PTX3 structure determination using a hybrid cryoelectron microscopy and AlphaFold approach offers insights into ligand binding and complement activation.

Pattern recognition molecules (PRMs) form an important part of innate immunity, where they facilitate the response to infections and damage by triggering processes such as inflammation. The pentraxin family of soluble PRMs comprises long and short pentraxins, with the former containing unique N-terminal regions unrelated to other proteins or each other. No complete high-resolution structural information exists about long pentraxins, unlike the short pentraxins, where there is an abundance of both X-ray and cryoelectron microscopy (cryo-EM)-derived structures. This study presents a high-resolution structure of the prototypical long pentraxin, PTX3. Cryo-EM yielded a 2.5-Å map of the C-terminal pentraxin domains that revealed a radically different quaternary structure compared to other pentraxins, comprising a glycosylated D4 symmetrical octameric complex stabilized by an extensive disulfide network. The cryo-EM map indicated α-helices that extended N terminal of the pentraxin domains that were not fully resolved. AlphaFold was used to predict the remaining N-terminal structure of the octameric PTX3 complex, revealing two long tetrameric coiled coils with two hinge regions, which was validated using classification of cryo-EM two-dimensional averages. The resulting hybrid cryo-EM/AlphaFold structure allowed mapping of ligand binding sites, such as C1q and fibroblast growth factor-2, as well as rationalization of previous biochemical data. Given the relevance of PTX3 in conditions ranging from COVID-19 prognosis, cancer progression, and female infertility, this structure could be used to inform the understanding and rational design of therapies for these disorders and processes.

Cryo-Electron Microscopy and Biochemical Analysis Offer Insights Into the Effects of Acidic pH, Such as Occur During Acidosis, on the Complement Binding Properties of C-Reactive Protein.

The pentraxin family of proteins includes C-reactive protein (CRP), a canonical marker for the acute phase inflammatory response. As compared to normal physiological conditions in human serum, under conditions associated with damage and inflammation, such as acidosis and the oxidative burst, CRP exhibits modulated biochemical properties that may have a structural basis. Here, we explore how pH and ligand binding affect the structure and biochemical properties of CRP. Cryo-electron microscopy was used to solve structures of CRP at pH 7.5 or pH 5 and in the presence or absence of the ligand phosphocholine (PCh), which yielded 7 new high-resolution structures of CRP, including pentameric and decameric complexes. Structures previously derived from crystallography were imperfect pentagons, as shown by the variable angles between each subunit, whereas pentameric CRP derived from cryoEM was found to have C5 symmetry, with subunits forming a regular pentagon with equal angles. This discrepancy indicates flexibility at the interfaces of monomers that may relate to activation of the complement system by the C1 complex. CRP also appears to readily decamerise in solution into dimers of pentamers, which obscures the postulated binding sites for C1. Subtle structural rearrangements were observed between the conditions tested, including a putative change in histidine protonation that may prime the disulphide bridges for reduction and enhanced ability to activate the immune system. Enzyme-linked immunosorbent assays showed that CRP had markedly increased association to the C1 complex and immunoglobulins under conditions associated with acidosis, whilst a reduction in the Ca2+ concentration lowered this pH-sensitivity for C1q, but not immunoglobulins, suggesting different modes of binding. These data suggest a model whereby a change in the ionic nature of CRP and immunological proteins can make it more adhesive to potential ligands without large structural rearrangements.

A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation across Biological Membranes.

Protein translocation is a fundamental process in biology. Major gaps in our understanding of this process arise due the poor sensitivity, low time resolution and irreproducibility of translocation assays. To address this, we applied NanoLuc split-luciferase to produce a new strategy for measuring protein transport. The system reduces the timescale of data collection from days to minutes and allows for continuous acquisition with a time resolution in the order of seconds, yielding kinetics parameters suitable for mechanistic elucidation and mathematical fitting. To demonstrate its versatility, we implemented and validated the assay in vitro and in vivo for the bacterial Sec system and the mitochondrial protein import apparatus. Overall, this technology represents a major step forward, providing a powerful new tool for fundamental mechanistic enquiry of protein translocation and for inhibitor (drug) screening, with an intensity and rigor unattainable through classical methods.