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. 2021 Dec 16:12:757633.
doi: 10.3389/fimmu.2021.757633. eCollection 2021.

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

Dylan P Noone  1 , Tijn T van der Velden  1 , Thomas H Sharp  1
Affiliations

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

Dylan P Noone et al. Front Immunol. .

Abstract

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.

Keywords: CRP; ELISA; acidosis; complement; cryoEM; structural biology.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Measuring CRP binding to protein ligands at pH 7.5 and pH 5 in the presence of 2 mM calcium. (A) Binding of CRP to immobilised C1q, gC1q, and human and rabbit IgG (hIgG and rIgG, respectively). (B) Binding of CRP to immobilised HSA and BSA. CRP binding to gC1q at pH 5 and the spermidine coat are present in both panels for reference. Error bars show the standard deviation of 3 independent replicates.
Figure 2
Figure 2
Assessing the influence of calcium on the binding properties of CRP. (A, B) Measuring CRP binding at 50 µM Ca2+ to (A) C1q and gC1q, (B) human and rabbit IgG. (C, D) Measuring CRP binding at 0 mM Ca2+ and 5 mM EDTA to (C) C1q and gC1q, (D) human and rabbit IgG. Binding is measured at pH 7.5 and pH 5. CRP binding to gC1q at pH 5 and spermidine (coat) is present in all panels for reference. Error bars show the standard deviation of 3 independent replicates.
Figure 3
Figure 3
CryoEM of CRP reveals the co-existence of pentameric and decameric structures. (A) Class averages show pentameric and decameric structures in solution within the same sample (pH 7.5). (B) EM maps of CRP pentamers solved at pH 7.5 with both C1 (orange) and C5 (green) symmetry applied. Examples of sidechain density are shown above a schematic of the CRP pentamer as a disk with 2 opposing A and B faces. (C) EM map of a CRP decamer solved at pH 7.5, with examples of sidechain density and a schematic showing A-face stacking between the dimers of pentamers. (D) Top view of the atomic model of the CRP pentamer solved at pH 7.5. (E) Atomic model of the CRP decamer solved at pH 7.5, shown from the top and side. Panels (B, C, E) show both side and top-down views of pentameric and decameric maps or models related to one another via a 90° rotation.
Figure 4
Figure 4
Comparing the similarity of CRP models and maps. (A) Comparing CRP pentamers refined into C1 and C5 symmetric maps at pH 7.5 and 5, ± PCh. RMSD is used to compare models, and cross-correlation coefficients (CCC) used to compare maps. (B) Comparing CRP decamers and pentamers at pH 7.5 and 5, ± PCh. In all panels, blue indicates high similarity (low RMSD, high CCC), red indicates divergence (high RMSD, low CCC).
Figure 5
Figure 5
Monomers associate via ionic bonds within CRP pentamers and decamers. (A) Monomer-monomer interfaces are shown by the red regions. Five ionic bonds act as a zipper between subunits: Arg6–Asp169, Glu101–Lys201, Glu197–Lys123 and Arg116–Glu42–Lys119. Images are taken from C5 symmetric pentameric maps and models. (B) Glu197 shifts away from Lys123 at pH 5, breaking this ionic bond. Residues in green and orange represent pH 7.5 and pH 5, resp. Both are derived from the C5-symmetric pentameric models. (C) Pentamer-pentamer interfaces are shown in red. Five ionic bonds interact between pentamers. Schematic shows the subunit layout of the decameric models. Ionic bonds mediate pentamer-pentamer contacts. All images shown are from the pH 7.5 decamer.
Figure 6
Figure 6
Comparison of inter-monomer and inter-pentamer ionic bond lengths in EM and X-ray derived models. The indicated bonds and interfaces within each model were measured and plotted (colored marks). Averages of these bonds were also calculated (black horizonal bars). (A) Comparison of the EM-derived pentameric CRP model at pH 5, and PDB depositions 1B09, 3LY2, 1GNH, 3PVO and 3PVN (–3, 40). (B) Comparison of monomer-monomer ionic bonds between all pentameric CRP cryoEM-derived models. (C) Comparison of monomer-monomer ionic bonds between all decameric cryoEM derived protein models. Measurements from the pentameric CRP cryoEM-derived model at pH 5 is shown for reference in panels (A–C) for reference. (D) Comparison of interpentameric ionic bonds present in cryoEM-derived decameric models, and decameric CRP found in PDB entries 3LY2 and 3PVN. CryoEM derived decameric subunit labelling was used (A–J).
Figure 7
Figure 7
Differences in pH and ligand binding revealed by cryoEM. (A) Both Ca2+calcium binding sites shown in maps of CRP decamers at pH 5. Maps show density differences in the absence and presence of phosphocholine ligand (PCh). (B) Density corresponding to coordinated water molecules are present in both pH 7.5 and pH 5 (top), and display different density maps at higher isosurface thresholds (bottom).
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