MyosinA is a druggable target in the widespread protozoan parasite Toxoplasma gondii.

Toxoplasma gondii is a widespread apicomplexan parasite that can cause severe disease in its human hosts. The ability of T. gondii and other apicomplexan parasites to invade into, egress from, and move between cells of the hosts they infect is critical to parasite virulence and disease progression. An unusual and highly conserved parasite myosin motor (TgMyoA) plays a central role in T. gondii motility. The goal of this work was to determine whether the parasite's motility and lytic cycle can be disrupted through pharmacological inhibition of TgMyoA, as an approach to altering disease progression in vivo. To this end, we first sought to identify inhibitors of TgMyoA by screening a collection of 50,000 structurally diverse small molecules for inhibitors of the recombinant motor's actin-activated ATPase activity. The top hit to emerge from the screen, KNX-002, inhibited TgMyoA with little to no effect on any of the vertebrate myosins tested. KNX-002 was also active against parasites, inhibiting parasite motility and growth in culture in a dose-dependent manner. We used chemical mutagenesis, selection in KNX-002, and targeted sequencing to identify a mutation in TgMyoA (T130A) that renders the recombinant motor less sensitive to compound. Compared to wild-type parasites, parasites expressing the T130A mutation showed reduced sensitivity to KNX-002 in motility and growth assays, confirming TgMyoA as a biologically relevant target of KNX-002. Finally, we present evidence that KNX-002 can slow disease progression in mice infected with wild-type parasites, but not parasites expressing the resistance-conferring TgMyoA T130A mutation. Taken together, these data demonstrate the specificity of KNX-002 for TgMyoA, both in vitro and in vivo, and validate TgMyoA as a druggable target in infections with T. gondii. Since TgMyoA is essential for virulence, conserved in apicomplexan parasites, and distinctly different from the myosins found in humans, pharmacological inhibition of MyoA offers a promising new approach to treating the devastating diseases caused by T. gondii and other apicomplexan parasites.

Structural and mechanistic insights into the function of the unconventional class XIV myosin MyoA from Toxoplasma gondii.

Parasites of the phylum Apicomplexa are responsible for significant morbidity and mortality on a global scale. Central to the virulence of these pathogens are the phylum-specific, unconventional class XIV myosins that power the essential processes of parasite motility and host cell invasion. Notably, class XIV myosins differ from human myosins in key functional regions, yet they are capable of fast movement along actin filaments with kinetics rivaling previously studied myosins. Toward establishing a detailed molecular mechanism of class XIV motility, we determined the 2.6-Å resolution crystal structure of the Toxoplasma gondii MyoA (TgMyoA) motor domain. Structural analysis reveals intriguing strategies for force transduction and chemomechanical coupling that rely on a divergent SH1/SH2 region, the class-defining "HYAG"-site polymorphism, and the actin-binding surface. In vitro motility assays and hydrogen-deuterium exchange coupled with MS further reveal the mechanistic underpinnings of phosphorylation-dependent modulation of TgMyoA motility whereby localized regions of increased stability and order correlate with enhanced motility. Analysis of solvent-accessible pockets reveals striking differences between apicomplexan class XIV and human myosins. Extending these analyses to high-confidence homology models of Plasmodium and Cryptosporidium MyoA motor domains supports the intriguing potential of designing class-specific, yet broadly active, apicomplexan myosin inhibitors. The successful expression of the functional TgMyoA complex combined with our crystal structure of the motor domain provides a strong foundation in support of detailed structure-function studies and enables the development of small-molecule inhibitors targeting these devastating global pathogens.

Dissecting the molecular assembly of the Toxoplasma gondii MyoA motility complex.

