During the second funding period, we have discovered that KAHRP initially also binds to the ankyrin bridges (ABs) before it relocalizes to the actin-based JCs, where it becomes a major component of the spiral (Fig. 1C).
For the third funding period, we plan to uncover the exact mechanism underlying KAHRP relocation and how it shapes the spiral scaffold. Our main hypotheses are that KAHRP-phosphorylation leads to the observed relocalization and that the spiral is built mainly from a host protein. To test these hypotheses, we will continue our KAHRP mutational analysis, based on site-directed mutagenesis of phosphorylation sites and an alanine linker scan. We have already generated 24 different endogenous KAHRP mutants in the parasite, which are currently being characterized. To causatively link pheno- and genotypes with loss of binding function, we will investigate these mutants using super-resolution microscopy.
We will further continue our efforts to reveal the molecular nature of the knob spiral and the role KAHRP plays in spiral formation by purifying spirals, performing proximity labelling and mass spectrometry, and resolving the structure using single-particle cryo-electron microscopy.