Cloning, Expression, and Characterization of a Novel Molecular Motor, Leishmania Myosin-XXI

Batters, Christopher; Woodall, Katy; Toseland, Christopher P.; Hundschell, Christian; Veigel, Claudia

doi:10.1074/jbc.m112.381301

Cited by 17 publications

(23 citation statements)

References 38 publications

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“…An unexpected finding was revealed by sequence analysis. The motor has eight potential calmodulinbinding sites, only one of which bound human calmodulin in our previous study (10). We have now identified a binding site close to the converter domain that is located within an extended dimerization site covering the entire C terminus of the molecule from the converter region to the C-terminal end.…”

Section: Discussionmentioning

confidence: 80%

“…The SEC study showed that FL-XXI heavy chain, coexpressed with high levels of calmodulin virus, was monomeric. Using the approach of Coluccio (16), we had found in a previous study that the stoichiometry between myosin-XXI heavy chain and human calmodulin was 1:1 for these conditions of expression (10). In contrast, in the absence of added calmodulin virus, most of the purified myosin-XXI sample eluted earlier, indicating the formation of dimers or higher oligomers (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The converter domain (645-747) is thought to transform small conformational changes in the nucleotide-binding pocket to changes in the orientation of the calmodulin-binding neck domain that serves as a mechanical lever arm. Intriguingly, the sequence suggests a coiled-coil domain (11,12) with a strong propensity to dimerize between the end of the motor domain and what we previously thought to be the first calmodulin-binding IQ motif (809-823) (10). This predicted coiled-coil region (730-810) revealed a further potential calmodulin-binding site (754-769, yellow in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In a previous study we identified six potential calmodulinbinding IQ motifs outside the motor domain of myosin XXI. The data suggested that only the motif closest to the motor domain bound Xenopus/human calcium-calmodulin (10). Intriguingly, sequence analysis indicated a coiled-coil domain (11,12) with a strong propensity to dimerize in between the end of the motor domain and what we initially thought to be the first calmodulinbinding IQ motif.…”

mentioning

confidence: 93%

“…binding was not required for the ATPase activity of the motor, but for myosin XXI in vitro motility (10).…”

Section: Significancementioning

confidence: 99%

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Calmodulin regulates dimerization, motility, and lipid binding of Leishmania myosin XXI

Batters

Ellrich

Helbig

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Myosin XXI is the only myosin expressed in Leishmania parasites. Although it is assumed that it performs a variety of motile functions, the motor's oligomerization states, cargo-binding, and motility are unknown. Here we show that binding of a single calmodulin causes the motor to adopt a monomeric state and to move actin filaments. In the absence of calmodulin, nonmotile dimers that cross-linked actin filaments were formed. Unexpectedly, structural analysis revealed that the dimerization domains include the calmodulin-binding neck region, essential for the generation of force and movement in myosins. Furthermore, monomeric myosin XXI bound to mixed liposomes, whereas the dimers did not. Lipid-binding sections overlapped with the dimerization domains, but also included a phox-homology domain in the converter region. We propose a mechanism of myosin regulation where dimerization, motility, and lipid binding are regulated by calmodulin. Although myosin-XXI dimers might act as nonmotile actin cross-linkers, the calmodulin-binding monomers might transport lipid cargo in the parasite.unconventional myosin | motor properties

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Section: Discussionmentioning

confidence: 80%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 93%

“…binding was not required for the ATPase activity of the motor, but for myosin XXI in vitro motility (10).…”

Section: Significancementioning

confidence: 99%

See 3 more Smart Citations

Calmodulin regulates dimerization, motility, and lipid binding of Leishmania myosin XXI

Batters

Ellrich

Helbig

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Fluorescence to Study the ATPase Mechanism of Motor Proteins

Toseland

2014

Experientia Supplementum

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This chapter provides an overview of different methodologies to dissect the ATPase mechanism of motor proteins. The use of ATP is fundamental to how these molecular engines work and how they can use the energy to perform various cellular roles. Rapid reaction and single-molecule techniques will be discussed to monitor reactions in real time through the application of fluorescence intensity, anisotropy and FRET. These approaches utilise fluorescent nucleotides and biosensors. While not every technique may be suitable for your motor protein, the different ways to determine the ATPase mechanism should allow a good evaluation of the kinetic parameters.

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Biology of Human Pathogenic Trypanosomatids: Epidemiology, Lifecycle and Ultrastructure

Rodrigues

Godinho

Souza

2013

Subcellular Biochemistry

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Leishmania and Trypanosoma belong to the Trypanosomatidae family and cause important human infections such as leishmaniasis, Chagas disease, and sleeping sickness. Leishmaniasis, caused by protozoa belonging to Leishmania, affects about 12 million people worldwide and can present different clinical manifestations, i.e., visceral leishmaniasis (VL), cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), diffuse cutaneous leishmaniasis (DCL), and post-kala-azar dermal leishmaniasis (PKDL). Chagas disease, also known as American trypanosomiasis, is caused by Trypanosoma cruzi and is mainly prevalent in Latin America but is increasingly occurring in the United States, Canada, and Europe. Sleeping sickness or human African trypanosomiasis (HAT), caused by two sub-species of Trypanosoma brucei (i.e., T. b. rhodesiense and T. b. gambiense), occurs only in sub-Saharan Africa countries. These pathogenic trypanosomatids alternate between invertebrate and vertebrate hosts throughout their lifecycles, and different developmental stages can live inside the host cells and circulate in the bloodstream or in the insect gut. Trypanosomatids have a classical eukaryotic ultrastructural organization with some of the same main organelles found in mammalian host cells, while also containing special structures and organelles that are absent in other eukaryotic organisms. For example, the mitochondrion is ramified and contains a region known as the kinetoplast, which houses the mitochondrial DNA. Also, the glycosomes are specialized peroxisomes containing glycolytic pathway enzymes. Moreover, a layer of subpellicular microtubules confers mechanic rigidity to the cell. Some of these structures have been investigated to determine their function and identify potential enzymes and metabolic pathways that may constitute targets for new chemotherapeutic drugs.

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Cloning, Expression, and Characterization of a Novel Molecular Motor, Leishmania Myosin-XXI

Cited by 17 publications

References 38 publications

Calmodulin regulates dimerization, motility, and lipid binding of Leishmania myosin XXI

Calmodulin regulates dimerization, motility, and lipid binding of Leishmania myosin XXI

Fluorescence to Study the ATPase Mechanism of Motor Proteins

Biology of Human Pathogenic Trypanosomatids: Epidemiology, Lifecycle and Ultrastructure

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