Department of Material Science and Chemical Engineering, Stony Brook University, Stony Brook, NY, United States.
Biology Department, Brookhaven National Laboratory, Upton, NY, United States.
Methods Enzymol. 2022;676:103-131. doi: 10.1016/bs.mie.2022.07.026. Epub 2022 Sep 8.
Metacaspases are highly conserved in plants and play essential roles in mediating programmed cell death, biotic and abiotic stress responses, and damage-induced innate immunity. Ca signaling induced by plant damage leads to activation of metacaspase 4 from Arabidopsis thaliana (AtMC4), which subsequently processes a plant elicitor peptide to trigger downstream immuno-response. To understand the structural basis of AtMC4 activation by Ca, we previously determined its crystal structure and performed in-crystal Ca treatment to probe activation-associated conformational changes. To enable structure determination and in-crystal Ca activation analysis, we used microcrystals and related methods which were essential for our successful approach. Here we describe in detail the methods that we used for determination of AtMC4 structure using single-wavelength isomorphous replacement with anomalous signals assembled from 22 microcrystals. We also describe the method for in-crystal Ca soaking, microcrystal data collection, data assembly and analysis to obtain the activated structure of AtMC4 from 91 micro-sized crystals. The described methods may be useful to study other plant metacaspases and more broadly other plant enzymes for their structure determination and in-crystal functional characterization.
植物中的类半胱天冬酶高度保守，在介导程序性细胞死亡、生物和非生物胁迫反应以及损伤诱导的先天免疫中发挥重要作用。植物损伤诱导的钙信号导致拟南芥（Arabidopsis thaliana）的类半胱天冬酶 4（AtMC4）的激活，随后该酶处理植物激发肽以触发下游免疫反应。为了了解 Ca 对 AtMC4 激活的结构基础，我们先前确定了其晶体结构，并进行了晶体中的 Ca 处理以探测激活相关的构象变化。为了能够进行结构测定和晶体中的 Ca 激活分析，我们使用了微晶体和相关方法，这对我们的成功方法至关重要。在这里，我们详细描述了使用来自 22 个微晶体的单波长同晶置换与异常信号组装来确定 AtMC4 结构的方法。我们还描述了用于晶体中 Ca 浸泡、微晶体数据收集、数据组装和分析的方法，以从 91 个微晶体中获得激活的 AtMC4 结构。所描述的方法可用于研究其他植物类半胱天冬酶，更广泛地用于研究其他植物酶的结构测定和晶体中的功能表征。
