell.2008.11.049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2706395PMC
February 2009

Helix sliding in the stalk coiled coil of dynein couples ATPase and microtubule binding.

Nat Struct Mol Biol 2009 Mar 8;16(3):325-33. Epub 2009 Feb 8.

Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba 3-8-1, Tokyo 153-8902, Japan.

Coupling between ATPase and track binding sites is essential for molecular motors to move along cytoskeletal tracks. In dynein, these sites are separated by a long coiled coil stalk that must mediate communication between them, but the underlying mechanism remains unclear. Here we show that changes in registration between the two helices of the coiled coil can perform this function. We locked the coiled coil at three specific registrations using oxidation to disulfides of paired cysteine residues introduced into the two helices. These trapped ATPase activity either in a microtubule-independent high or low state, and microtubule binding activity either in an ATP-insensitive strong or weak state, depending on the registry of the coiled coil. Our results provide direct evidence that dynein uses sliding between the two helices of the stalk to couple ATPase and microtubule binding activities during its mechanochemical cycle.
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http://dx.doi.org/10.1038/nsmb.1555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2757048PMC
March 2009

Evidence for the oligomeric state of 'elastic' titin in muscle sarcomeres.

J Mol Biol 2008 Dec 20;384(2):299-312. Epub 2008 Sep 20.

Institute for Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK.

The giant protein titin has important roles in muscle sarcomere integrity, elasticity and contractile activity. The key role in elasticity was highlighted in recent years by single-molecule mechanical studies, which showed a direct relationship between the non-uniform structure of titin and the hierarchical mechanism of its force-extension behavior. Further advances in understanding mechanisms controlling sarcomere structure and elasticity require detailed knowledge of titin arrangement and interactions in situ. Here we present data on the structure and self-interactive properties of an approximately 290 kDa ( approximately 100 nm long) tryptic fragment from the I-band part of titin that is extensible in situ. The fragment includes the conserved 'distal' tandem Ig segment of the molecule and forms side-by-side oligomers with distinctive 4 nm cross-striations. Comparisons between these oligomers and the end filaments seen at the tips of native thick filaments indicate identical structure. This shows that end-filaments are formed by the elastic parts of six titin molecules connecting each end of the thick filament to the Z-line. Self-association of elastic titin into stiff end-filaments adds a further hierarchical level in the mechanism of titin extensibility in muscle cells. Self-association of this part of titin may be required to prevent interference of the individual flexible molecules with myosin cross-bridges interacting with actin.
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http://dx.doi.org/10.1016/j.jmb.2008.09.030DOI Listing
December 2008

Folding and regulation in myosins II and V.

J Muscle Res Cell Motil 2007 22;28(7-8):363-70. Epub 2008 Apr 22.

Laboratory of Molecular Physiology, National Heart, Lung and Blood Institute, National Institutes of Health, Building 50, Room 3523, Bethesda, MD 20892, USA.

The enzymatic activity of many myosins is regulated by various means including calcium binding, phosphorylation or binding of receptor molecules. In this review we compare and contrast the regulation of smooth muscle myosin II and myosin Va with particular emphasis on the structural basis for the regulation. Both myosins adopt folded compact conformations in their off states, but the details of the conformations are markedly different. In the regulated smooth muscle myosin II, the key feature is an asymmetric interaction between the two heads of the molecule with contributions of specific tail-head interactions. In myosin V the key feature is an interaction between the heads and the globular tail domain.
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http://dx.doi.org/10.1007/s10974-008-9134-0DOI Listing
July 2008

Conservation of the regulated structure of folded myosin 2 in species separated by at least 600 million years of independent evolution.

Proc Natl Acad Sci U S A 2008 Apr 14;105(16):6022-6. Epub 2008 Apr 14.

Institute of Molecular and Cellular Biology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.

The myosin 2 family of molecular motors includes isoforms regulated in different ways. Vertebrate smooth-muscle myosin is activated by phosphorylation of the regulatory light chain, whereas scallop striated adductor-muscle myosin is activated by direct calcium binding to its essential light chain. The paired heads of inhibited molecules from myosins regulated by phosphorylation have an asymmetric arrangement with motor-motor interactions. It was unknown whether such interactions were a common motif for inactivation used in other forms of myosin-linked regulation. Using electron microscopy and single-particle image processing, we show that indistinguishable structures are indeed found in myosins and heavy meromyosins isolated from scallop striated adductor muscle and turkey gizzard smooth muscle. The similarities extend beyond the shapes of the heads and interactions between them: In both myosins, the tail folds into three segments, apparently at identical sites; all three segments are in close association outside the head region; and two segments are associated in the same way with one head in the asymmetric arrangement. Thus, these organisms, which have different regulatory mechanisms and diverged from a common ancestor >600 Myr ago, have the same quaternary structure. Conservation across such a large evolutionary distance suggests that this conformation is of fundamental functional importance.
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http://dx.doi.org/10.1073/pnas.0707846105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2329715PMC
April 2008

Calcium and cargoes as regulators of myosin 5a activity.

