Native dynamics and allosteric responses in PTP1B probed by high-resolution HDX-MS.

IF 4.5 3区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY Protein Science Pub Date : 2024-06-01 DOI:10.1002/pro.5024
Virgil A Woods, Rinat R Abzalimov, Daniel A Keedy
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Abstract

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.

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通过高分辨率 HDX-MS 探测 PTP1B 的原生动力学和异构反应。
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,异构抑制剂具有潜在的治疗用途,但对这种蛋白质的构象动力学和异构的不完全了解阻碍了它们的开发。在这里,我们利用高分辨率氢氘交换质谱(HDX-MS)这一新兴而强大的生物物理技术来研究 PTP1B 的溶液动力学和异构反应。利用 HDX-MS,我们获得了骨架酰胺交换的详细图谱,该图谱可作为 apo PTP1B 溶液动力学的替代物,揭示了蛋白质结构中穿插在更受约束的刚性区域中的几个柔性环路,以及从二级结构和溶剂可及性来看交换速度快于预期的局部区域。我们证明,在溶液中获得的 HDX 速率数据增加了从约 200 个 PTP1B 晶体结构构建的伪组合中得出的构象异质性估计值的价值。此外,我们还报告了 PTP1B 在活性位点与异位小分子抑制剂作用下的 HDX-MS 图谱。这些图谱表明,相对于apo形式,这些抑制剂对蛋白质动力学产生了不同且广泛的影响,包括在距离各自配体结合位点较远的位置(>35 Å)发生的变化。这些结果表明,PTP1B 的异构抑制剂可诱导出人意料的动态变化,这种变化超出了人们先前所理解的异构网络。总之,我们的数据提出了 PTP1B 中 BB3 异构模型,该模型结合了活性位点残基的构象限制和有助于熵平衡的远端残基的补偿性释放。总之,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异构效应方面的潜力,并强调了将 HDX-MS 与室温 X 射线晶体学、核磁共振光谱和分子动力学模拟等其他辅助方法结合起来指导新疗法开发的潜力。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Protein Science
Protein Science 生物-生化与分子生物学
CiteScore
12.40
自引率
1.20%
发文量
246
审稿时长
1 months
期刊介绍: Protein Science, the flagship journal of The Protein Society, is a publication that focuses on advancing fundamental knowledge in the field of protein molecules. The journal welcomes original reports and review articles that contribute to our understanding of protein function, structure, folding, design, and evolution. Additionally, Protein Science encourages papers that explore the applications of protein science in various areas such as therapeutics, protein-based biomaterials, bionanotechnology, synthetic biology, and bioelectronics. The journal accepts manuscript submissions in any suitable format for review, with the requirement of converting the manuscript to journal-style format only upon acceptance for publication. Protein Science is indexed and abstracted in numerous databases, including the Agricultural & Environmental Science Database (ProQuest), Biological Science Database (ProQuest), CAS: Chemical Abstracts Service (ACS), Embase (Elsevier), Health & Medical Collection (ProQuest), Health Research Premium Collection (ProQuest), Materials Science & Engineering Database (ProQuest), MEDLINE/PubMed (NLM), Natural Science Collection (ProQuest), and SciTech Premium Collection (ProQuest).
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