鸟苷酸结合蛋白(GBP)是必需的干扰素-γ激活的大型GTP酶,在宿主抵抗细胞内细菌和寄生虫的防御中起着至关重要的作用。虽然它们的保护功能依赖于蛋白质聚合,我们对这些多聚化状态的结构复杂性的理解仍然有限。为了弥合这种知识差距,我们提出了二聚体模型的人GBP1(hGBP1)和鼠GBP2和7(mGBP2和mGBP7)使用综合方法,掺入hGBP1的GTP酶结构域二聚体的晶体结构,交联质谱(XL-MS),小角度X射线散射(SAXS),蛋白质-蛋白质对接,和分子动力学(MD)模拟。我们的研究从比较hGBP1,mGBP2和mGBP7的蛋白质动力学开始。我们观察到所有三种蛋白质中的M/E结构域都表现出显著的迁移率和铰链运动,mGBP7在其GTPase结构域中显示出略不明显的运动,但具有更大的灵活性。这些动态差异可归因于mGBP7和hGBP1/mGBP2序列的变化,导致不同的二聚化模式。与hGBP1及其紧密的直系同源物mGBP2不同,它们仅通过其GTP酶结构域进行二聚化,我们发现mGBP7表现出三个同样可能的替代二聚体结构。mGBP7的GTPase结构域仅部分参与其二聚化,主要是由于负电荷的积累,允许MGBP7独立于GTP进行二聚化。相反,mGBP7在其茎上表现出反平行排列的强烈二聚化趋势。这项工作的结果超越了序列-结构-函数关系,由于mGBP7和mGBP2/hGBP1的序列差异不会导致不同的结构,而是不同的蛋白质动力学和二聚化。预期不同的GBP二聚体结构编码对于破坏病原体膜至关重要的特定功能。本文受版权保护。保留所有权利。
Guanylate-binding proteins (GBPs) are essential interferon-γ-activated large GTPases that play a crucial role in host defense against intracellular bacteria and parasites. While their protective functions rely on protein polymerization, our understanding of the structural intricacies of these multimerized states remains limited. To bridge this knowledge gap, we present dimer models for human GBP1 (hGBP1) and murine GBP2 and 7 (mGBP2 and mGBP7) using an integrative approach, incorporating the crystal structure of hGBP1\'s GTPase domain dimer, crosslinking mass spectrometry, small-angle X-ray scattering, protein-protein docking, and molecular dynamics simulations. Our investigation begins by comparing the protein dynamics of hGBP1, mGBP2, and mGBP7. We observe that the M/E domain in all three proteins exhibits significant mobility and hinge motion, with mGBP7 displaying a slightly less pronounced motion but greater flexibility in its GTPase domain. These dynamic distinctions can be attributed to variations in the sequences of mGBP7 and hGBP1/mGBP2, resulting in different dimerization modes. Unlike hGBP1 and its close ortholog mGBP2, which exclusively dimerize through their GTPase domains, we find that mGBP7 exhibits three equally probable alternative dimer structures. The GTPase domain of mGBP7 is only partially involved in its dimerization, primarily due to an accumulation of negative charge, allowing mGBP7 to dimerize independently of GTP. Instead, mGBP7 exhibits a strong tendency to dimerize in an antiparallel arrangement across its stalks. The results of this work go beyond the sequence-structure-function relationship, as the sequence differences in mGBP7 and mGBP2/hGBP1 do not lead to different structures, but to different protein dynamics and dimerization. The distinct GBP dimer structures are expected to encode specific functions crucial for disrupting pathogen membranes.