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Mapping the Binding Interactions between Human Gasdermin D and Human Caspase-1 Using Carbene Footprinting
James R Lloyd1, Antonio Biasutto2, Katharina L Dürr2
1School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, U.K.
JACS Au
|July 28, 2023
Summary
Carbene footprinting reveals direct binding of human Gasdermin D to human Caspase-1 active sites. This technique also shows how Gasdermin D inserts into lipid bilayers and how inhibitors interact with Caspase-1.
Area of Science:
- Biochemistry
- Chemical Biology
- Molecular Biology
Background:
- Protein-protein interactions are crucial for cellular processes.
- Caspase-1 and Gasdermin D are key regulators of inflammatory cell death.
- Understanding their interaction is vital for therapeutic development.
Purpose of the Study:
- To investigate the binding interactions between human Caspase-1 (C285A) and human Gasdermin D (hGSDMD) using carbene footprinting.
- To elucidate the role of hGSDMD in membrane pore formation.
- To study the interaction of Caspase-1 with the inhibitor VRT-043198.
Main Methods:
- Carbene footprinting, a mass spectrometry-based chemical labeling technique.
- Analysis of protein binding interfaces and conformational changes.
- Differential labeling of full-length hGSDMD and its N-terminal domain in liposomes.
Main Results:
- Identified masked regions on hCaspase-1 and hGSDMD upon binding, indicating active-site and exosite interactions.
- Provided the first direct evidence of hGSDMD occupying the hCaspase-1 active site.
- Demonstrated carbene labeling of the C285A mutant active site by the inhibitor VRT-043198.
- Showed masking of the hGSDMD N-terminal domain in liposomes, consistent with oligomerization and membrane insertion.
Conclusions:
- Carbene footprinting is effective for studying protein-protein interactions in the Caspase-1/GSDMD system.
- The study reveals novel insights into the molecular mechanisms of inflammatory cell death.
- Findings advance the understanding of Caspase-1 inhibition and Gasdermin D pore formation.
