The proteasome is a multi-subunit enzyme complex that is responsible for most of the non-lysosomal proteolysis in eukaryotic cells. The 20S proteasome core is comprised of four heptameric rings, two rings of β-subunits, which contain the catalytic sites, sandwiched by two α-rings, which control substrate access to the proteasome core. The malaria parasite, Plasmodium falciparum is highly reliant on its protein turnover machinery, thus the proteasome is a drug target in the treatment of malaria. The activity of the 20S proteasome is regulated by protein complexes, such as the 19S complex, that bind to the α-subunit rings. A less-well studied regulator is the PA28 (11S) activator, a heptameric protein which stimulates proteasome activity independent of ubiquitin or ATP.
Here, we characterized PA28 from P. falciparum (PfPA28). We showed that genetic deletion of PfPA28 renders parasites more sensitive to anti-malarial drugs, consistent with a role for PA28 in responding to proteotoxic stress. We solved the crystal structure of PfPA28, revealing a bell-shaped structure with a highly charged central channel. We purified Pf20S from parasite cultures and structurally characterized the PfPA28-Pf20S complex using single-particle cryoEM. We solved the structure of Pf20S in complex with one and two PfPA28 caps to a resolution of 3.92 Å and 3.82 Å respectively. These structures provide insight into the binding and activation mechanism of PfPA28 and provide further evidence that 11S activators employ a distinct mechanism of activation compared to the 19S complex. Analysis of the cryo-EM data also showed that PfPA28 and Pf20S form a dynamic complex, with PfPA28 undergoing large motions on Pf20S. We propose lateral transfer of proteasome products through this dynamic interface as an alternative mechanism of substrate egress, avoiding the need for products to traverse the PfPA28 pore.