Structural and dynamic insights into the HNH nuclease of divergent Cas9 species.
Structural and dynamic insights into the HNH nuclease of divergent Cas9 species.
复制标题
DOI：
10.1016/j.jsb.2021.107814
复制
发表时间：
2022-03
影响因子：
3
通讯作者：
Lisi GP
中科院分区：
文献类型：
--
作者：
Belato HB;D'Ordine AM;Nierzwicki L;Arantes PR;Jogl G;Palermo G;Lisi GP
CRISPR-Cas9是一种广泛使用的生化工具，应用于分子生物学和精准医学。 RNA引导的Cas9蛋白利用其HNH核酸内切酶结构域来切割与其内源引导RNA互补的DNA链。在这项研究中，来自两种不同生物体嗜热脂肪芽孢杆菌 (GeoHNH) 和化脓性链球菌 (SpHNH) 的新 HNH 构建体是从它们各自的全长 Cas9 蛋白改造而来的。尽管序列相似性较低，但这些构建体的 X 射线晶体结构表明，活性位点周围的 HNH 核心是保守的。使用 Phyre2 和 AlphaFold2 对全长 GeoCas9 蛋白的结构预测还表明，GeoHNH 的晶体结构代表了全长 GeoCas9 蛋白内的结构域。然而，在 GeoHNH 和 SpHNH 结构保守区域的溶液动力学中观察到显着差异，后者被证明利用这种分子运动来传播 DNA 裂解信号。事实上，分子模拟表明，驱动 SpHNH 功能的域内信号通路在 GeoHNH 中是非特异性的且形成不良。综上所述，这些结果表明嗜温和嗜热 Cas9 物种之间存在机制差异。
CRISPR-Cas9 is a widely used biochemical tool with applications in molecular biology and precision medicine. The RNA-guided Cas9 protein uses its HNH endonuclease domain to cleave the DNA strand complementary to its endogenous guide RNA. In this study, novel constructs of HNH from two divergent organisms, G. stearothermophilus (GeoHNH) and S. pyogenes (SpHNH) were engineered from their respective full-length Cas9 proteins. Despite low sequence similarity, the X-ray crystal structures of these constructs reveal that the core of HNH surrounding the active site is conserved. Structure prediction of the full-length GeoCas9 protein using Phyre2 and AlphaFold2 also showed that the crystallographic construct of GeoHNH represents the structure of the domain within the full-length GeoCas9 protein. However, significant differences are observed in the solution dynamics of structurally conserved regions of GeoHNH and SpHNH, the latter of which was shown to use such molecular motions to propagate the DNA cleavage signal. Indeed, molecular simulations show that the intradomain signaling pathways, drive SpHNH function, are non-specific and poorly formed in GeoHNH. Taken together, these outcomes suggest mechanistic differences between mesophilic and thermophilic Cas9 species.
登录
查看更多内容
影响因子：
64.8
作者：
Jumper J;Evans R;Pritzel A;Green T;Figurnov M;Ronneberger O;Tunyasuvunakool K;Bates R;Žídek A;Potapenko A;Bridgland A;Meyer C;Kohl SAA;Ballard AJ;Cowie A;Romera-Paredes B;Nikolov S;Jain R;Adler J;Back T;Petersen S;Reiman D;Clancy E;Zielinski M;Steinegger M;Pacholska M;Berghammer T;Bodenstein S;Silver D;Vinyals O;Senior AW;Kavukcuoglu K;Kohli P;Hassabis D
通讯作者：
Hassabis D
影响因子：
14.8
作者：
Kelley LA;Mezulis S;Yates CM;Wass MN;Sternberg MJ
通讯作者：
Sternberg MJ
影响因子：
64.8
作者：
Anders, Carolin;Niewoehner, Ole;Duerst, Alessia;Jinek, Martin
通讯作者：
Jinek, Martin
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
15
作者：
East, Kyle W.;Newton, Jocelyn C.;Lisi, George P.
通讯作者：
Lisi, George P.