Many pathogenic bacteria produce pore-forming toxins to attack and kill human cells. We have determined the 4.5 Å structure of the ~2.2 MDa pore complex of pneumolysin, the main virulence factor of Streptococcus pneumoniae, by cryoEM. The pneumolysin pore is a 400 Å ring of 42 membrane-inserted monomers. Domain D3 of the soluble toxin refolds into two ~85 Å β-hairpins that traverse the lipid bilayer and assemble into a 168-strand β-barrel. The pore complex is stabilized by salt bridges between β-hairpins of adjacent subunits and an internal α-barrel. The apolar outer barrel surface with large sidechains is immersed in the lipid bilayer, while the inner barrel surface is highly charged. Comparison of the cryoEM pore complex to the prepore structure obtained by electron cryo-tomography and the x-ray structure of the soluble form reveals the detailed mechanisms by which the toxin monomers insert into the lipid bilayer to perforate the target membrane.
Crystal structure of pneumolysin D168A mutantPublicly available at the RCSB Protein Data Bank (accession no: 5AOE).
Crystal structure of pneumolysin deletion mutant Delta146_147Publicly available at the RCSB Protein Data Bank (accession no: 5AOF).
CryoEM structure of the membrane pore complex of Pneumolysin at 4.5APublicly available at the EMBL-EBI Protein Data Bank (accession no: 5LY6).
CryoEM structure of the membrane pore complex of Pneumolysin at 4.5APublicly available at the EMBL-EBI Protein Data Bank (accession no: EMD-4118).
Crystal structure of wild type pneumolysin.Publicly available at the RCSB Protein Data Bank (accession no: 5AOD).
- Katharina van Pee
- Alexander Neuhaus
- Edoardo D'Imprima
- Deryck J Mills
- Werner Kühlbrandt
- Özkan Yildiz
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Sjors HW Scheres, MRC Laboratory of Molecular Biology,, United Kingdom
© 2017, van Pee et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Exocytosis of secretory vesicles requires the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and small GTPase Rabs. As a Rab3/Rab27 effector protein on secretory vesicles, Rabphilin 3A was implicated to interact with SNAP-25 to regulate vesicle exocytosis in neurons and neuroendocrine cells, yet the underlying mechanism remains unclear. In this study, we have characterized the physiologically relevant binding sites between Rabphilin 3A and SNAP-25. We found that an intramolecular interplay between the N-terminal Rab-binding domain and C-terminal C2AB domain enables Rabphilin 3A to strongly bind the SNAP-25 N-peptide region via its C2B bottom α-helix. Disruption of this interaction significantly impaired docking and fusion of vesicles with the plasma membrane in rat PC12 cells. In addition, we found that this interaction allows Rabphilin 3A to accelerate SNARE complex assembly. Furthermore, we revealed that this interaction accelerates SNARE complex assembly via inducing a conformational switch from random coils to α-helical structure in the SNAP-25 SNARE motif. Altogether, our data suggest that the promotion of SNARE complex assembly by binding the C2B bottom α-helix of Rabphilin 3A to the N-peptide of SNAP-25 underlies a pre-fusion function of Rabphilin 3A in vesicle exocytosis.
3′ end formation of most eukaryotic mRNAs is dependent on the assembly of a ~1.5 MDa multiprotein complex, that catalyzes the coupled reaction of pre-mRNA cleavage and polyadenylation. In mammals, the cleavage and polyadenylation specificity factor (CPSF) constitutes the core of the 3′ end processing machinery onto which the remaining factors, including cleavage stimulation factor (CstF) and poly(A) polymerase (PAP), assemble. These interactions are mediated by Fip1, a CPSF subunit characterized by high degree of intrinsic disorder. Here, we report two crystal structures revealing the interactions of human Fip1 (hFip1) with CPSF30 and CstF77. We demonstrate that CPSF contains two copies of hFip1, each binding to the zinc finger (ZF) domains 4 and 5 of CPSF30. Using polyadenylation assays we show that the two hFip1 copies are functionally redundant in recruiting one copy of PAP, thereby increasing the processivity of RNA polyadenylation. We further show that the interaction between hFip1 and CstF77 is mediated via a short motif in the N-terminal ‘acidic’ region of hFip1. In turn, CstF77 competitively inhibits CPSF-dependent PAP recruitment and 3′ polyadenylation. Taken together, these results provide a structural basis for the multivalent scaffolding and regulatory functions of hFip1 in 3′ end processing.