The Filament Forming Mechanism of SgrAI Endonuclease‐Structural and Kinetic Analysis
The Filament Forming Mechanism of SgrAI Endonuclease‐Structural and Kinetic Analysis
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SgrAI核酸内切酶的丝形成机制——结构和动力学分析
DOI：
10.1096/fasebj.2020.34.s1.00728
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发表时间：
2020
期刊：
影响因子：
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通讯作者：
Horton, Nancy C.
中科院分区：
文献类型：
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作者：
Horton, Nancy C.
近年来，酶的纤维形成已成为酶调控的新范式。酶在细胞中广泛分布的可逆自组装也被证明发生在生命的所有分支和各种代谢和生物途径中。我们使用SgrAI模型系统来研究成丝酶的机制。由SgrAI形成的细丝已经用低温电子显微镜进行了结构表征2，完整的反应途径包括所有的速率常数已经用单转换酶反应、FRET和全局动力学模型确定3 - 4。结构研究揭示了SgrAI通过丝的形成激活酶活性的机制，以及SgrAI在丝内底物特异性异常扩大的原因。SgrAI二聚体的构象变化，由丝中蛋白质-蛋白质和蛋白质- DNA接触稳定，直接导致活性位点的移动，假设导致第二个催化Mg2+离子的更强结合。假设DNA的结构和能量学控制着SgrAI的底物特异性和丝的形成，因此二级DNA序列只有在SgrAI与一级DNA序列形成的丝结合时才会被SgrAI切割。完整的动力学模型显示，在细丝形成过程中有一个缓慢的二级步骤，该步骤被证明可以控制酶的活性，使得只有入侵的噬菌体DNA被切割，而宿主基因组不受影响。该模型还表明，在初始识别初级DNA序列时，纤维形成机制在快速激活酶活性方面具有优势。使用SgrAI内切酶作为模型系统来研究成丝酶，我们发现该酶是通过在丝内形成的接触稳定激活的构象而激活的。通过DNA结构能量学控制低活性、非丝状状态和高活性、丝状状态之间的平衡，导致SgrAI通过丝状形成的DNA序列特异性异常明显地扩大。利用全动力学途径的模拟显示，在响应入侵噬菌体DNA的激活速度方面，长丝机制比其他非长丝机制具有优势，这给这种长丝形成机制带来了强大的进化优势。支持或资助信息本研究由美国国家科学基金会（nsf）资助。MCB‐1410355.1Park等人（2019）“生物物理评论结构、功能和成丝酶的机制：酶丝化的复兴”arXiv:1909.13141.2Polley等人（2019）“DNA的间接读出控制序列特异性内切酶的成丝和激活”，Structure 27,1 - 13 barahona等人（2019）“速度的需要：杨志强，等（2018），“低聚物丝状酶酶活性的研究进展”，中国生物防治学报，32(5),344 - 344。第2部分：全DNA裂解途径的动力学建模[J]，生物工程学报，1993(3):14599 - 14615。
Filament formation by enzymes has recently emerged as a new paradigm of enzyme regulation 1.Wide‐spread reversible self‐assembly of enzymes in cells has also been shown to occur in all branches of life and diverse metabolic and biological pathways. We use the model system, SgrAI, to investigate the filament forming enzyme mechanism.Filaments formed by SgrAI have been structurally characterized using cryo‐electron microscopy 2 and the full reaction pathway including all rate constants has been determined using single turnover enzyme reactions, FRET, and global kinetic modeling 3– 4.The structural studies uncover the mechanism of activation of enzyme activity in SgrAI by filament formation, as well as the origin of the unusual expansion of substrate specificity in SgrAI within the filament. A conformational change in the SgrAI dimer, stabilized by protein‐protein and protein‐DNA contacts in the filament, leads directly to a shift in the active site, hypothesized to lead to stronger binding of a second catalytic Mg2+ ion. DNA structure and energetics is hypothesized to control substrate specificity and filament formation by SgrAI such that the secondary class of DNA sequences are cleaved by SgrAI only upon joining a filament formed by SgrAI bound to a primary class of DNA sequences. The full kinetic model showed a slow second order step in filament formation which is shown to control enzyme activity such that only invading phage DNA is cleaved, leaving the host genome untouched. This model also shows that the filament forming mechanism is superior in fast activation of enzyme activity upon initial recognition of primary DNA sequences.Using the SgrAI endonuclease as a model system to study filament forming enzymes, we have discovered that the enzyme is activated by stabilization of an activated conformation by contacts made within the filament. Control of the equilibrium between a low activity, non‐filamentous state and the high activity, filamentous state by DNA structural energetics gives rise to the unusual apparent expansion of DNA sequence specificity of SgrAI by filament formation. Simulations using the full kinetic pathway reveal an advantage in the filament mechanism over other non‐filamentous mechanisms in the speed of activation in response to invading phage DNA, giving strong evolutionary advantage to this filament forming mechanism.Support or Funding InformationThis work was supported by the National Science Foundation under Grant No. MCB‐1410355.1Park, et al ( 2019) “ Biophysical Reviews Structures, Functions, and Mechanisms of Filament Forming Enzymes: A Renaissance of Enzyme Filamentation” arXiv:1909.13141.2Polley, et al ( 2019) “ Indirect Readout of DNA Controls Filamentation and Activation of a Sequence-Specific Endonuclease”, Structure 27, 1– 11.3Barahona, et al ( 2019) “ The Need for Speed: Run-On Oligomer Filament formation provides Maximum Speed with Maximum Sequestration of Activity”, J. Virol, 93 ( 5), e01647– 18.4Park, et al ( 2018) “ The Run-on Oligomer Filament Enzyme Mechanism of SgrAI. Part 2: Kinetic Modeling of the Full DNA Cleavage Pathway”, J Biol Chem, 293( 38): 14599– 14615.
DOI：
10.1101/585943
发表时间：
2019-03
期刊：
bioRxiv
影响因子：
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作者：
Smarajit Polley;D. Lyumkis;N. Horton
通讯作者：
Smarajit Polley;D. Lyumkis;N. Horton
影响因子：
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作者：
Park, Chad K;Horton, Nancy C
通讯作者：
Horton, Nancy C