Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Aug 16;119(33):e2208144119.
doi: 10.1073/pnas.2208144119. Epub 2022 Aug 8.

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

Dylan P Noone  1 , Douwe J Dijkstra  2 , Teun T van der Klugt  1 , Peter A van Veelen  3 , Arnoud H de Ru  3 , Paul J Hensbergen  3 , Leendert A Trouw  2 , Thomas H Sharp  1
Affiliations

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

Dylan P Noone et al. Proc Natl Acad Sci U S A. .

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.

Keywords: AlphaFold; COVID19; Long pentraxin; complement; cryoEM.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Purification of recombinant human PTX3. (A) Schematic of the PTX3 monomer, indicating sites of glycosylation (N220) and disulfide bonds. Intramonomeric disulfide bonds are shown as closed loops. Numbering of residues is shown in gray. (B) SEC elution profile of PTX3 after Ni-NTA affinity and SEC purification. (C) Gel electrophoretic profiles based on TCE staining (S) and Western blots (WB) of PTX3 after purification under in the presence of SDS and/or DTT. Arrows indicate standard protein markers of the indicated molecular weight.
Fig. 2.
Fig. 2.
Cryo-EM structure of human PTX3. (A) Class averages show octameric structures in solution with less well-defined density protruding from the octameric core. (Scale bar, 110 Å.) (B) Cryo-EM map of the octameric PTX domains of PTX3 resolved with D4 symmetry applied. Each monomer is colored differently. (Scale bar, 50 Å.) (C) Noisy density was visible at the N-terminal regions at lower isosurface thresholds, which a low-pass filter reveals to be structured density protruding from the PTX domain core. (Scale bar, 50 Å, Right, and 10 Å, Left.) (D) The C1 (no applied symmetry, Upper) and D4 symmetrized (Lower) maps both provided sufficient density to model side chain rotamers. (E) Subunit schematic showing PTX domains arranged in a dice-like structure. (F) Model of the PTX domains and partial N-terminal coiled-coil region. (Scale bar, 50 Å.)
Fig. 3.
Fig. 3.
Cysteine bond network of the PTX domain of PTX3. (A) Octameric arrangement of the PTX domains with cysteine sulfur atoms shown as yellow spheres and disulfide bonds labeled. (B) C317/C318 intertetramer disulfide bond with density mesh showing fit for all atoms except VGG of the glycine linker. The map was low-pass–filtered to 5 Å to aid model building. (C) Reduced resolution in this region does not exclude other potential configurations of the C317/C318 bond network. Shown are a schematic (Top), and two views of the disulfide patch rotated 90° (Middle and Bottom).
Fig. 4.
Fig. 4.
Heterogenous N-linked glycosylation of PTX3 at N220. (A) Treatment of monomeric PTX3 with PNGase F (Upper) and neuraminidase (Lower). (B) MS analysis of different glycoforms of the PTX3 tryptic peptide ATDVLNK containing the N-glycosylation site (N220, underlined) in the lower and higher molecular-weight band 1 (Left) and band 2 (Right), respectively. (C) Summed mass spectra (20 to 24 min) from the LC-MS analysis of tryptic peptides from PTX3, corresponding to the lower and higher molecular weight bands (band 1, Upper; and band 2, Lower) shown in A. Predicted species are shown in schematic form. (D) D4 symmetrical map lowpass filtered to 5 Å showing density not accounted for by the protein, with a schematic of the core glycan and detailed view of the boxed region in E. (E) Location of glycans and N220 (cyan) on the low-pass–filtered (4 Å) D4 symmetrical map of the PTX octameric complex. (Scale bar, 50 Å.) The key for the monosaccharides species in C is applicable to D (sialic acid, galactose, mannose, fucose, and N-acetylglucosamine are represented by Neu5Ac, Gal, Man, Fuc, and GlcNAc respectively).
Fig. 5.
Fig. 5.
AlphaFold-based structure prediction and validation of the flexible N-terminal region. (A) Structural data from AlphaFold was used to model the entire tetrameric coiled coil N-terminal tail. Cysteine and proline residues in the N-terminal region are represented by yellow and cyan spheres, respectively. (B) Variability analysis of cryo-EM images with masking either side of the pentraxin domain used to visualize the N-terminal region. (Scale bar, 100 nm.) (C) Focused 2D variability analysis with selective masking of one N-terminal region. The atomic model of the N-terminal region could then be modeled onto the 2D classes. Green density in the Lower panels in B and C highlight the N-terminal domain in the 2D classes. (Scale bar in upper panels, 100 nm. Scale bar in lower panel, 50 nm.) (D) Enlarged view of the disordered region showing two possible configurations of C47 and C49 disulfide bonding. (E) Enlarged views of the second hinge region with the two possible configurations of C103-mediated disulfide bonding shown. (F) Binding sites for the ligands shown mapped onto the full-length PTX3 structure. (Scale bar, 50 nm.)
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Garlanda C., Bottazzi B., Bastone A., Mantovani A., Pentraxins at the crossroads between innate immunity, inflammation, matrix deposition, and female fertility. Annu. Rev. Immunol. 23, 337–366 (2005). - PubMed
    1. Reading P. C., et al. , Antiviral activity of the long chain pentraxin PTX3 against influenza viruses. J. Immunol. 180, 3391–3398 (2008). - PubMed
    1. Doni A., et al. , An acidic microenvironment sets the humoral pattern recognition molecule PTX3 in a tissue repair mode. J. Exp. Med. 212, 905–925 (2015). - PMC - PubMed
    1. Salustri A., et al. , PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization. Development 131, 1577–1586 (2004). - PubMed
    1. Noone D. P., van der Velden T. T., Sharp T. H., 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. Front Immunol. 12, 757633 (2021). - PMC - PubMed

Publication types

LinkOut - more resources