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Abstract：
The rapid identification of protein-protein interactions has been significantly enabled by mass spectrometry (MS) proteomics-based methods, including affinity purification-MS, crosslinking-MS, and proximity-labeling proteomics. While these methods can reveal networks of interacting proteins, they cannot reveal how specific protein-protein interactions alter cell signaling or protein function. For instance, when two proteins interact, there can be emergent signaling processes driven purely by the individual activities of those proteins being co-localized. Alternatively, protein-protein interactions can allosterically regulate function, enhancing or suppressing activity in response to binding. In this work, we investigate the interaction between the tyrosine phosphatase PTP1B and the adaptor protein Grb2, which have been annotated as binding partners in a number of proteomics studies. This interaction has been postulated to co-localize PTP1B with its substrate IRS-1 by forming a ternary complex, thereby enhancing the dephosphorylation of IRS-1 to suppress insulin signaling. Here, we report that Grb2 binding to PTP1B also allosterically enhances PTP1B catalytic activity. We show that this interaction is dependent on the proline-rich region of PTP1B, which interacts with the C-terminal SH3 domain of Grb2. Using NMR spectroscopy and hydrogen-deuterium exchange mass spectrometry (HDX-MS) we show that Grb2 binding alters PTP1B structure and/or dynamics. Finally, we use MS proteomics to identify other interactors of the PTP1B proline-rich region that may also regulate PTP1B function similarly to Grb2. This work presents one of the first examples of a protein allosterically regulating the enzymatic activity of PTP1B and lays the foundation for discovering new mechanisms of PTP1B regulation in cell signaling.
摘要：
通过基于质谱（MS）蛋白质组学的方法，可以显着快速鉴定蛋白质-蛋白质相互作用，包括亲和纯化-MS，交联-MS，和邻近标记蛋白质组学。虽然这些方法可以揭示相互作用蛋白质的网络，它们无法揭示特定的蛋白质-蛋白质相互作用如何改变细胞信号或蛋白质功能。例如,当两种蛋白质相互作用时，可能有紧急的信号传导过程，纯粹是由这些蛋白质的个体活动共同定位驱动的。或者，蛋白质-蛋白质相互作用可以变构调节功能，增强或抑制响应于结合的活性。在这项工作中,我们研究了酪氨酸磷酸酶PTP1B和衔接蛋白Grb2之间的相互作用，在许多蛋白质组学研究中已将其注释为结合伴侣。假定这种相互作用通过形成三元复合物来使PTP1B与其底物IRS-1共定位，从而增强IRS-1的去磷酸化以抑制胰岛素信号传导。这里,我们报道了Grb2与PTP1B的结合也可以变构地增强PTP1B的催化活性。我们表明，这种相互作用依赖于PTP1B的富含脯氨酸的区域，它与Grb2的C端SH3结构域相互作用。使用NMR光谱和氢-氘交换质谱（HDX-MS），我们表明Grb2结合会改变PTP1B的结构和/或动力学。最后,我们使用MS蛋白质组学来鉴定PTP1B富含脯氨酸区域的其他相互作用因子，这些相互作用因子也可能与Grb2相似地调节PTP1B的功能。这项工作提出了蛋白质变构调节PTP1B酶活性的第一个实例，并为发现PTP1B在细胞信号传导中的调节新机制奠定了基础。
■蛋白质-蛋白质相互作用对于细胞信号传导至关重要。磷酸酶PTP1B和衔接蛋白Grb2之间的相互作用将PTP1B与其底物共定位，从而增强它们的去磷酸化。我们表明，Grb2结合还通过涉及PTP1B富含脯氨酸区域的变构机制直接调节PTP1B活性。我们的研究揭示了一种通过蛋白质-蛋白质相互作用调节PTP1B的新模式，该模式可能被这种重要信号酶的其他细胞相互作用者利用。
■蛋白质-蛋白质相互作用对于细胞信号传导至关重要。磷酸酶PTP1B和衔接蛋白Grb2之间的相互作用将PTP1B与其底物共定位，从而增强它们的去磷酸化。我们表明，Grb2结合还通过涉及PTP1B富含脯氨酸区域的变构机制直接调节PTP1B活性。我们的研究揭示了一种通过蛋白质-蛋白质相互作用调节PTP1B的新模式，该模式可能被这种重要信号酶的其他细胞相互作用者利用。
