An atypical heterotrimeric Gα protein has substantially reduced nucleotide binding but retains nucleotide-independent interactions with its cognate RGS protein and Gβγ dimer

Plants uniquely have a family of proteins called extra-large G proteins (XLG) that share homology in their C-terminal half with the canonical Gα subunits; we carefully detail here that Arabidopsis XLG2 lacks critical residues requisite for nucleotide binding and hydrolysis which is consistent with our quantitative analyses. Based on microscale thermophoresis, Arabidopsis XLG2 binds GTPγS with an affinity 100 times lower than that to canonical Gα subunits. This means that given the concentration range of guanine nucleotide in plant cells, XLG2 is not likely bound by GTP <i>in vivo</i>. Homology modeling and molecular dynamics simulations provide a plausible mechanism for the poor nucleotide binding affinity of XLG2. Simulations indicate substantially stronger salt bridge networks formed by several key amino-acid residues of AtGPA1 which are either misplaced or missing in XLG2. These residues in AtGPA1 not only maintain the overall shape and integrity of the apoprotein cavity but also increase the frequency of favorable nucleotide-protein interactions in the nucleotide-bound state. Despite this loss of nucleotide dependency, XLG2 binds the RGS domain of AtRGS1 with an affinity similar to the Arabidopsis AtGPA1 in its apo-state and about 2 times lower than AtGPA1 in its transition state. In addition, XLG2 binds the Gβγ dimer with an affinity similar to that of AtGPA1. XLG2 likely acts as a dominant negative Gα protein to block G protein signaling. We propose that XLG2, independent of guanine nucleotide binding, regulates the active state of the canonical G protein pathway directly by sequestering Gβγ and indirectly by promoting heterodimer formation. Communicated by Ramaswamy H. Sarma

植物独有一类被称为超大G蛋白（extra-large G proteins, XLG）的蛋白家族，其C端半区与经典Gα亚基具有同源性。本文详细阐述了拟南芥XLG2缺失核苷酸结合与水解所需的关键氨基酸残基，这一结果与我们的定量分析相一致。基于微量热泳动（microscale thermophoresis）实验结果，拟南芥XLG2结合GTPγS的亲和力比其与经典Gα亚基的结合亲和力低100倍。这意味着，考虑到植物细胞内鸟嘌呤核苷酸的浓度范围，XLG2在体内（in vivo）不太可能结合GTP。同源建模与分子动力学模拟为XLG2较弱的核苷酸结合亲和力提供了合理的机制解释：模拟结果显示，拟南芥G蛋白α亚基1（AtGPA1）的多个关键氨基酸残基形成了显著更强的盐桥网络，而这些残基在XLG2中要么位置异常，要么完全缺失。AtGPA1中的这些残基不仅维持了脱辅基蛋白结合腔的整体形态与完整性，还提升了核苷酸结合状态下核苷酸-蛋白间有利相互作用的发生频率。尽管失去了核苷酸依赖性，XLG2仍能结合拟南芥G蛋白信号调控因子1（AtRGS1）的RGS结构域，其结合亲和力与处于脱辅基状态的AtGPA1相当，且仅比处于过渡态的AtGPA1低约2倍。此外，XLG2结合Gβγ二聚体的亲和力与AtGPA1相当。XLG2可能作为显性负效Gα蛋白，阻断G蛋白信号通路。我们提出，XLG2无需结合鸟嘌呤核苷酸，即可通过两种方式调控经典G蛋白通路的激活状态：直接螯合Gβγ二聚体，以及间接促进异二聚体的形成。由Ramaswamy H. Sarma转交。