PDF(Pubmed)
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.
摘要：
转谷氨酰胺酶2（TG2）是一种GTP结合酶，蛋白质交联酶已被研究为乳糜泻的治疗靶标，神经系统疾病,和侵袭性癌症。TG2已被建议采用调节其功能的两种构象状态：GTP结合，闭合构象，和钙结合，交联活性开放构象。组成型采用开放构象的TG2突变体对癌细胞具有细胞毒性。因此，结合和稳定TG2开放构象的小分子可以提供一种新的治疗策略。这里,我们研究了TG2，使用静态和时间分辨小角度X射线散射（SAXS）和单粒子冷冻电子显微镜（cryo-EM），以确定负责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可有效杀死胶质母细胞瘤细胞，并使用SAXS研究LM11如何影响TG2的构象状态。使用SAXS和低温EM，我们显示鸟嘌呤核苷酸结合并稳定单体闭合构象，而钙结合到可以形成更高阶寡聚体的开放状态。SAXS分析提示组成型采用开放状态的TG2突变体如何通过与野生型TG2的替代机制结合核苷酸。此外，我们使用时间分辨SAXS来显示LM11增加钙结合和稳定开放构象的能力，鸟嘌呤核苷酸是不可逆的，对癌细胞有细胞毒性。一起来看,我们的发现表明，TG2的构象动力学比以前提出的更复杂，并强调了LM11对TG2的构象稳定如何维持TG2处于细胞毒性构象状态。
