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. 2024 Sep 19;13(3):77.
doi: 10.3390/antib13030077.

Residue-Specific Epitope Mapping of the PD-1/Nivolumab Interaction Using X-ray Footprinting Mass Spectrometry

Line G Kristensen  1 , Sayan Gupta  1 , Yan Chen  2 , Christopher J Petzold  2 , Corie Y Ralston  3
Affiliations

Residue-Specific Epitope Mapping of the PD-1/Nivolumab Interaction Using X-ray Footprinting Mass Spectrometry

Line G Kristensen et al. Antibodies (Basel). .

Abstract

X-ray footprinting coupled with mass spectrometry (XFMS) presents a novel approach in structural biology, offering insights into protein conformation and dynamics in the solution state. The interaction of the cancer-immunotherapy monoclonal antibody nivolumab with its antigen target PD-1 was used to showcase the utility of XFMS against the previously published crystal structure of the complex. Changes in side-chain solvent accessibility, as determined by the oxidative footprint of free PD-1 versus PD-1 bound to nivolumab, agree with the binding interface side-chain interactions reported from the crystal structure of the complex. The N-linked glycosylation sites of PD-1 were confirmed through an LC-MS/MS-based deglycosylation analysis of asparagine deamidation. In addition, subtle changes in side-chain solvent accessibility were observed in the C'D loop region of PD-1 upon complex formation with nivolumab.

Keywords: ICI; PD-1; X-ray footprinting mass spectrometry (XFMS); epitope mapping; hydroxyl radical footprinting; nivolumab; programmed cell death protein 1.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Schematic representation of the X-ray hydroxyl radical footprinting mass spectrometry workflow. (A) Synchrotron irradiation of a dilute protein sample in an aqueous, buffered solution produces hydroxyl radicals as a result of radiolysis of water, and hydroxyl radicals covalently label solvent-exposed side-chains if they are produced in proximity to the side-chain. The interface between antigen and antibody provides protection from labeling which leads to a different oxidative footprint from that of the free antigen. (B) Bottom-up LC-MS/MS analysis of protease-digested samples produces chromatograms of modified and unmodified peptides for each exposure time. (C) Fraction unmodified, calculated on the basis of the peak areas under the curve (AUC), is plotted as a function of exposure time. The dose response plot is fitted to a first-order exponential equation which generates the hydroxyl radical reactivity rate constant, k(s−1). The ratio of hydroxyl radical reactivity rate constants is independent of the intrinsic reactivity of the residue and the ratio therefore represents the relative change in solvent accessibility of a particular residue.
Figure 2
Figure 2
PD-1 residue-specific change in solvent accessibility (SA) upon PD-1/nivolumab complex formation. The height of each column corresponds to the ratio (R) of the hydroxyl radical reactivity rate constant of free PD-1 to the hydroxyl radical reactivity rate constant of PD-1 bound to nivolumab. Unless noted, the residue modification represents a hydroxylated product with a +16 Da mass shift. Co-eluting, modified peptide isomers are shown as mixed modifications. The bar chart color scheme reflects changes in solvent accessibility between free PD-1 and the PD-1/nivolumab complex. Gray-colored bars indicate the modification observed, but minimal change in solvent accessibility. Error bars represent the SD of the ratio [28]. The sequence locations of N-linked glycosylation sites and PD-1 structural loops are indicated with green triangles and purple arrows respectively.
Figure 3
Figure 3
XFMS data mapped onto the structure of PD-1 bound to nivolumab-Fab (PDB: 5WT9). The blue color gradient for footprinted PD-1 residues indicates the change in solvent accessibility upon complex formation, with deep blue representing a greater than three-fold decrease in solvent accessibility. Labeled side-chains in gray showed modification but minimal change in solvent accessibility. The modified residues D85, R86, P89, and C93 could not be visualized in the structural model due to the missing C′D loop. This figure was prepared with ChimeraX [29].
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