Apicomplexan parasites such as Toxoplasma gondii rely on a unique form of locomotion known as gliding motility. Generating the mechanical forces to support motility are divergent class XIV myosins (MyoA) coordinated by accessory proteins known as light chains. Although the importance of the MyoA-light chain complex is well-established, the detailed mechanisms governing its assembly and regulation are relatively unknown. To establish a molecular blueprint of this dynamic complex, we first mapped the adjacent binding sites of light chains MLC1 and ELC1 on the MyoA neck (residues 775-818) using a combination of hydrogen-deuterium exchange mass spectrometry and isothermal titration calorimetry. We then determined the 1.85 Å resolution crystal structure of MLC1 in complex with its cognate MyoA peptide. Structural analysis revealed a bilobed architecture with MLC1 clamping tightly around the helical MyoA peptide, consistent with the stable 10 nm Kd measured by isothermal titration calorimetry. We next showed that coordination of calcium by an EF-hand in ELC1 and prebinding of MLC1 to the MyoA neck enhanced the affinity of ELC1 for the MyoA neck 7- and 8-fold, respectively. When combined, these factors enhanced ELC1 binding 49-fold (to a Kd of 12 nm). Using the full-length MyoA motor (residues 1-831), we then showed that, in addition to coordinating the neck region, ELC1 appears to engage the MyoA converter subdomain, which couples the motor domain to the neck. These data support an assembly model where staged binding events cooperate to yield high-affinity complexes that are able to maximize force transduction.

A Toxoplasma gondii class XIV myosin, expressed in Sf9 cells with a parasite co-chaperone, requires two light chains for fast motility.

Many diverse myosin classes can be expressed using the baculovirus/Sf9 insect cell expression system, whereas others have been recalcitrant. We hypothesized that most myosins utilize Sf9 cell chaperones, but others require an organism-specific co-chaperone. TgMyoA, a class XIVa myosin from the parasite Toxoplasma gondii, is required for the parasite to efficiently move and invade host cells. The T. gondii genome contains one UCS family myosin co-chaperone (TgUNC). TgMyoA expressed in Sf9 cells was soluble and functional only if the heavy and light chain(s) were co-expressed with TgUNC. The tetratricopeptide repeat domain of TgUNC was not essential to obtain functional myosin, implying that there are other mechanisms to recruit Hsp90. Purified TgMyoA heavy chain complexed with its regulatory light chain (TgMLC1) moved actin in a motility assay at a speed of ∼1.5 µm/s. When a putative essential light chain (TgELC1) was also bound, TgMyoA moved actin at more than twice that speed (∼3.4 µm/s). This result implies that two light chains bind to and stabilize the lever arm, the domain that amplifies small motions at the active site into the larger motions that propel actin at fast speeds. Our results show that the TgMyoA domain structure is more similar to other myosins than previously appreciated and provide a molecular explanation for how it moves actin at fast speeds. The ability to express milligram quantities of a class XIV myosin in a heterologous system paves the way for detailed structure-function analysis of TgMyoA and identification of small molecule inhibitors.

Identification and characterization of an Nrf2-mediated ARE upstream of the rat glutamate cysteine ligase catalytic subunit gene (GCLC).

The antioxidant response element (ARE) is an essential component of upstream regulatory sequences present on genes for most phase II detoxification enzymes, including the glutamate cysteine ligase catalytic subunit (GCLC). NF-E2-related factor 2 (Nrf2) is a principal transcription factor that binds to the ARE and plays a key role in cellular responses to stress via the Keap1-Nrf2-ARE pathway. However, the ARE that mediates human GCLC gene expression has not been found in the rat. Thus, how the ARE-mediated Keap1-Nrf2-ARE pathway regulates glutathione homeostasis in the rat remains a puzzle. We have identified a putative ARE sequence approximately 4 kb upstream in the rat GCLC. We further defined the rat GCLC-ARE in the category with the most ARE characters, that is, this rat GCLC-ARE is a sequence-specific site that significantly enhances promoter activity in reporter genes. The rat GCLC-ARE is an Nrf2-mediated element to which binding has been demonstrated in nuclear extracts and induced by tert-butylhydroquinone. Given the central role that rat models play in toxicology and pathology, this first discovery of the rat GCLC-ARE enhancer similar to that found in the human gene has broad implications for the study of antioxidant defenses and their regulation in a number of different fields.