Lu Zhuoyang, Wang Yun, Chen Fang, Tong Huimin, Reddy M V V V Sekhar, Luo Lin, Seshadrinathan Suchithra, Zhang Lei, Holthauzen Luis Marcelo F, Craig Ann Marie, Ren Gang, Rudenko Gabby
the Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California 94720, the School of Life Science and Technology and Frontier Institute of Science and Technology, Xi'an Jiaotong University, Xi'an 710049, China.
the University of Michigan, Ann Arbor, Michigan 48109.
J Biol Chem. 2014 Dec 12;289(50):34530-42. doi: 10.1074/jbc.M114.606806. Epub 2014 Oct 28.
Calsyntenin 3 (Cstn3 or Clstn3), a recently identified synaptic organizer, promotes the development of synapses. Cstn3 localizes to the postsynaptic membrane and triggers presynaptic differentiation. Calsyntenin members play an evolutionarily conserved role in memory and learning. Cstn3 was recently shown in cell-based assays to interact with neurexin 1α (n1α), a synaptic organizer that is implicated in neuropsychiatric disease. Interaction would permit Cstn3 and n1α to form a trans-synaptic complex and promote synaptic differentiation. However, it is contentious whether Cstn3 binds n1α directly. To understand the structure and function of Cstn3, we determined its architecture by electron microscopy and delineated the interaction between Cstn3 and n1α biochemically and biophysically. We show that Cstn3 ectodomains form monomers as well as tetramers that are stabilized by disulfide bonds and Ca(2+), and both are probably flexible in solution. We show further that the extracellular domains of Cstn3 and n1α interact directly and that both Cstn3 monomers and tetramers bind n1α with nanomolar affinity. The interaction is promoted by Ca(2+) and requires minimally the LNS domain of Cstn3. Furthermore, Cstn3 uses a fundamentally different mechanism to bind n1α compared with other neurexin partners, such as the synaptic organizer neuroligin 2, because Cstn3 does not strictly require the sixth LNS domain of n1α. Our structural data suggest how Cstn3 as a synaptic organizer on the postsynaptic membrane, particularly in tetrameric form, may assemble radially symmetric trans-synaptic bridges with the presynaptic synaptic organizer n1α to recruit and spatially organize proteins into networks essential for synaptic function.
钙连接蛋白3（Cstn3或Clstn3）是最近发现的一种突触组织者，可促进突触的发育。Cstn3定位于突触后膜并触发突触前分化。钙连接蛋白成员在记忆和学习中发挥着进化保守的作用。最近在基于细胞的实验中发现，Cstn3与神经连接蛋白1α（n1α）相互作用，n1α是一种与神经精神疾病有关的突触组织者。这种相互作用将使Cstn3和n1α形成跨突触复合物并促进突触分化。然而，Cstn3是否直接结合n1α仍存在争议。为了了解Cstn3的结构和功能，我们通过电子显微镜确定了其结构，并从生化和生物物理角度描绘了Cstn3与n1α之间的相互作用。我们发现，Cstn3胞外域形成单体以及由二硫键和Ca(2+)稳定的四聚体，两者在溶液中可能都是灵活的。我们进一步表明，Cstn3和n1α的细胞外域直接相互作用，并且Cstn3单体和四聚体均以纳摩尔亲和力结合n1α。这种相互作用由Ca(2+)促进，并且至少需要Cstn3的LNS结构域。此外，与其他神经连接蛋白伴侣（如突触组织者神经配体-2）相比，Cstn3使用一种根本不同的机制来结合n1α，因为Cstn3并不严格需要n1α的第六个LNS结构域。我们的结构数据表明，作为突触后膜上的突触组织者，尤其是四聚体形式的Cstn如何与突触前突触组织者n1α组装成径向对称的跨突触桥，以招募蛋白质并将其空间组织成突触功能所必需的网络。
