RAPID: Structure, Function and Dynamics of SARS Coronavirus-2 Main Protease 3CLpro Determined with Mix-and-Inject Serial XFEL Crystallography

威斯康星大学密尔沃基分校(UWM)获奖，以实时研究一种基本冠状病毒蛋白酶反应的分子机制。这个项目解决了SARS冠状病毒-2(CoV-2)如何扩散的基本问题。冠状病毒2型是新冠肺炎全球大流行的源头，严重影响公共卫生和国家繁荣。CoV-2主要蛋白水解酶，也称为3CLPro，催化CoV-2感染性颗粒组装所必需的反应。如果3CLPro被阻断，病毒就无法组装，其传播也被有效抑制。我们的目标是用X射线结构表征3CLPro在常温下的催化循环。这项研究利用了X射线自由电子激光(XFELs)的机会，例如加利福尼亚州门洛帕克斯坦福直线加速器中心的直线加速器相干光源(LCLS)。XFELs是世界上最强的X射线源，可以在生物相关的温度和时间范围内以接近原子的分辨率捕获分子反应中间产物。在XFELS上的独特机会将促进对3CLPro催化及其在病毒扩散中的作用的理解，并有助于消除大流行。该项目将涉及密歇根大学的研究生和博士后研究人员，他们将在XFELS接受最新的数据收集和数据分析方法的培训。在宿主细胞中，SARS-CoV-2‘S RNA基因组由宿主核糖体翻译成一条长的多肽链，必须切割成功能蛋白。这是由CoV-2 3CLPro实现的。如果3CLPro被抑制，新形成的病毒颗粒就不能正确组装，变得不具传染性。该项目从结构上描述了3CLPro在XFELS上的催化循环。XFEL是一种非常强大的飞秒脉冲X射线源，十年前开始在更广泛的社区使用。在XFELs中，基本上没有辐射损伤的晶体结构可以在环境(接近生理)温度下确定。在3CLPRO微晶内，催化循环将由底物的扩散启动。由于晶体很小，扩散不受速度限制。微晶在被注入X射线束之前，以不同的时间延迟与基片混合，这种方法被称为“混合-注入”系列结晶学(MISC)。MISC将用于实时跟踪3CLPro的酶反应，并获得反应中间产物的X-射线结构。抑制3CLPro的小分子化合物的结合将在相关温度下以近原子分辨率进行探测。结果将(I)有助于设计和发现新的抑制化合物，影响这种基本的蛋白酶的功能并防止感染性病毒颗粒的形成，以及(Ii)有助于MISC的发展，作为XFELS的适用方法，用于表征具有生物学意义的分子的反应的结构特征。这一快速奖项由生物基础设施部(DBI)利用冠状病毒援助、救济和经济安全法案(CARE)的资金颁发。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

An award is made to the University of Wisconsin-Milwaukee (UWM) to investigate the molecular mechanism of an essential coronavirus protease reaction in real time. This project addresses fundamental questions how the SARS coronavirus-2 (CoV-2) proliferates. CoV-2 is the source of the worldwide COVID-19 pandemic, which severely impacts public health and national prosperity. The CoV-2 main protease, also called 3CLpro, catalyzes an essential reaction for assembly of CoV-2 infectious particles. If the 3CLpro is blocked, the virus cannot assemble, and its spread is effectively suppressed. The goal is to characterize the 3CLpro’s catalytic cycle at ambient temperatures with X-ray structures. This research takes advantage of opportunities at X-ray Free Electron Lasers (XFELs) such as the Linac Coherent Light Source (LCLS) at Stanford Linear Accelerator Center in Menlo Park, CA. XFELs are the strongest X-ray sources in the world that make it possible to capture molecular reaction intermediates at near atomic resolution within biologically relevant temperatures and time scales. The unique opportunities at XFELs will advance the understanding of 3CLpro catalysis and its function in the proliferation of the virus and contribute to the elimination of the pandemic. This project will involve UWM graduate students and postdoctoral researchers who will be trained in newest data collection and data analysis methods at XFELs.In host cells the SARS-CoV-2’s RNA genome is translated by host ribosomes into a long polypeptide strand that must be cleaved into functional proteins. This is achieved by the CoV-2 3CLpro. If the 3CLpro is inhibited, the newly formed virus particles cannot assemble correctly and become non-infectious. This project structurally characterizes the catalytic cycle of the 3CLpro at XFELs. XFELs are extremely powerful, femtosecond-pulsed X-ray sources, which became available to a wider community a decade ago. At XFELs, crystal structures that are essentially free of radiation damage can be determined at ambient (near physiological) temperatures. Within 3CLpro microcrystals the catalytic cycle will be initiated by diffusion of substrate. Since the crystals are so small, diffusion is not rate-limiting. Microcrystals are mixed with substrate at various time delays before the mixture is injected into the X-ray beam, a method known as ‘mix-and-inject’ serial crystallography (MISC). MISC will be used to follow the 3CLpro enzymatic reaction in real time with X-ray structures of reaction intermediates. The binding of small compounds that inhibit the 3CLpro will be probed at near atomic resolution at relevant temperatures. Results will (i) aid the design and discovery of new inhibitory compounds that affect the function of this essential protease and prevent the formation of infectious viral particles, and (ii) contribute to the development of MISC as an applicable method at XFELs, to be used for the structural characterization of reactions in biologically significant molecules.This RAPID award is made by the Division of Biological Infrastructure (DBI) using funds from the Coronavirus Aid, Relief, and Economic Security (CARES) Act.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.