Native State of Complement Protein C3d Analysed via Hydrogen Exchange and Conformational Sampling

Abstract

Hydrogen/deuterium exchange detected by mass spectrometry (HDXMS) provides valuable information on protein structure and dynamics. Although HDX-MS data is often interpreted using crystal structures, it was suggested that conformational ensembles produced by molecular dynamics simulations yield more accurate interpretations. In this paper, we analyse the complement protein C3d by performing an HDX-MS experiment, and evaluate several interpretation methodologies using an existing prediction model to derive HDX-MS data from protein structure. To interpret and refine C3d's HDX-MS data, we look for a conformation (or conformational ensemble) of C3d that allows computationally replicating this data. We confirm that crystal structures are not a good choice and suggest that conformational ensembles produced by molecular dynamics simulations might not always be satisfactory either. Finally, we show that coarse-grained conformational sampling of C3d produces a conformation from which its HDX-MS data can be replicated and refined.

Keywords: X-ray crystallography; coarse-grained conformational sampling; complement protein C3d; conformational ensembles; hydrogen exchange; mass spectrometry; molecular dynamics; native state; protein conformational sampling; protein structures.

Figure 1

Deriving HDX-MS data from two conformations of C3d. Histograms of differences (for the 81 peptides extracted from C3d) obtained when assessing the goodness-of-fit between the experimental HDX-MS data obtained for C3d and the HDX-MS data derived from the PDB conformation of C3d, or from a minimized version of this conformation.

Figure 2

Deriving HDX-MS data from MD simulations of C3d. Histograms of differences (for the 81 peptides extracted from C3d) obtained when assessing the goodness-of-fit between the experimentally-observed HDX-MS data and the HDX-MS data derived from ensembles of conformations extracted from three MD simulations of C3d.

Figure 3

Deriving HDX-MS data from MD conformations of C3d. Histograms of differences (for the 81 peptides of C3d) obtained when assessing the goodness-of-fit between the experimentally-observed HDX-MS data and the HDX-MS data derived from single conformations extracted from three MD simulations of C3d.

Figure 4

Deriving HDX-MS data from high-temperature MD simulations of C3d. Histograms of differences obtained when assessing the goodness-of-fit between the experimentally-observed HDX-MS data and the HDX-MS data derived from single conformations extracted from MD simulations of C3d at 400 K, 450 K and 500 K respectively.

Figure 5

Deriving HDX-MS data from coarse-grained conformational sampling of C3d. Histograms of differences obtained when assessing the goodness-of-fit between the experimentally-observed HDX-MS data of C3d and the HDX-MS data derived from its PDB conformation or its HDX conformation (i.e., the conformation produced by coarse-grained conformational sampling).

Figure 6

PDB and HDX conformations of C3d depicted using the ribbon model.

Figure 7

Heat-map visualization of the protection factors of C3d’s residues. Protection factors are derived from, and depicted on, the HDX conformation of C3d. Prolines are coloured in green. Other residues are coloured using a spectrum corresponding to the range of protection values.