Background

Human HINT2 is an important mitochondrial enzyme involved in many processes such as apoptosis and bioenergetics, but its endogenous substrates and the three-dimensional structure of the full-length protein have not been identified yet.

Methods

An HPLC assay was used to test the hydrolytic activity of HINT2 against various adenosine, guanosine, and 2′-deoxyguanosine derivatives containing phosphate bonds of different types and different leaving groups. Data on binding affinity were obtained by microscale thermophoresis (MST). Crystal structures of HINT2, in its apo form and with a dGMP ligand, were resolved to atomic resolution.

Results

HINT2 substrate specificity was similar to that of HINT1, but with the major exception of remarkable discrimination against substrates lacking the 2′-hydroxyl group. The biochemical results were consistent with binding affinity measurements. They showed a similar binding strength of AMP and GMP to HINT2, and much weaker binding of dGMP, in contrast to HINT1. A non-hydrolyzable analog of Ap4A (JB419) interacted with both proteins with similar K_d and Ap4A is the signaling molecule that can interact with hHINT1 and regulate the activity of some transcription factors.

Conclusions

Several forms of homo- and heterodimers of different lengths of N-terminally truncated polypeptides resulting from degradation of the full-length protein were described. Ser144 in HINT2 appeared to be functionally equivalent to Ser107 in HINT1 by supporting the protonation of the leaving group in the hydrolytic mechanism of HINT2.

Significance

Our results should be considered in future studies on the natural function of HINT2 and its role in nucleotide prodrug processing.

中文翻译：

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组氨酸三联体核苷酸结合蛋白 2 与不同配体（包括 Ap4A 的不可水解类似物）的生化、晶体学和生物物理表征

背景

人类HINT2是一种重要的线粒体酶，参与细胞凋亡和生物能学等许多过程，但其内源性底物和全长蛋白的三维结构尚未确定。

方法

HPLC测定用于测试HINT2对含有不同类型和不同离去基团的磷酸键的各种腺苷、鸟苷和2'-脱氧鸟苷衍生物的水解活性。结合亲和力数据通过微量热泳 (MST) 获得。HINT2 的晶体结构，以其 apo 形式并带有 dGMP 配体，被解析为原子分辨率。

结果

HINT2 底物特异性与 HINT1 相似，但主要的例外是对缺乏 2'-羟基的底物的显着区分。生化结果与结合亲和力测量结果一致。与 HINT1 相比，它们显示 AMP 和 GMP 与 HINT2 的结合强度相似，而 dGMP 的结合要弱得多。与具有相似 K _d和 Ap4A 的两种蛋白质相互作用的 Ap4A 的不可水解类似物 (JB419)是可以与 hHINT1 相互作用并调节某些转录因子活性的信号分子。

结论

描述了由全长蛋白质降解产生的不同长度的 N 端截短多肽的同二聚体和异二聚体的几种形式。通过支持 HINT2 水解机制中离去基团的质子化，HINT2 中的 Ser144 在功能上与 HINT1 中的 Ser107 等效。

意义

我们的结果应该在未来关于 HINT2 的自然功能及其在核苷酸前药加工中的作用的研究中加以考虑。