Structural study of the Ly49A T cell recognition domain

先前的晶体研究表明，Ly49A同二聚体以不对称的相互作用结合一个MHC分子，而Ly49C同二聚体以对称的方式结合两个MHC分子。此外，结合受体采用明显不同的同型二聚体形式：Ly49A为封闭状态，Ly49C为开放状态。MHC分子之间的空间冲突会阻止封闭的Ly49A二聚体像开放的Ly49C二聚体那样与两个MHC结合。为了解决单个Ly49受体是否可以经历构象开关，使它们以不同的方式结合MHC，我们在偶极偶联技术的帮助下，对未结合的Ly49A进行了溶液核磁共振研究。本研究表明，在溶液中，未结合的Ly49A采用与Ly49C相似的对称开态同二聚体构象。因此，Ly49A可以同时处于闭合和开放状态。为了解决Ly49A二聚体在溶液中是否可以结合两个MHC分子，除了在晶体中观察到的一个MHC的结合，我们进行了分析性的超离心实验。速度沉降表明Ly49A二聚体可以与溶液中的两个MHC结合，这与NMR结果一致，表明未结合的Ly49A主要以开放状态存在。

Previous crystal studies revealed that the Ly49A homodimer binds one MHC molecule in an asymmetric interaction, whereas the Ly49C homodimer binds two MHC in a symmetrical fashion. Moreover, the bound receptors adopt distinctly different homodimeric forms: a closed state for Ly49A and an open state for Ly49C. Steric clashes between MHC molecules would preclude the closed Ly49A dimer from engaging two MHC in the manner of the open Ly49C dimer. To address whether individual Ly49 receptors can undergo a conformational switch enabling them to bind MHC in different ways, we carried out a solution NMR study of unbound Ly49A, aided by dipolar coupling technology. This study reveals that in solution unligated Ly49A adopts a symmetric, open state, homodimer conformation similar to that previously seen for Ly49C. Hence, Ly49A can assume both closed and open states. To address whether the Ly49A dimer can bind two MHC molecules in solution, besides the binding of one MHC observed in the crystal, we carried out analytical ultracentrifugation experiments. Velocity sedimentation demonstrates that the Ly49A dimer can engage two MHC in solution, in agreement with NMR results showing that unbound Ly49A exists predominantly in the open state.