Allosteric Influence of Extremophile Hairpin Motif Mutations on the Protein Splicing Activity of a Hyperthermophilic Intein
Allosteric Influence of Extremophile Hairpin Motif Mutations on the Protein Splicing Activity of a Hyperthermophilic Intein
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DOI：
10.1021/acs.biochem.0c00348
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发表时间：
2020-07-07
期刊：
影响因子：
2.9
通讯作者：
Mills，Kenneth
中科院分区：
文献类型：
--
作者：
Chiarolanzio，Kathryn C.;Pusztay，Jennifer M.;Mills，Kenneth
蛋白质剪接是由内含肽介导的翻译后过程，其中内含肽从前体蛋白中切除自身，同时连接两个侧翼多肽。在极端超嗜热菌Pyrococcus abyssi中，中断DNA聚合酶II的内含肽具有延伸内含肽中央β折叠的β发夹。这种β-发夹主要存在于古细菌和嗜盐真细菌的内含肽中，因此被称为嗜极发夹（EXH）基序。EXH通过多种有利的相互作用来稳定，包括涉及Glu 29、Glu 31和Arg 40的静电相互作用。这些残基的突变可能通过干扰剪接步骤的协调来降低N-末端切割的程度和蛋白质剪接的程度。这些相同的突变降低了整体稳定性的内含肽倍，所测得的敏感性嗜热菌蛋白酶裂解。15 N-1H杂合单量子相干表明，这些突变改变了活性位点残基，如His 93（B-块组氨酸）和Ser 166（F-块残基4）的化学环境。这项工作再次强调了内含肽构象和动力学的连接和协调性质，其中远程突变可以干扰微调的相互作用网络以抑制或增强蛋白质剪接。
Protein splicing is a post-translational process mediated by an intein, whereby the intein excises itself from a precursor protein with concomitant ligation of the two flanking polypeptides. The intein that interrupts the DNA polymerase II in the extreme hyperthermophilePyrococcus abyssihas a β-hairpin that extends the central β-sheet of the intein. This β-hairpin is mostly found in inteins from archaea, as well as halophilic eubacteria, and is thus called the extremophile hairpin (EXH) motif. The EXH is stabilized by multiple favorable interactions, including electrostatic interactions involving Glu29, Glu31, and Arg40. Mutations of these residues diminish the extent of N-terminal cleavage and the extent of protein splicing, likely by interfering with the coordination of the steps of splicing. These same mutations decrease the global stability of the intein fold as measured by susceptibility to thermolysin cleavage.15N–1H heteronuclear single-quantum coherence demonstrated that these mutations altered the chemical environment of active site residues such as His93 (B-block histidine) and Ser166 (F-block residue 4). This work again underscores the connected and coordinated nature of intein conformation and dynamics, where remote mutations can disturb a finely tuned interaction network to inhibit or enhance protein splicing.