Exploring CRISPR-Cas9 HNH-Domain-Catalyzed DNA Cleavage Using Accelerated Quantum Mechanical Molecular Mechanical Free Energy Simulation.

IF 2.9 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY
Biochemistry Biochemistry Pub Date : 2025-01-07 Epub Date: 2024-12-16 DOI:10.1021/acs.biochem.4c00651
Richard Van, Xiaoliang Pan, Saadi Rostami, Jin Liu, Pratul K Agarwal, Bernard Brooks, Rakhi Rajan, Yihan Shao
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引用次数: 0

Abstract

The target DNA (tDNA) cleavage catalyzed by the CRISPR Cas9 enzyme is a critical step in the Cas9-based genome editing technologies. Previously, the tDNA cleavage from an active SpyCas9 enzyme conformation was modeled by Palermo and co-workers (Nierzwicki et al., Nat. Catal. 2022 5, 912) using ab initio quantum mechanical molecular mechanical (ai-QM/MM) free energy simulations, where the free energy barrier was found to be more favorable than that from a pseudoactive enzyme conformation. In this work, we performed ai-QM/MM simulations based on another catalytically active conformation (PDB 7Z4J) of the Cas9 HNH domain from cryo-electron microscopy experiments. For the wildtype enzyme, we acquired a free energy profile for the tDNA cleavage that is largely consistent with the previous report. Furthermore, we explored the role of the active-site K866 residue on the catalytic efficiency by modeling the K866A mutant and found that the K866A mutation increased the reaction free energy barrier, which is consistent with the experimentally observed reduction in the enzyme activity.

利用加速量子力学分子机械自由能模拟探索 CRISPR-Cas9 HNH-Domain 催化的 DNA 裂解。
由CRISPR Cas9酶催化的靶DNA (tDNA)切割是基于Cas9的基因组编辑技术的关键步骤。先前,Palermo及其同事(Nierzwicki et al., Nat. catalal . 2022 5,912)使用从头算量子力学分子力学(ai-QM/MM)自由能模拟模拟了活性SpyCas9酶构象的tDNA切割,其中发现自由能垒比假活性酶构象更有利。在这项工作中,我们基于低温电子显微镜实验中Cas9 HNH结构域的另一个催化活性构象(PDB 7Z4J)进行了ai-QM/MM模拟。对于野生型酶,我们获得了tDNA切割的自由能谱,这与之前的报道基本一致。此外,我们通过模拟K866A突变体来探索活性位点K866残基对催化效率的作用,发现K866A突变体增加了反应的自由能垒,这与实验观察到的酶活性降低一致。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Biochemistry Biochemistry
Biochemistry Biochemistry 生物-生化与分子生物学
CiteScore
5.50
自引率
3.40%
发文量
336
审稿时长
1-2 weeks
期刊介绍: Biochemistry provides an international forum for publishing exceptional, rigorous, high-impact research across all of biological chemistry. This broad scope includes studies on the chemical, physical, mechanistic, and/or structural basis of biological or cell function, and encompasses the fields of chemical biology, synthetic biology, disease biology, cell biology, nucleic acid biology, neuroscience, structural biology, and biophysics. In addition to traditional Research Articles, Biochemistry also publishes Communications, Viewpoints, and Perspectives, as well as From the Bench articles that report new methods of particular interest to the biological chemistry community.
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