Abstract
IA-2 (insulinoma-associated protein 2) is a protein-tyrosine phosphatase receptor located in secretory granules of neuroendocrine cells. Initially, it attracted attention due to its involvement in the autoimmune response associated to diabetes. Later it was found that upon exocytosis, the cytoplasmic domain of IA-2 is cleaved and relocated to the nucleus, where it enhances the transcription of the insulin gene. A concerted functioning of the whole receptor is to be expected. However, very little is known about the structure and function of the transmembrane and extracellular domains of IA-2. To address this issue, we solved the x-ray structure of the mature ectodomain of IA-2 (meIA-2) to 1.30A resolution. The fold of meIA-2 is related to the SEA (sea urchin sperm protein, enterokinase, agrin)) domains of mucins, suggesting its participation in adhesive contacts to the extracellular matrix and providing clues on how this kind of molecule may associate and form homo- and heterodimers. Moreover, we discovered that meIA-2 is self-proteolyzed in vitro by reactive oxygen species, suggesting the possibility of a new shedding mechanism that might be significant in normal function or pathological processes. Knowledge of meIA-2 structure should facilitate the search of its possible ligands and molecular interactions.
Highlights
Tion, and metabolism [1]
Crystal Structure—MeIA-2 crystallized in the orthorhombic space group P212121, and the structure was refined to 1.30 Å
There was no electron density for residues 1–19 and 111–128, which cannot be the result of local disorder for the presence of the missing residues would lead to unrealistic crystal density
Summary
Miscellaneous—Unless otherwise indicated, reagents were from Sigma. The broad protease inhibitor mixture was Set III (AEBSF, aprotinin, bestatin, E-64, leupeptin, and pepstatin A; Calbiochem). Soluble Escherichia coli proteins were subjected to ionic exchange chromatography on a 1.5 ϫ 5.0-cm column Mono Q (GE Healthcare) equilibrated at 20 °C with 20 mM Tris-HCl, pH 8.0, and eluted with a salt gradient (20 ml, 0 – 0.2 M NaCl in equilibration buffer). Two data sets were collected on a single crystal: one at low energy (7.8 keV, 1.589 Å) for phasing purposes and one at high energy (12.6 keV, 0.979 Å) for refinement. The initial model was manually completed and refined with the high energy data using COOT [31] and REFMAC5 [32], respectively. This procedure allowed the identification of three calcium ions and 176 water molecules in the asymmetric unit. Molecular graphics were displayed with MOLMOL [33] and PyMOL
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
