Structural and mechanistic analysis of Ca 2+ -dependent regulation of transglutaminase 2 activity using a Ca 2+ -bound intermediate state

Author:

Sewa Agnele S.1ORCID,Besser Harrison A.23,Mathews Irimpan I.4,Khosla Chaitan256ORCID

Affiliation:

1. Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305

2. Department of Chemistry, Stanford University, Stanford, CA 94305

3. Stanford Medical Scientist Training Program, Stanford University School of Medicine, Stanford, CA 94305

4. Stanford Synchrotron Radiation Lightsource, Stanford Linear Accelerator Center National Accelerator Laboratory, Menlo Park, CA 94025

5. Department of Chemical Engineering, Stanford University, Stanford, CA 94305

6. Sarafan Chemistry, Engineering Medicine and Human Health, Stanford University, Stanford, CA 94305

Abstract

Mammalian transglutaminases, a family of Ca 2+ -dependent proteins, are implicated in a variety of diseases. For example, celiac disease (CeD) is an autoimmune disorder whose pathogenesis requires transglutaminase 2 (TG2) to deamidate select glutamine residues in diet-derived gluten peptides. Deamidation involves the formation of transient γ-glutamyl thioester intermediates. Recent studies have revealed that in addition to the deamidated gluten peptides themselves, their corresponding thioester intermediates are also pathogenically relevant. A mechanistic understanding of this relevance is hindered by the absence of any structure of Ca 2+ -bound TG2. We report the X-ray crystallographic structure of human TG2 bound to an inhibitory gluten peptidomimetic and two Ca 2+ ions in sites previously designated as S1 and S3. Together with additional structure-guided experiments, this structure provides a mechanistic explanation for how S1 regulates formation of an inhibitory disulfide bond in TG2, while also establishing that S3 is essential for γ-glutamyl thioester formation. Furthermore, our crystallographic findings and associated analyses have revealed that i) two interacting residues, H305 and E363, play a critical role in resolving the thioester intermediate into an isopeptide bond (transamidation) but not in thioester hydrolysis (deamidation); and ii) residues N333 and K176 stabilize preferred TG2 substrates and inhibitors via hydrogen bonding to nonreactive backbone atoms. Overall, the intermediate-state conformer of TG2 reported here represents a superior model to previously characterized conformers for both transition states of the TG2-catalyzed reaction.

Funder

HHS | National Institutes of Health

Publisher

Proceedings of the National Academy of Sciences

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