Native dynamics and allosteric responses in PTP1B probed by high-resolution HDX-MS.
Native dynamics and allosteric responses in PTP1B probed by high-resolution HDX-MS.
复制标题
通过高分辨率 HDX-MS 探测 PTP1B 的天然动力学和变构反应。
DOI：
10.1101/2023.07.12.548582
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Keedy,DanielA
中科院分区：
文献类型：
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作者：
Woods,VirgilA;Abzalimov,RinatR;Keedy,DanielA
蛋白酪氨酸磷酸酶1B(PTP1B)是肥胖症、糖尿病和某些类型癌症的有效治疗靶点。特别是，变构抑制剂具有治疗用途的潜力，但对这种蛋白质的构象动力学和变构的不完全了解阻碍了它们的发展。在这里，我们使用高分辨率氢-氚交换质谱仪(HDX-MS)来研究PTP1B中的溶液动力学和变构反应。使用HDX-MS，我们获得了作为apo PTP1B溶液动力学代理的主链酰胺交换的详细图，揭示了散布在蛋白质结构中更受约束和刚性区域之间的几个柔性环，以及从其二级结构和溶剂可及性出发交换速度快于预期的局部区域。我们证明，我们在溶液中获得的HDX速率数据增加了由~200个PTP1B晶体结构构建的赝系综所得到的构象不均质性的估计值。此外，我们还报道了具有活性中心的PTP1B与变构小分子抑制剂的HDX-MS图谱。这些图谱显示了与载脂蛋白形式相关的对蛋白质动力学的明显和广泛的影响，包括距离各自配体结合位点远端(>；35 ä)的位置的变化。这些结果说明，PTP1B的变构抑制剂可以引起超出先前所知的变构网络的动力学的意外变化。综上所述，我们的数据表明了PTP1B中BB3别构的模型，该模型结合了活性位点残基的构象限制和远端残基的补偿性释放，有助于熵平衡。总体而言，我们的工作展示了HDX-MS在阐明蛋白质构象动力学和小分子配体的变构效应方面的潜力，并强调了将HDX-MS与其他互补方法(如室温X射线结晶学、核磁共振光谱和分子动力学模拟)相结合的潜力，以指导新疗法的开发。
Protein tyrosine phosphatase 1B (PTP1B) is a validated therapeutic target for obesity, diabetes, and certain types of cancer. In particular, allosteric inhibitors hold potential for therapeutic use, but an incomplete understanding of conformational dynamics and allostery in this protein has hindered their development. Here, we interrogate solution dynamics and allosteric responses in PTP1B using high‐resolution hydrogen‐deuterium exchange mass spectrometry (HDX‐MS), an emerging and powerful biophysical technique. Using HDX‐MS, we obtain a detailed map of backbone amide exchange that serves as a proxy for the solution dynamics of apo PTP1B, revealing several flexible loops interspersed among more constrained and rigid regions within the protein structure, as well as local regions that exchange faster than expected from their secondary structure and solvent accessibility. We demonstrate that our HDX rate data obtained in solution adds value to estimates of conformational heterogeneity derived from a pseudo‐ensemble constructed from ~200 crystal structures of PTP1B. Furthermore, we report HDX‐MS maps for PTP1B with active‐site versus allosteric small‐molecule inhibitors. These maps suggest distinct and widespread effects on protein dynamics relative to the apo form, including changes in locations distal (>35 Å) from the respective ligand binding sites. These results illuminate that allosteric inhibitors of PTP1B can induce unexpected changes in dynamics that extend beyond the previously understood allosteric network. Together, our data suggest a model of BB3 allostery in PTP1B that combines conformational restriction of active‐site residues with compensatory liberation of distal residues that aid in entropic balancing. Overall, our work showcases the potential of HDX‐MS for elucidating aspects of protein conformational dynamics and allosteric effects of small‐molecule ligands and highlights the potential of integrating HDX‐MS alongside other complementary methods, such as room‐temperature X‐ray crystallography, NMR spectroscopy, and molecular dynamics simulations, to guide the development of new therapeutics.