Dynamic Long-Range Interactions Influence Substrate Binding and Catalysis by Human Histidine Triad Nucleotide-Binding Proteins (HINTs), Key Regulators of Multiple Cellular Processes and Activators of Antiviral ProTides.
Dynamic Long-Range Interactions Influence Substrate Binding and Catalysis by Human Histidine Triad Nucleotide-Binding Proteins (HINTs), Key Regulators of Multiple Cellular Processes and Activators of Antiviral ProTides.
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DOI：
10.1021/acs.biochem.2c00506
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发表时间：
2022-12-06
期刊：
影响因子：
2.9
通讯作者：
Wagner, Carston R.
中科院分区：
文献类型：
--
作者：
Strom, Alexander;Shah, Rachit;Dolot, Rafal;Rogers, Melanie S.;Tong, Cher-Ling;Wang, David;Xia, Youlin;Lipscomb, John D.;Wagner, Carston R.
人组氨酸三联体核苷酸结合（hHINT）蛋白催化核苷酸磷酰胺酶和酰基磷酸酶反应，这些反应对于激活抗病毒磷酰胺酯前体（如索非布韦和Remdesivir）至关重要。hHINT 1和hHINT 2是高度同源的，但作为阿片耐受性（hHINT 1）和线粒体活性（hHINT 2）的调节剂表现出不同的作用。hHINT 1的NMR研究揭示了一对动态表面残基（Q62，E100），其门控保守的水通道，该水通道通向13 ° C外的活性位点。hHINT2晶体结构鉴定类似残基（R99、D137）和水通道。hHINT 1 Q62变体显著改变了荧光底物（TpAd）周转的稳态kcat和Km，而停流动力学表明KD也发生了变化。hHINT2，像hHINT1一样，在限速水解和核苷酸释放之前，表现出腺苷酸化的爆发期，通过荧光色胺释放监测。hHINT2表现出比hHINT1小得多的脉冲串相位幅度，这在hHINT2 R99Q中进一步减小。动力学模拟表明，幅度的变化可以解释为一个可变的荧光产量的E·S复合物的环境中的变化结合TpAd。抑制剂结合的等温滴定量热法测量显示，这些hHINT变体也改变热力学结合概况。我们建议，这些改变的表面残基产生长距离的动态变化，影响绑定配体的方向，改变hHINT活性位点功能的热力学和动力学特性。因此，hHINT的细胞作用和磷酰胺酯前药激活潜力的研究应考虑远程相互作用和可能的蛋白质结合表面远离活性位点的重要性。
Human histidine triad nucleotide binding (hHINT) proteins catalyze nucleotide phosphoramidase and acyl-phosphatase reactions that are essential for the activation of antiviral proTides, such as Sofosbuvir and Remdesivir. hHINT1 and hHINT2 are highly homologous but exhibit disparate roles as regulators of opioid tolerance (hHINT1) and mitochondrial activity (hHINT2). NMR studies of hHINT1 reveal a pair of dynamic surface residues (Q62, E100) which gate a conserved water channel leading to the active site 13 Å away. hHINT2 crystal structures identify analogous residues (R99, D137) and water channel. hHINT1 Q62 variants significantly alter the steady-state kcat and Km for turnover of the fluorescent substrate (TpAd), while stopped-flow kinetics indicate the KD also changes. hHINT2, like hHINT1, exhibits a burst-phase of adenylation, monitored by fluorescent tryptamine release, prior to rate-limiting hydrolysis and nucleotide release. hHINT2 exhibits a much smaller burst-phase amplitude than hHINT1, which is further diminished in hHINT2 R99Q. Kinetic simulations suggest that amplitude variations can be accounted for by a variable fluorescent yield of the E•S complex from changes in the environment of bound TpAd. Isothermal titration calorimetry measurements of inhibitor binding shows that these hHINT variants also alter the thermodynamic binding profile. We propose that these altered surface residues engender long-range dynamic changes that affect the orientation of bound ligands, altering the thermodynamic and kinetic characteristics of hHINT active site function. Thus, studies of the cellular roles and proTide activation potential by hHINTs should consider the importance of long-range interactions and possible protein binding surfaces far from the active site.
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