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Abstract The glutaminase enzymes GAC and GLS2 catalyze the hydrolysis of glutamine to glutamate, satisfying the ‘glutamine addiction’ of cancer cells. They are the targets of anti-cancer drugs; however, their mechanisms of activation and catalytic activity have been unclear. Here we demonstrate that the ability of GAC and GLS2 to form filaments is directly coupled to their catalytic activity and present their cryo-EM structures which provide a view of the conformational states essential for catalysis. Filament formation guides an ‘activation loop’ to assume a specific conformation that works together with a ‘lid’ to close over the active site and position glutamine for nucleophilic attack by an essential serine. Our findings highlight how ankyrin repeats on GLS2 regulate enzymatic activity, while allosteric activators stabilize, and clinically relevant inhibitors block, filament formation that enables glutaminases to catalyze glutaminolysis and support cancer progression.
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Distinct conformational states enable transglutaminase 2 to promote cancer cell survival versus cell death
Abstract Transglutaminase 2 (TG2) is a GTP-binding, protein-crosslinking enzyme that has been investigated as a therapeutic target for Celiac disease, neurological disorders, and aggressive cancers. TG2 has been suggested to adopt two conformational states that regulate its functions: a GTP-bound, closed conformation, and a calcium-bound, crosslinking-active open conformation. TG2 mutants that constitutively adopt an open conformation are cytotoxic to cancer cells. Thus, small molecules that bind and stabilize the open conformation of TG2 could offer a new therapeutic strategy. Here, we investigate TG2, using static and time-resolved small-angle X-ray scattering (SAXS) and single-particle cryoelectron microscopy (cryo-EM), to determine the conformational states responsible for conferring its biological effects. We also describe a newly developed TG2 inhibitor, LM11, that potently kills glioblastoma cells and use SAXS to investigate how LM11 affects the conformational states of TG2. Using SAXS and cryo-EM, we show that guanine nucleotides bind and stabilize a monomeric closed conformation while calcium binds to an open state that can form higher order oligomers. SAXS analysis suggests how a TG2 mutant that constitutively adopts the open state binds nucleotides through an alternative mechanism to wildtype TG2. Furthermore, we use time resolved SAXS to show that LM11 increases the ability of calcium to bind and stabilize an open conformation, which is not reversible by guanine nucleotides and is cytotoxic to cancer cells. Taken together, our findings demonstrate that the conformational dynamics of TG2 are more complex than previously suggested and highlight how conformational stabilization of TG2 by LM11 maintains TG2 in a cytotoxic conformational state.
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- Award ID(s):
- 1719875
- PAR ID:
- 10532895
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Communications Biology
- Volume:
- 7
- Issue:
- 1
- ISSN:
- 2399-3642
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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