Tissue transglutaminase 2 (TG2) plays a vital role in stabilizing extracellular matrix (ECM) proteins through enzymatic crosslinking during tissue growth, repair, and inflammation. TG2 also binds non-covalently to fibronectin (FN), an essential component of the ECM, facilitating cell adhesion, migration, proliferation, and survival. However, the interaction between TG2 and fibrillar FN remains poorly understood, as most studies have focused on soluble or surface-adsorbed FN or FN fragments, which differ in their conformations from insoluble FN fibers. Using a well-established in vitro FN fiber stretch assay, we discovered that the binding of a crosslinking enzyme to ECM fibers is mechano-regulated. TG2 binding to FN is tuned by the mechanical tension of FN fibers, whereby TG2 predominantly co-localizes to low-tension FN fibers, while fiber stretching reduces their affinity for TG2. This mechano-regulated binding relies on the proximity between the N-terminal β-sandwich and C-terminal β-barrels of TG2. Crosslinking mass spectrometry (XL-MS) revealed a novel TG2-FN synergy site within TG2’s C-terminal β-barrels that interacts with FN regions located outside of the canonical gelatin binding domain, specifically FNI₂ and FNIII_14-15. Combining XL-MS distance restraints with molecular docking revealed the mechano-regulated binding mechanism between TG2 and modules FNI_7-9 by which mechanical forces regulate TG2-FN interactions. This highlights a previously unrecognized role of TG2 as a tension sensor for FN fibers. This novel interaction mechanism has significant implications in physiology and mechanobiology, including how forces regulate cell adhesion, spreading, migration, phenotype modulation, depending on the tensional state of ECM fibers. Data are available via ProteomeXchange with identifier PXD043976.

组织转谷氨酰胺酶 2 (TG2) 在组织生长、修复和炎症过程中通过酶交联在稳定细胞外基质 (ECM) 蛋白方面发挥着至关重要的作用。TG2 还与纤连蛋白 (FN)（ECM 的重要成分）非共价结合，促进细胞粘附、迁移、增殖和存活。然而，TG2 和纤维状 FN 之间的相互作用仍然知之甚少，因为大多数研究都集中在可溶性或表面吸附的 FN 或 FN 片段上，它们的构象与不溶性 FN 纤维不同。使用成熟的体外FN 纤维拉伸测定，我们发现交联酶与 ECM 纤维的结合是机械调节的。TG2 与 FN 的结合通过 FN 纤维的机械张力进行调节，其中 TG2 主要共定位于低张力 FN 纤维，而纤维拉伸会降低其对 TG2 的亲和力。这种机械调节的结合依赖于 TG2 N 端 β 三明治和 C 端 β 桶之间的邻近性。交联质谱 (XL-MS) 揭示了 TG2 C 端 β 桶内的一个新的 TG2-FN 协同位点，该位点与位于规范明胶结合结构域之外的 FN 区域相互作用，特别是 FNI ₂和 FNIII _14-15。_{将 XL-MS 距离限制与分子对接相结合，揭示了 TG2 和模块 FNI 7-9}之间的机械调节结合机制，通过机械力调节 TG2-FN 相互作用。这凸显了 TG2 作为 FN 光纤张力传感器的一个先前未被认识到的作用。这种新颖的相互作用机制对生理学和力学生物学具有重要意义，包括力如何根据 ECM 纤维的张力状态调节细胞粘附、扩散、迁移、表型调节。数据可通过 ProteomeXchange 获得，标识符为 PXD043976。