Department of Chemistry and Chemical Biology, Cornell University, 14853, Ithaca, NY, USA.
Department of Molecular Medicine, Cornell University, 14853, Ithaca, NY, USA.
Commun Biol. 2024 Aug 13;7(1):982. doi: 10.1038/s42003-024-06672-x.
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.
转谷氨酰胺酶 2（TG2）是一种 GTP 结合、蛋白交联酶，已被研究作为乳糜泻、神经紊乱和侵袭性癌症的治疗靶点。有人提出，TG2 可以采用两种构象状态来调节其功能：一种是与 GTP 结合的、关闭的构象，另一种是与钙结合的、交联活性的开放构象。持续采用开放构象的 TG2 突变体对癌细胞具有细胞毒性。因此，与 TG2 的开放构象结合并稳定其的小分子可能提供一种新的治疗策略。在这里，我们使用静态和时间分辨小角 X 射线散射（SAXS）和单颗粒冷冻电子显微镜（cryo-EM）研究 TG2，以确定赋予其生物学效应的构象状态。我们还描述了一种新开发的 TG2 抑制剂 LM11，它能够有效地杀死神经胶质瘤细胞，并使用 SAXS 研究 LM11 如何影响 TG2 的构象状态。我们使用 SAXS 和 cryo-EM 表明，鸟嘌呤核苷酸结合并稳定单体封闭构象，而钙结合到可以形成更高阶寡聚体的开放状态。SAXS 分析表明，持续采用开放状态的 TG2 突变体如何通过替代机制结合核苷酸，而不是野生型 TG2。此外，我们使用时间分辨 SAXS 表明，LM11 增加了钙结合和稳定开放构象的能力，这不能被鸟嘌呤核苷酸逆转，并且对癌细胞具有细胞毒性。总之，我们的研究结果表明，TG2 的构象动力学比以前认为的更为复杂，并强调了 LM11 如何通过稳定 TG2 的构象来维持 TG2 处于细胞毒性构象。
