The Recombinant Inhibitor of DNA Binding Id2 Forms Multimeric Structures via the Helix-Loop-Helix Domain and the Nuclear Export Signal

Abstract

The inhibitor of DNA binding and cell differentiation 2 (Id2) is a helix-loop-helix (HLH) protein that acts as negative dominant regulator of basic-HLH transcription factors during development and in cancer. The structural properties of Id2 have been investigated so far by using synthetic or recombinant fragments reproducing single domains (N-terminus, HLH, C-terminus): the HLH domain tends to dimerize into a four-helix bundle, whereas the flanking regions are flexible. In this work, the intact protein was expressed in E. coli, solubilized from inclusion bodies with urea, purified and dissolved in water at pH~4. Under these conditions, Id2 was obtained with both cysteine residues disulfide-bonded to β-mercaptoethanol that was present during the solubilization process. Moreover, it existed in a self-assembled state, in which the N-terminus remained highly flexible, while the HLH domain and, surprisingly, part of the C-terminus, which corresponds to the nuclear export signal (NES), both were involved in slowly tumbling, rigid structures. The protein oligomers also formed twisted fibrils that were several micrometers long and up to 80 nm thick. These results show that self-assembly decreases the backbone flexibility of those two protein regions (HLH and NES) that are important for interaction with basic-HLH transcription factors or for nucleocytoplasmic shuttling.

Inhibitor of Differentiation-2 Protein Ameliorates DSS-Induced Ulcerative Colitis by Inhibiting NF-κB Activation in Neutrophils

“…The results showed that FITC-hID2 could be effectively phagocytosed by neutrophils and macrophages, especially by neutrophils. It was also reported that the ID2 protein could form a dimer (57), which was consistent with our expressed recombinant hID2, and the ID2 protein in cells could shuttle from the nucleus to the cytoplasm (58), but its detailed cytoplasmic function is not yet clear. Neutrophils are important for controlling colitis, and recent findings suggest that neutrophils are the major contributor to the development of UC (59); therefore, we sought to investigate the relationship between hID2 and neutrophils.…”

bHLH–PAS Proteins: Their Structure and Intrinsic Disorder

“…As all the in vitro analyses results obtained for the MET/C [75] were consistent with the in silico studies presented above (Figure 5C), we hypothesize that the disorder character of the bHLH–PAS proteins subfamily C-terminal fragments can be a more common characteristic and also be very important for their functionality. Previously, the importance of the disordered character of regions flanking the bHLH domain of bHLH transcription factors was shown [77,78,79].…”