Biochem Biophys Res Commun 2008 Apr 3;369(1):176-81. Epub 2007 Dec 3.

Laboratory of Molecular Physiology, NHLBI, National Institutes of Health, Bethesda, MD 20892-1762, USA.

Myosin 5a is a two-headed actin-dependent motor that transports various cargoes in cells. Its enzymology and mechanochemistry have been extensively studied in vitro. It is a processive motor that takes multiple 36nm steps on actin. The enzymatic activity of myosin 5 is regulated by an intramolecular folding mechanism whereby its lever arms fold back against the coiled-coil tail such that the motor domains directly bind the globular tail domains. We show that the structure seen in individual folded molecules is consistent with electron density map of two-dimensional crystals of the molecule. In this compact state, the actin-activated MgATPase activity of the molecule is markedly inhibited and the molecule cannot move processively on surface bound actin filaments. The actin-activated MgATPase activity of myosin 5a is activated by increasing the calcium concentration or by binding of a cargo-receptor molecule, melanophilin, in vitro. However, calcium binding to the calmodulin light chains results in dissociation of some of the calmodulin which disrupts the ability of myosin 5a to move on actin filaments in vitro. Thus we propose that the physiologically relevant activation pathway in vivo involves binding of cargo-receptor proteins.
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http://dx.doi.org/10.1016/j.bbrc.2007.11.109DOI Listing
April 2008

Structures of smooth muscle myosin and heavy meromyosin in the folded, shutdown state.

J Mol Biol 2007 Oct 21;372(5):1165-78. Epub 2007 Jul 21.

Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, LS2 9JT, UK.

Remodelling the contractile apparatus within smooth muscle cells allows effective contractile activity over a wide range of cell lengths. Thick filaments may be redistributed via depolymerisation into inactive myosin monomers that have been detected in vitro, in which the long tail has a folded conformation. Using negative stain electron microscopy of individual folded myosin molecules from turkey gizzard smooth muscle, we show that they are more compact than previously described, with heads and the three segments of the folded tail closely packed. Heavy meromyosin (HMM), which lacks two-thirds of the tail, closely resembles the equivalent parts of whole myosin. Image processing reveals a characteristic head region morphology for both HMM and myosin, with features identifiable by comparison with less compact molecules. The two heads associate asymmetrically: the tip of one motor domain touches the base of the other, resembling the blocked and free heads of this HMM when it forms 2D crystals on lipid monolayers. The tail of HMM lies between the heads, contacting the blocked motor domain, unlike in the 2D crystal. The tail of whole myosin is bent sharply and consistently close to residues 1175 and 1535. The first bend position correlates with a skip in the coiled coil sequence, the second does not. Tail segments 2 and 3 associate only with the blocked head, such that the second bend is near the C-lobe of the blocked head regulatory light chain. Quantitative analysis of tail flexibility shows that the single coiled coil of HMM has an apparent Young's modulus of about 0.5 GPa. The folded tail of the whole myosin is less flexible, indicating interactions between the segments. The folded tail does not modify the compact head arrangement but stabilises it, indicating a structural mechanism for the very low ATPase activity of the folded molecule.
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http://dx.doi.org/10.1016/j.jmb.2007.07.014DOI Listing
October 2007

Load-dependent mechanism of nonmuscle myosin 2.

Proc Natl Acad Sci U S A 2007 Jun 4;104(24):9994-9. Epub 2007 Jun 4.

Laboratory of Molecular Physiology, National Heart, Lung, and Blood Institute/NIH, Bethesda, MD 20892-8015, USA.

Loads on molecular motors regulate and coordinate their function. In a study that directly measures properties of internally strained myosin 2 heads bound to actin, we find that human nonmuscle myosins 2A and 2B show marked load-dependent changes in kinetics of ADP release but not in nucleotide binding. We show that the ADP release rate constant is increased 4-fold by the assisting load on one head and decreased 5-fold (for 2A) or 12-fold (for 2B) by the resisting load on the other. Thus these myosins, especially 2B, have marked mechanosensitivity of product release. By regulating the actin attachment of myosin heads, this provides a basis for energy-efficient tension maintenance without obstructing cellular contractility driven by other motors such as smooth muscle myosin. Whereas forward load accelerates the cycle of interaction with actin, resistive load increases duty ratio to favor tension maintenance by two-headed attachment.
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http://dx.doi.org/10.1073/pnas.0701181104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1885822PMC
June 2007