Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC, USA.
Waters Corporation, Milford, MA, USA.
Protein J. 2020 Oct;39(5):461-471. doi: 10.1007/s10930-020-09926-9. Epub 2020 Oct 26.
An abundance of protein structures has been solved in the last six decades that are paramount in defining the function of such proteins. For unsolved protein structures, however, predictions based on sequence and phylogenetic similarity can be useful for identifying key domains of interaction. Here, we describe expression and purification of a recombinant plant LRR-RLK ectodomain MIK1 using a modified baculovirus-mediated expression system with subsequent N-linked glycosylation analysis using LC-MS/MS and computational sequence-based analyses. Though highly ubiquitous, glycosylation site specificity and the degree of glycosylation influenced by genetic and exogenous factors are still largely unknown. Our experimental analysis of N-glycans on MIK1 identified clusters of glycosylation that may explicate the regions involved in MIK1 ectodomain binding. Whether these glycans are necessary for function is yet to be determined. Phylogenetic comparison using multiple sequence alignment between MIK1 and other LRR-RLKs, namely TDR in Arabidopsis thaliana, revealed conserved structural motifs that are known to play functional roles in ligand and receptor binding.
在过去的六十年中，已经解决了大量的蛋白质结构，这些结构对于定义这些蛋白质的功能至关重要。然而，对于未解决的蛋白质结构，可以基于序列和系统发育相似性的预测来识别关键的相互作用结构域。在这里，我们描述了使用改良的杆状病毒介导的表达系统表达和纯化重组植物 LRR-RLK 胞外结构域 MIK1，随后使用 LC-MS/MS 和基于计算序列的分析进行 N 连接糖基化分析。尽管高度普遍存在，但糖基化位点特异性和遗传及外源因素影响的糖基化程度在很大程度上仍不清楚。我们对 MIK1 上 N-聚糖的实验分析确定了糖基化簇，这些糖基化簇可能解释了参与 MIK1 胞外结构域结合的区域。这些糖基是否对功能是必需的，还有待确定。使用拟南芥中的 TDR 等 MIK1 和其他 LRR-RLK 之间的多重序列比对进行系统发育比较，揭示了已知在配体和受体结合中发挥功能作用的保守结构基序。
