Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, United States.
Department of Biological Chemistry and Molecular Pharmacology and Department of Pediatrics, Harvard Medical School, Boston, United States.
Elife. 2021 Dec 2;10:e73359. doi: 10.7554/eLife.73359.
Integrin conformational ensembles contain two low-affinity states, bent-closed and extended-closed, and an active, high-affinity, extended-open state. It is widely thought that integrins must be activated before they bind ligand; however, one model holds that activation follows ligand binding. As ligand-binding kinetics are not only rate limiting for cell adhesion but also have important implications for the mechanism of activation, we measure them here for integrins α4β1 and α5β1 and show that the low-affinity states bind substantially faster than the high-affinity state. On- and off-rates are similar for integrins on cell surfaces and as ectodomain fragments. Although the extended-open conformation's on-rate is ~20-fold slower, its off-rate is ~25,000-fold slower, resulting in a large affinity increase. The tighter ligand-binding pocket in the open state may slow its on-rate. Low-affinity integrin states not only bind ligand more rapidly, but are also more populous on the cell surface than high-affinity states. Thus, our results suggest that integrin binding to ligand may precede, rather than follow, activation by 'inside-out signaling.'
整联蛋白构象集合包含两种低亲和力状态，弯曲-关闭和延伸-关闭，以及一种活性的、高亲和力的、延伸-开放状态。人们普遍认为，整联蛋白在结合配体之前必须被激活；然而，有一种模型认为激活发生在配体结合之后。由于配体结合动力学不仅对细胞黏附具有限速作用，而且对激活机制也有重要影响，我们在这里测量了 α4β1 和 α5β1 整联蛋白的动力学，并表明低亲和力状态的结合速度明显快于高亲和力状态。整联蛋白在细胞表面和作为胞外结构域片段上的结合速率和解离速率相似。尽管延伸-开放构象的结合速率慢约 20 倍，但解离速率慢约 25000 倍，导致亲和力显著增加。开放状态下配体结合口袋的紧密性可能会降低其结合速率。低亲和力整联蛋白状态不仅更快地结合配体，而且在细胞表面上比高亲和力状态更为普遍。因此，我们的结果表明，整联蛋白与配体的结合可能先于通过“内-外信号”激活，而不是之后。
