Abstract
Myosin B (MyoB) is a class 14 myosin expressed in all invasive stages of the malaria parasite, Plasmodium falciparum. It is not associated with the glideosome complex that drives motility and invasion of host cells. During red blood cell invasion, it remains at the apical tip of the merozoite but is no longer observed once invasion is completed. MyoB is not essential for parasite survival but, when it is knocked out, merozoites are delayed in the initial stages of red blood cell invasion, giving rise to a growth defect that correlates with reduced invasion success. Here, we have expressed and purified functional MyoB with the help of parasite-specific chaperones Hsp90 and Unc45, characterized its binding to actin and its known light chain MLC-B using biochemical and biophysical methods, and determined its low-resolution structure in solution using small-angle X-ray scattering. In addition to MLC-B, four other putative regulatory light chains were found to bind to the MyoB IQ2 motif in vitro. The purified recombinant MyoB adopted the overall shape of a myosin, exhibited actin-activated ATPase activity, and moved actin filaments in vitro. The ADP release rate was faster than the ATP turnover number, and thus, does not appear to be rate-limiting. This, together with the observed high affinity to actin and the specific localization of MyoB, may point towards a role in tethering and/or force sensing during early stages of invasion.
Competing Interest Statement
The authors have declared no competing interest.
Abbreviations and nomenclature
- CaMLD
- calmodulin like-domain
- ELC
- essential light chain
- HCM
- hypertrophic cardiomyopathy
- Hsp
- heat-shock protein
- MLC-B
- myosin light chain B
- MTIP
- myosin A-tail domain interacting protein Myo myosin
- NPI
- N-(1-pyrene)iodoacetamide
- Pf
- Plasmodium falciparum
- PLC
- putative light chain
- PTM
- post translational modifications
- SAXS
- small-angle X-ray scattering
- SDS-PAGE
- sodium dodecyl sulfate polyacrylamide gel electrophoresis
- SEC
- size-exclusion chromatography
- Sf
- Spodoptera frugiperda
- SR-CD
- synchrotron radiation circular dichroism spectroscopy
- TCEP
- tris(2-carboxyethyl)phosphine
- TEV
- tobacco etch virus protease Unc45 uncoordinated mutant number 45