Schlenker Oliver, Rittinger Katrin
Medical Research Council National Institute for Medical Research, The Ridgeway, London, UK.
J Mol Biol. 2009 Feb 20;386(2):280-9. doi: 10.1016/j.jmb.2008.12.050. Epub 2008 Dec 30.
GIT (G protein-coupled receptor kinase-interacting protein) and PIX (p21-activated kinase-interacting exchange factor) family proteins integrate signaling pathways involving Arf and Rho family GTPases. GIT1 and beta-PIX form a constitutively associated complex that acts as a scaffold to allow the formation of large multiprotein assemblies that regulate synaptogenesis, cell polarity and cell migration among other physiological processes. Complex formation is mediated by the GIT binding domain (GBD) in beta-PIX, which recognizes the Spa homology domain of GIT1. Both binding domains are adjacent to predicted coiled-coil segments that allow homo-oligomerization of GIT1 and beta-PIX, respectively. Oligomerization of GIT and PIX proteins is important for their physiological functions, and deletion of the coiled-coil domains interferes with correct subcellular localization and the GEF (guanine nucleotide exchange factor) activity of PIX. We have solved the crystal structures of the CC domains of GIT1 and beta-PIX and determined the stoichiometry of complex formation between the two proteins in order to understand the molecular architecture of the GIT1-beta-PIX complex. The crystal structure of the CC domain of GIT1 solved at 1.4 A resolution shows a dimeric, parallel CC that spans 67 A in length. Unexpectedly, and in contrast to prevalent dimeric models, the structure of the CC region of beta-PIX determined at 2.8 A resolution, combined with hydrodynamic studies, reveals that this protein forms a parallel trimer. Furthermore, we demonstrate that dimeric GIT and trimeric PIX form an unusual high-affinity heteropentameric complex in which each Spa homology domain of the GIT1 dimer recognizes one GBD of the beta-PIX trimer, leaving one GBD unoccupied. These results can serve as a basis to better understand oligomerization-dependent GIT1-beta-PIX-regulated signaling events and provide an insight into the architecture of large signaling complexes involving GIT1 and beta-PIX.
GIT（G蛋白偶联受体激酶相互作用蛋白）和PIX（p21激活激酶相互作用交换因子）家族蛋白整合涉及Arf和Rho家族GTP酶的信号通路。GIT1和β-PIX形成一种组成型相关复合物，该复合物作为一种支架，允许形成大型多蛋白组装体，这些组装体在突触形成、细胞极性和细胞迁移等生理过程中发挥调节作用。复合物的形成由β-PIX中的GIT结合结构域（GBD）介导，该结构域识别GIT1的Spa同源结构域。两个结合结构域都与预测的卷曲螺旋片段相邻，这些片段分别允许GIT1和β-PIX进行同源寡聚化。GIT和PIX蛋白的寡聚化对其生理功能很重要，卷曲螺旋结构域的缺失会干扰PIX正确的亚细胞定位和鸟嘌呤核苷酸交换因子（GEF）活性。我们解析了GIT1和β-PIX的卷曲螺旋结构域的晶体结构，并确定了这两种蛋白之间复合物形成的化学计量比，以便了解GIT1-β-PIX复合物的分子结构。以1.4埃分辨率解析的GIT1卷曲螺旋结构域的晶体结构显示出一个二聚体、平行的卷曲螺旋，长度为67埃。出乎意料的是，与普遍的二聚体模型不同，以2.8埃分辨率解析的β-PIX卷曲螺旋区域的结构，结合流体动力学研究，揭示该蛋白形成一个平行三聚体。此外，我们证明二聚体GIT和三聚体PIX形成一种不寻常的高亲和力异戊五聚体复合物，其中GIT1二聚体的每个Spa同源结构域识别β-PIX三聚体的一个GBD，留下一个GBD未被占据。这些结果可作为更好地理解寡聚化依赖性GIT1-β-PIX调节的信号事件的基础，并为涉及GIT1和β-PIX的大型信号复合物的结构提供见解。
