Structure of the Inhibited State of the Sec Translocon.

Protein secretion in eukaryotes and prokaryotes involves a universally conserved protein translocation channel formed by the Sec61 complex. Unrelated small-molecule natural products and synthetic compounds inhibit Sec61 with differential effects for different substrates or for Sec61 from different organisms, making this a promising target for therapeutic intervention. To understand the mode of inhibition and provide insight into the molecular mechanism of this dynamic translocon, we determined the structure of mammalian Sec61 inhibited by the Mycobacterium ulcerans exotoxin mycolactone via electron cryo-microscopy. Unexpectedly, the conformation of inhibited Sec61 is optimal for substrate engagement, with mycolactone wedging open the cytosolic side of the lateral gate. The inability of mycolactone-inhibited Sec61 to effectively transport substrate proteins implies that signal peptides and transmembrane domains pass through the site occupied by mycolactone. This provides a foundation for understanding the molecular mechanism of Sec61 inhibitors and reveals novel features of translocon function and dynamics.

Cep120 promotes microtubule formation through a unique tubulin binding C2 domain.

Centrioles are microtubule-based structures that play essential roles in cell division and cilia biogenesis. Cep120 is an important protein for correct centriole formation and mutations in the Cep120 gene cause severe human diseases like Joubert syndrome and complex ciliopathies. Here, we show that Cep120 contains three consecutive C2 domains that are followed by a coiled-coil dimerization domain. Surprisingly, unlike the classical C2 domains, all three Cep120 C2 domains lack calcium- and phospholipid-binding activities. However, biophysical and biochemical assays revealed that the N-terminal Cep120 C2 domain (C2A) binds to both tubulin and microtubules, and promotes microtubule formation. Structural analyses coupled with mutagenesis identified a highly conserved, positively charged residue patch on the surface of Cep120 C2A, which mediates the interaction with tubulin and microtubules. Together, our results establish Cep120 C2A as a unique microtubule-binding domain. They further provide insights into the molecular mechanism of Cep120 during centriole biogenesis.

Structural basis for inhibition of Plasmodium vivax invasion by a broadly neutralizing vaccine-induced human antibody.

The most widespread form of malaria is caused by Plasmodium vivax. To replicate, this parasite must invade immature red blood cells through a process requiring interaction of the P. vivax Duffy binding protein (PvDBP) with its human receptor, the Duffy antigen receptor for chemokines. Naturally acquired antibodies that inhibit this interaction associate with clinical immunity, suggesting PvDBP as a leading candidate for inclusion in a vaccine to prevent malaria due to P. vivax. Here, we isolated a panel of monoclonal antibodies from human volunteers immunized in a clinical vaccine trial of PvDBP. We screened their ability to prevent PvDBP from binding to the Duffy antigen receptor for chemokines, and their capacity to block red blood cell invasion by a transgenic Plasmodium knowlesi parasite genetically modified to express PvDBP and to prevent reticulocyte invasion by multiple clinical isolates of P. vivax. This identified a broadly neutralizing human monoclonal antibody that inhibited invasion of all tested strains of P. vivax. Finally, we determined the structure of a complex of this antibody bound to PvDBP, indicating the molecular basis for inhibition. These findings will guide future vaccine design strategies and open up possibilities for testing the prophylactic use of such an antibody.