Structural and mechanistic analysis of Ca2+- dependent regulation of transglutaminase 2 activity using a Ca2+- bound intermediate state

• 成果类型: Article
• 署名作者: Sewa, Agnele S.; Besser, Harrison A.; Mathews, Irimpan I.; Khosla, Chaitan
• 署名单位: Stanford University; Stanford University; Stanford University; Stanford University; United States Department of Energy (DOE); SLAC National Accelerator Laboratory; Stanford University; Stanford University
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-12577
• DOI: 10.1073/pnas.2407066121
• 发表日期: 2024-07-09
• 关键词: tissue transglutaminase; celiac-disease; binding; transamidation

摘要

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 gamma- 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 gamma- 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.