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

Significance

Long pentraxins are soluble pattern-recognition molecules. There is a wealth of functional data linking them to processes as varied as innate immune defense, female fertility, and neurobiology. However, structural information is missing. Here, we present a complete high-resolution model of a long pentraxin, PTX3, using a hybrid structural biology approach combining cryoelectron microscopy, mass spectrometry, and AlphaFold-based artificial intelligence structure prediction, which was able to model flexible regions of the complex. This gives insights into the functions of PTX3, such as immune defense, as well as exemplifying a method to resolving flexible domains of protein complexes.

Abstract

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.