Protein-Protein Interactions in Crystals of the Human Receptor-Type Protein Tyrosine Phosphatase ICA512 Ectodomain

ICA512 (or IA-2) is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Initially, it was identified as one of the main antigens of autoimmune diabetes. Later, it was found that during insulin secretion, the cytoplasmic domain of ICA512 is cleaved and relocated to the nucleus, where it stimulates the transcription of the insulin gene. The role of the other parts of the receptor in insulin secretion is yet to be unveiled. The structures of the intracellular pseudocatalytic and mature extracellular domains are known, but the transmembrane domain and several intracellular and extracellular parts of the receptor are poorly characterized. Moreover the overall structure of the receptor remains to be established. We started to address this issue studying by X-ray crystallography the structure of the mature ectodomain of ICA512 (ME ICA512) and variants thereof. The variants and crystallization conditions were chosen with the purpose of exploring putative association interfaces, metal binding sites and all other structural details that might help, in subsequent works, to build a model of the entire receptor. Several structural features were clarified and three main different association modes of ME ICA512 were identified. The results provide essential pieces of information for the design of new experiments aimed to assess the structure in vivo.ICA512（又称IA-2）是一种定位于神经内分泌细胞（neuroendocrine cells）分泌颗粒（secretory granules）的跨膜蛋白酪氨酸磷酸酶（transmembrane protein-tyrosine phosphatase）。最初，该蛋白被鉴定为自身免疫性糖尿病（autoimmune diabetes）的主要抗原之一。后续研究发现，在胰岛素分泌过程中，ICA512的胞质结构域（cytoplasmic domain）会被切割并易位至细胞核，在此处刺激胰岛素基因的转录。该受体其余结构域在胰岛素分泌中的功能仍有待阐明。目前已知其胞内伪催化结构域（intracellular pseudocatalytic domain）与成熟胞外结构域（mature extracellular domain）的三维结构，但该受体的跨膜结构域（transmembrane domain）及部分胞内、胞外区段的结构特征尚未被充分解析。此外，该受体的完整整体结构仍有待确定。为此，我们通过X射线晶体学（X-ray crystallography）技术解析了ICA512成熟胞外结构域（ME ICA512）及其突变体的结构。我们通过筛选突变体与结晶条件，旨在探索潜在的相互作用界面、金属结合位点（metal binding sites）及其他所有结构细节，以期在后续研究中构建该受体的完整三维模型。本研究阐明了多项结构特征，并确定了ME ICA512的三种主要组装模式。本研究结果为后续设计用于解析其体内（in vivo）结构的全新实验提供了关键依据。