蛋白质的翻译后修饰作为表观遗传的重要一环，可以通过在目标蛋白中引入修饰改变其特性，在生命周期和细胞命运决定中发挥了至关重要的作用。而在不同类型的翻译后修饰中，甲基化修饰是最为重要且常见的修饰类型之一。而随着近些年来的研究逐渐深入，越来越多的非组蛋白甲基化修饰以及非赖氨酸、精氨酸甲基化修饰也被逐渐鉴定出来，并被证实在诸多生命过程中发挥着重要的作用。组氨酸甲基化修饰就是其中的典型代表。近些年的研究逐渐发掘出了组氨酸甲基化对于大量生命活动的影响。组氨酸侧链咪唑环的N1（π）或N3（τ）位置的甲基化会赋予被修饰的组氨酸新的特征，比如：使其分子体积和疏水性增加，并且固定了组氨酸质子化和互变异构体状态。同样的，组氨酸甲基化会影响金属离子结合蛋白结合二价金属离子，如Zn2+, Fe2+, Cu2+的能力。比如目前已经有研究指出，β-actin上His73位的甲基化会促进肌肉收缩和肌动蛋白纤维固定。同样的，组氨酸甲基化是肌肽（carnosine）转变为鹅肌肽（anserine）的关键步骤。除了这两个组氨酸甲基化外，近几年的高通量蛋白组学研究更是在蛋白质组中鉴定出了几百个组氨酸甲基化位点，证明了组氨酸甲基化可能作为一种重要的修饰对于生命活动发挥了大量至关重要的调控功能。METTL9催化了大量含有“His-x-His”基序的蛋白质底物的N1-甲基化。我们通过结构和生物化学手段的研究，揭示了METTL9可以特异性的将“HxH”基序的第二个组氨酸甲基化，而会利用第一个组氨酸作为识别特征性位点。我们观察到了METTL9和五肽底物的高度紧贴式的结合，而小侧链残基“x”则被深埋固定在了底物结合口袋中，在酶与底物的相互作用中，被甲基化的组氨酸侧链上N3原子被METTL9的天冬氨酸的侧链通过氢键固定，从而使N1原子可以直接被S-腺苷甲硫氨酸进行甲基化。同时，METTL9会对于多个并列的“HxH”基序进行倾向性的连续“C端到N端”甲基化，而多个连续的“HxH”基序正是很多METTL9底物的序列特征。并且我们证实了METTL9可以将多个蛋白质底物甲基化。综上所述，我们的工作揭示了METTL9在催化生理条件下大量存在的含“HxH”基序蛋白质并且阐述了其进行特异性N1-甲基化的分子机制设计，进一步扩展和延伸了对于组氨酸甲基化生物学的理解以其重要性的认识。
As an important part of epigenetic regulation, post-translational modifications of proteins play a critical role in life cycle and cell fate decision by introducing altered traits of the modified proteins. Among the different types of post-translational modifications, methylation modifications are one of the most important and common types of modifications. Extensive studies have demonstrated the importance of histone lysine and arginine methylation for biological regulation and cellular responses. More and more non-histone, non-lysine and arginine methylations, have been identified and proved important biologically. A typical example is the histidine methylation.Recent studies have shined light on the significant role of histidine methylation involved in various biological pathways. The methylation of histidine side chain brings about new biochemical properties of the modified histidine, such as: increase in molecular volume and hydrophobicity as well as the fixation of histidine's protonation and tautomerization states. Methylation of histidine may affect the chelation of divalent metal ions such as Zn2+, Cu2+, and Fe2+ by metal binding proteins. It has been reported that methylation of His73 on β-actin promotes smooth muscle contraction and actin filament stabilization. Moreover, histidine methylation serves as a key step for converting carnosine to anserine, both are bioactive dipeptides in the brain and muscle.As a newly identified histidine methyltransferase, METTL9 catalyzes N1-methylation of protein substrates containing the “His-x-His” motif (HxH, x denotes small side chain residue). Here our structural and biochemical studies revealed that METTL9 specifically methylates the second histidine of the “HxH” motif, while exploiting the first one as a recognition signature. We observed an intimate engagement between METTL9 and a pentapeptide motif, where the small “x” residue is embedded and confined within the substrate pocket. Upon complex formation, the N3 atom of histidine imidazole ring is stabilized by an aspartate residue such that the N1 atom is presented to S-adenosylmethionine for methylation. Moreover, METTL9 displayed a feature in preferred consecutive and “C-to-N” directional methylation of tandem “HxH” repeats that exist in many METTL9 substrates. And we have also identified several proteins as substrate of METTL9.Collectively, our work illustrates the molecular design of METTL9 in N1-specific methylation of the broadly existing “HxH” motifs containing proteins, and further expands and highlights the understanding of histidine methylation biology and its importance.
