EG5

EG5 (PF3D7_0317500) is a plus-end-directed kinesin-5 microtubule motor that operates during the atypical cell divisions of the Plasmodium life cycle. In P. berghei, EG5 localizes to the spindle apparatus and spindle poles during both atypical mitosis and meiosis, co-localizing with α-tubulin and centrin-2 and partially overlapping with the kinetochore marker NDC80 during blood-stage schizogony, with close association with NDC80 during male gametogony but not with the basal body/axoneme marker kinesin-8B; genetic deletion selectively reduces motile sporozoite production in oocysts without visible defects at other proliferative stages, defining a stage-specific requirement in the mosquito phase (PMID:33154955). Cryo-EM of the P. falciparum EG5 motor domain bound to microtubules establishes that it uses classical kinesin mechanochemistry, including ATP-induced subdomain rearrangement and cover-neck bundle formation consistent with plus-end-directed motility (PMID:34375637). The purified P. falciparum and P. vivax motor domains possess ATPase activity, and a loop5 insertion in the motor domain creates a structurally distinct environment at the human kinesin-5 drug-binding site that supports selective allosteric inhibition—exemplified by MMV666693, which inhibits Plasmodium EG5 without competing at the ATP site or disrupting microtubule binding (PMID:34375637, PMID:24737313).

1. 2014 Medium

   To test whether Plasmodium EG5 is a druggable target distinct from its human counterpart, its motor-domain ATPase was screened against small molecules, establishing that a selective allosteric inhibitor could act without competing at the conserved ATP or microtubule sites.

   Evidence Small-molecule ATPase screen of ~2000 compounds with parallel human kinesin-5 counter-screen and substrate-competition assays using P. falciparum and P. vivax motor domains

   PMID:24737313

   Open questions at the time

   • In vitro enzymatic assay only; no cellular validation that MMV666693 inhibits parasite EG5 in situ
   • Structural basis of the allosteric site not defined by this biochemical study
   • No demonstration of anti-parasitic activity or selectivity margin in living parasites
2. 2020 High

   To determine where and when EG5 acts in the parasite, its subcellular localization was mapped across proliferative stages and the gene was deleted, establishing it as a spindle/spindle-pole motor with a stage-specific requirement for sporozoite production.

   Evidence Live-cell GFP imaging, fixed-cell IFA, dual-color live imaging with tubulin/centrin/NDC80/kinesin-8B markers, microtubule inhibitor treatment, and gene deletion in P. berghei

   PMID:33154955

   Open questions at the time

   • Molecular mechanism linking spindle-pole localization to sporozoite motility not resolved
   • Why deletion affects oocyst-stage sporozoites but not other proliferative stages is unexplained
   • Direct interaction partners at the spindle not biochemically identified
3. 2021 High

   To explain how EG5 generates force and how it could be selectively targeted, its microtubule-bound motor domain was solved by cryo-EM, revealing conserved kinesin mechanochemistry and a loop5 insertion that structurally distinguishes the Plasmodium drug-binding site.

   Evidence Cryo-EM structure determination of the microtubule-bound P. falciparum motor domain with nucleotide-dependent 3D classification

   PMID:34375637

   Open questions at the time

   • No co-structure with an inhibitor such as MMV666693 bound at the loop5 site
   • Structure limited to the isolated motor domain; full-length motor architecture and oligomerization not addressed
   • Motility parameters (velocity, processivity) not measured

• The mechanistic chain connecting EG5 spindle motor activity to infectious sporozoite formation, and validation of allosteric inhibitors against parasites in the mosquito stage, remains open.
• No cellular or in vivo proof that inhibiting EG5 ATPase blocks sporozoite production
• Spindle interactome and regulation of EG5 recruitment not characterized
• Species-specific differences between P. berghei function and P. falciparum/P. vivax biochemistry not reconciled