Related Experiment Video
Updated: Jun 12, 2025
Identifying PD-1/PD-L1 Inhibitors with Surface Plasmon Resonance Technology
Published on: May 2, 2025
Residue-Specific Epitope Mapping of the PD-1/Nivolumab Interaction Using X-ray Footprinting Mass Spectrometry
Line G Kristensen1, Sayan Gupta1, Yan Chen2
1Lawrence Berkeley National Laboratory, Molecular Biophysics and Integrated Bioimaging Division, Berkeley, CA 94720, USA.
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.
Insights
X-ray footprinting coupled with mass spectrometry (XFMS) reveals protein dynamics in solution. This method validated nivolumab-PD-1 interactions, agreeing with crystal structure data and identifying glycosylation sites.
Area of Science:
- Structural biology
- Biochemistry
- Biophysics
Background:
- Protein conformation and dynamics are crucial for biological function.
- Understanding antibody-antigen interactions is key in drug development, particularly for cancer immunotherapies.
- X-ray footprinting coupled with mass spectrometry (XFMS) is an emerging technique for studying biomolecular structures in solution.
Purpose of the Study:
- To demonstrate the utility of X-ray footprinting coupled with mass spectrometry (XFMS) for structural biology.
- To analyze the interaction between the monoclonal antibody nivolumab and its target PD-1 in solution.
- To compare XFMS data with existing crystal structure data of the nivolumab-PD-1 complex.
Main Methods:
- X-ray footprinting coupled with mass spectrometry (XFMS) was employed to assess solvent accessibility.
- Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for deglycosylation analysis.
- Oxidative footprinting was performed on free PD-1 and PD-1 bound to nivolumab.
Main Results:
- Changes in side-chain solvent accessibility upon nivolumab binding to PD-1 were consistent with the crystal structure interface.
- N-linked glycosylation sites on PD-1 were confirmed via LC-MS/MS analysis of asparagine deamidation.
- Subtle alterations in solvent accessibility were detected in the C'D loop of PD-1 upon complex formation.
Conclusions:
- XFMS is a valuable tool for characterizing protein conformation and dynamics in solution.
- The study validates XFMS findings against established structural data for the nivolumab-PD-1 complex.
- XFMS provides insights into the molecular details of antibody-antigen interactions relevant to immunotherapy.
