Mapping the Binding Interactions between Human Gasdermin D and Human Caspase-1 Using Carbene Footprinting

Lloyd, James R.; Biasutto, Antonio; Dürr, Katharina L.; Jazayeri, Ali; Hopper, Jonathan T.S.; Oldham, Neil J.

Abstract

Carbene footprinting is a recently developed mass spectrometry-based chemical labeling technique that probes protein interactions and conformation. Here, we use the methodology to investigate binding interactions between the protease human Caspase-1 (C285A) and full-length human Gasdermin D (hGSDMD), which are important in inflammatory cell death. GSDMD is cleaved by Caspase-1, releasing its N-terminal domain which oligomerizes in the membrane to form large pores, resulting in lytic cell death. Regions of reduced carbene labeling (masking), caused by protein binding, were observed for each partner in the presence of the other and were consistent with hCaspase-1 exosite and active-site interactions. Most notably, the results showed direct occupancy of hCaspase-1 (C285A) active-site by hGSDMD for the first time. Differential carbene labeling of full-length hGSDMD and the pore-forming N-terminal domain assembled in liposomes showed masking of the latter, consistent with oligomeric assembly and insertion into the lipid bilayer. Interactions between Caspase-1 and the specific inhibitor VRT-043198 were also studied by this approach. In wild-type hCaspase-1, VRT-043198 modifies the active-site Cys285 through the formation of a S,O-hemiacetal. Here, we showed by carbene labeling that this inhibitor can noncovalently occupy the active site of a C285A mutant. These findings add considerably to our knowledge of the hCaspase-1-hGSDMD system.

Citation

Lloyd, J. R., Biasutto, A., Dürr, K. L., Jazayeri, A., Hopper, J. T., & Oldham, N. J. (2023). Mapping the Binding Interactions between Human Gasdermin D and Human Caspase-1 Using Carbene Footprinting. JACS Au, 3(7), 2025-2035. https://doi.org/10.1021/jacsau.3c00236

• Journal Article Type: Article
• Acceptance Date: Jun 9, 2023
• Online Publication Date: Jun 23, 2023
• Publication Date: Jun 23, 2023
• Deposit Date: Jul 2, 2023
• Publicly Available Date: Jul 4, 2023
• Journal: JACS Au
• Print ISSN: 2691-3704
• Electronic ISSN: 2691-3704
• Publisher: American Chemical Society
• Peer Reviewed: Peer Reviewed
• Volume: 3
• Issue: 7
• Pages: 2025-2035
• DOI: https://doi.org/10.1021/jacsau.3c00236
• Keywords: Carbene labeling, diazirines, protein footprinting, gasdermin D, caspase-1, pyroptosis
• Public URL: https://nottingham-repository.worktribe.com/output/22454938
• Publisher URL: https://pubs.acs.org/doi/10.1021/jacsau.3c00236