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Mapping the Binding Interactions Between human Gasdermin D and human Caspase-1 Using Carbene Footprinting

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 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 Caspase-1, VRT-043198 alkylates the active-site C285. Here we showed by carbene labeling that this inhibitor can occupy the active-site of a C285A mutant reversibly. These findings add considerably to our knowledge of the hCaspase-1-hGSDMD system.

INSTRUMENT(S): LTQ FT

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: James Lloyd

LAB HEAD: Neil Oldham

PROVIDER: PXD040212 | Pride | 2023-08-14

REPOSITORIES: Pride

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			Action	DRS
	JRL_040522_GSDMD_CHYMOTRYPSIN.RAW	Raw
	JRL_040522_GSDMD_CHYMOTRYPSIN.t.xml	Xml
	JRL_040522_GSDMD_GLUC.RAW	Raw
	JRL_040522_GSDMD_GLUC.t.xml	Xml
	JRL_040522_GSDMD_TRYPSIN.RAW	Raw

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Mapping the Binding Interactions Between human Gasdermin D and human Caspase-1 Using Carbene Footprinting

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