Liu Yanshun, Gotte Giovanni, Libonati Massimo, Eisenberg David
Howard Hughes Medical Institute, UCLA-DOE Laboratory of Structural Biology and Molecular Medicine, Department of Chemistry, University of California, Los Angeles, California 90095, USA.
Protein Sci. 2002 Feb;11(2):371-80. doi: 10.1110/ps.36602.
When concentrated in mildly acidic solutions, bovine pancreatic ribonuclease (RNase A) forms long-lived oligomers including two types of dimer, two types of trimer, and higher oligomers. In previous crystallographic work, we found that the major dimeric component forms by a swapping of the C-terminal beta-strands between the monomers, and that the minor dimeric component forms by swapping the N-terminal alpha-helices of the monomers. On the basis of these structures, we proposed that a linear RNase A trimer can form from a central molecule that simultaneously swaps its N-terminal helix with a second RNase A molecule and its C-terminal strand with a third molecule. Studies by dissociation are consistent with this model for the major trimeric component: the major trimer dissociates into both the major and the minor dimers, as well as monomers. In contrast, the minor trimer component dissociates into the monomer and the major dimer. This suggests that the minor trimer is cyclic, formed from three monomers that swap their C-terminal beta-strands into identical molecules. These conclusions are supported by cross-linking of lysyl residues, showing that the major trimer swaps its N-terminal helix, and the minor trimer does not. We verified by X-ray crystallography the proposed cyclic structure for the minor trimer, with swapping of the C-terminal beta-strands. This study thus expands the variety of domain-swapped oligomers by revealing the first example of a protein that can form both a linear and a cyclic domain-swapped oligomer. These structures permit interpretation of the enzymatic activities of the RNase A oligomers on double-stranded RNA.
当牛胰核糖核酸酶（RNase A）在弱酸性溶液中浓缩时，会形成寿命较长的寡聚体，包括两种类型的二聚体、两种类型的三聚体以及更高阶的寡聚体。在之前的晶体学研究中，我们发现主要的二聚体组分是通过单体之间C端β链的交换形成的，而次要的二聚体组分是通过单体的N端α螺旋的交换形成的。基于这些结构，我们提出线性RNase A三聚体可以由一个中心分子形成，该中心分子同时与第二个RNase A分子交换其N端螺旋，并与第三个分子交换其C端链。解离研究与主要三聚体组分的这一模型一致：主要三聚体解离为主要和次要二聚体以及单体。相比之下，次要三聚体组分解离为单体和主要二聚体。这表明次要三聚体是环状的，由三个将其C端β链交换到相同分子中的单体形成。这些结论得到了赖氨酰残基交联的支持，表明主要三聚体交换其N端螺旋，而次要三聚体不交换。我们通过X射线晶体学验证了次要三聚体的环状结构，即C端β链的交换。因此，这项研究通过揭示一种既能形成线性又能形成环状结构域交换寡聚体的蛋白质的首个例子，扩展了结构域交换寡聚体的种类。这些结构有助于解释RNase A寡聚体对双链RNA的酶活性。
