Integrin α5β1 mediates cell adhesion to the extracellular matrix (ECM) by binding fibronectin (Fn). Selectivity for Fn by α5β1 is achieved through recognition of an RGD motif in the 10th type-III Fn domain (Fn10) and the synergy site in the 9th type-III Fn domain (Fn9). However, details of the interaction dynamics are unknown. Here, we compared synergy-site and Fn-truncation mutations for their α5β1-binding affinities and stabilities. We also interrogated binding of the α5β1 ectodomain headpiece fragment to Fn using hydrogen deuterium exchange mass spectrometry (HDX MS) to probe binding sites and sites of integrin conformational change. Our results suggest the synergistic effect of Fn9 requires both specific residues and a folded domain. We found some residues considered important for synergy are required for stability. Additionally, we show decreases in fibronectin HDX are localized to a synergy peptide containing contacting residues in two β-strands, an intervening loop in Fn9, and the RGD-containing loop in Fn10, indicative of binding sites. We also identified binding sites in the α5-subunit β-propeller domain for the Fn9 synergy site and in the β1-subunit βI domain for Fn10 based on decreases in α5β1 HDX. Interestingly, the dominant effect of Fn binding was an increase in α5β1 deuterium exchange distributed over multiple sites that undergo changes in conformation or solvent accessibility and appear to be sites where energy is stored in the higher-energy, open-integrin conformation. Together, our results highlight regions important for α5β1 binding to Fn and dynamics associated with this interaction.

整合素 α5β1 通过结合纤连蛋白 (Fn) 介导细胞与细胞外基质 (ECM) 的粘附。α5β1 对 Fn 的选择性是通过识别第 10 个 III 型 Fn 结构域 (Fn10) 中的 RGD 基序和第 9 个 III 型 Fn 结构域 (Fn9) 中的协同位点来实现的。然而，相互作用动力学的细节是未知的。在这里，我们比较了协同位点和 Fn 截断突变的 α5β1 结合亲和力和稳定性。我们还使用氢氘交换质谱 (HDX MS) 询问了 α5β1 胞外域头件片段与 Fn 的结合，以探测结合位点和整合素构象变化的位点。我们的结果表明 Fn9 的协同作用需要特定的残基和折叠结构域。我们发现一些被认为对协同作用很重要的残基是稳定性所必需的。此外，我们显示纤连蛋白 HDX 的减少定位于协同肽，该肽含有两条 β 链中的接触残基、Fn9 中的中间环和 Fn10 中含有 RGD 的环，表明结合位点。基于 α5β1 HDX 的减少，我们还确定了 Fn9 协同位点的 α5-亚基 β-螺旋桨结构域和 Fn10 的 β1-亚基 βI 结构域中的结合位点。有趣的是，Fn 结合的主要作用是增加分布在多个位点的 α5β1 氘交换，这些位点经历构象或溶剂可及性的变化，并且似乎是能量以更高能量的开放整合素构象存储的位点。总之，我们的结果突出了对 α5β1 与 Fn 结合的重要区域以及与这种相互作用相关的动力学。