Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
J Biol Chem. 2022 Aug;298(8):102230. doi: 10.1016/j.jbc.2022.102230. Epub 2022 Jul 1.
The protease inhibitor α-macroglobulin (A2M) is a member of the ancient α-macroglobulin superfamily (A2MF), which also includes structurally related proteins, such as complement factor C3. A2M and other A2MF proteins undergo an extensive conformational change upon cleavage of their bait region by proteases. However, the mechanism whereby cleavage triggers the change has not yet been determined. We have previously shown that A2M remains functional after completely replacing its bait region with glycine and serine residues. Here, we use this tabula rasa bait region to investigate several hypotheses for the triggering mechanism. When tabula rasa bait regions containing disulfide loops were elongated by reducing the disulfides, we found that A2M remained in its native conformation. In addition, cleavage within a disulfide loop did not trigger the conformational change until after the disulfide was reduced, indicating that the introduction of discontinuity into the bait region is essential to the trigger. Previously, A2MF structures have shown that the C-terminal end of the bait region (a.k.a. the N-terminal region of the truncated α chain) threads through a central channel in native A2MF proteins. Bait region cleavage abolishes this plug-in-channel arrangement, as the bait region retracts from the channel and the channel itself collapses. We found that mutagenesis of conserved plug-in-channel residues disrupted the formation of native A2M. These results provide experimental evidence for a structural hypothesis in which retraction of the bait region from this channel following cleavage and the channel's subsequent collapse triggers the conformational change of A2M and other A2MF proteins.
蛋白酶抑制剂 α-巨球蛋白（A2M）是古老的 α-巨球蛋白超家族（A2MF）的成员，该超家族还包括结构相关的蛋白质，如补体因子 C3。A2M 和其他 A2MF 蛋白在其诱饵区域被蛋白酶切割时会发生广泛的构象变化。然而，触发这种变化的机制尚未确定。我们之前已经表明，A2M 在完全用甘氨酸和丝氨酸残基替换其诱饵区域后仍然具有功能。在这里，我们使用这个无诱饵区域来研究触发机制的几个假设。当含有二硫键环的无诱饵区域通过还原二硫键而延长时，我们发现 A2M 仍然保持其天然构象。此外，直到二硫键被还原后，二硫键环内的切割才会触发构象变化，这表明在诱饵区域中引入不连续性对于触发是必不可少的。以前，A2MF 结构表明，诱饵区域的 C 末端（又名截断 α 链的 N 末端）穿过天然 A2MF 蛋白中的中央通道。诱饵区域切割会破坏这种插件通道排列，因为诱饵区域从通道缩回，并且通道本身坍塌。我们发现保守的插件通道残基的突变破坏了天然 A2M 的形成。这些结果为以下结构假设提供了实验证据：在切割后，诱饵区域从该通道缩回，并且通道随后坍塌，这触发了 A2M 和其他 A2MF 蛋白的构象变化。
