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

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.

Cloning, expression, and characterization of a novel molecular motor, Leishmania myosin-XXI.

The genome of the Leishmania parasite contains two classes of myosin. Myosin-XXI, seemingly the only myosin isoform expressed in the protozoan parasite, has been detected in both the promastigote and amastigote stages of the Leishmania life cycle. It has been suggested to perform a variety of functions, including roles in membrane anchorage, but also long-range directed movements of cargo. However, nothing is known about the biochemical or mechanical properties of this motor. Here we designed and expressed various myosin-XXI constructs using a baculovirus expression system. Both full-length (amino acids 1-1051) and minimal motor domain constructs (amino acids 1-800) featured actin-activated ATPase activity. Myosin-XXI was soluble when expressed either with or without calmodulin. In the presence of calcium (pCa 4.1) the full-length motor could bind a single calmodulin at its neck domain (probably amino acids 809-823). Calmodulin binding was required for motility but not for ATPase activity. Once bound, calmodulin remained stably attached independent of calcium concentration (pCa 3-7). In gliding filament assays, myosin-XXI moved actin filaments at ∼15 nm/s, insensitive to both salt (25-1000 mm KCl) and calcium concentrations (pCa 3-7). Calmodulin binding to the neck domain might be involved in regulating the motility of the myosin-XXI motor for its various cellular functions in the different stages of the Leishmania parasite life cycle.

Molecular architecture: construction of self-assembled organophosphonate duplexes and their electrochemical characterization.

Self-assembled monolayers of phosphonates (SAMPs) of 11-hydroxyundecylphosphonic acid, 2,6-diphosphonoanthracene, 9,10-diphenyl-2,6-diphosphonoanthracene, and 10,10'-diphosphono-9,9'-bianthracene and a novel self-assembled organophosphonate duplex ensemble were synthesized on nanometer-thick SiO(2)-coated, highly doped silicon electrodes. The duplex ensemble was synthesized by first treating the SAMP prepared from an aromatic diphosphonic acid to form a titanium complex-terminated one; this was followed by addition of a second equivalent of the aromatic diphosphonic acid. SAMP homogeneity, roughness, and thickness were evaluated by AFM; SAMP film thickness and the structural contributions of each unit in the duplex were measured by X-ray reflection (XRR). The duplex was compared with the aliphatic and aromatic monolayer SAMPs to determine the effect of stacking on electrochemical properties; these were measured by impedance spectroscopy using aqueous electrolytes in the frequency range 20 Hz to 100 kHz, and data were analyzed using resistance-capacitance network based equivalent circuits. For the 11-hydroxyundecylphosphonate SAMP, C(SAMP) = 2.6 ± 0.2 µF/cm(2), consistent with its measured layer thickness (ca. 1.1 nm). For the anthracene-based SAMPs, C(SAMP) = 6-10 µF/cm(2), which is attributed primarily to a higher effective dielectric constant for the aromatic moieties (ε = 5-10) compared to the aliphatic one; impedance spectroscopy measured the additional capacitance of the second aromatic monolayer in the duplex (2ndSAMP) to be C(Ti/2ndSAMP) = 6.8 ± 0.7 µF/cm(2), in series with the first.