Journal of Molecular Biology
Solution Structure of ASPP2 N-terminal Domain (N-ASPP2) Reveals a Ubiquitin-like Fold
Introduction
The tumour suppressor p53 is a tetrameric multidomain transcription factor that has several complex functions in the cell. In response to oncogenic stresses, p53-mediated transcriptional activation induces cell cycle arrest or apoptosis.1., 2., 3., 4.
It is still poorly understood how cells decide between cell cycle arrest and apoptosis during p53 response. Important factors seem to be cell type, degree of DNA damage, and the presence of survival factors, but the exact mechanism remains unknown.5., 6. Recently, the ASPP protein family was identified that specifically regulates p53-mediated apoptosis but not cell cycle arrest.7., 8., 9. The family comprises two members, ASPP1 and ASPP2, which are pro-apoptotic9., 10., 11., 12., 13. and one member, iASPP, which is anti-apoptotic.14., 15., 16. However, the molecular basis for their different responses upon p53 activation is currently unknown.
The domain organization of ASPP proteins consists of an N-terminal domain, which is unique to ASPP1 and ASPP2 and was described as α-helical,9 a predicted coiled-coil domain,17 and a proline-rich region followed by ankyrin repeats and an SH3 domain.18 The latter C-terminal domain is highly conserved among all ASPP family members.9., 15. Several interaction partners including p53, BCL2, protein phosphatase 1, and RELA/p65 bind to this region in vivo and in vitro.11., 18., 19., 20., 21., 22.
The N-terminus is only conserved in the pro-apoptotic members, ASPP1 and ASPP2, being absent from anti-apoptotic iASPP. In the case of ASPP2 the N-terminus is subject to alternative splicing. The shorter (and less frequent occurring) splice variant is lacking the N-terminal 123 residues and is less able to enhance the transcriptional and apoptotic functions of p53.12 Interestingly, the N-terminus also seems to play an important role for its cellular localization. While full-length ASPP1 and ASPP2 are predominantly cytoplasmic, removal of the N-terminal half of ASPP2 causes its C-terminal part, which contains a nuclear localization signal,23 to localize in the nucleus.12
In addition to its role in cellular localization, the N-terminus of ASPP proteins seems to be important for the transactivation function of p53 with regard to pro-apoptotic response elements like Bax or PIG3.12 While no ternary complex consisting of p53, DNA and the C-terminal domain of ASPP2 (53BP2) exists that could explain the selective up-regulation of pro-apoptotic response elements,22 it was shown that full-length ASPP proteins are required for maximal transactivation activity.12 This emphasizes the importance of the N-terminus and suggests that its binding to other cellular proteins might be an important factor in the apoptotic response network.
To gain insight into the role of ASPP N-termini, we solved the solution structure of the N-terminal domain of ASPP2 by NMR and characterized its properties using biophysical techniques.
Section snippets
The N-terminus of ASPP2 (N-ASPP2) constitutes a folded domain with high stability
Bioinformatics analysis suggested the existence of a folded domain within the N-terminus of ASPP2. This region is conserved amongst ASPP2 orthologues present in vertebrate genomes and shows a very high similarity to the N-terminus of its paralogue ASPP1 (Figure 1). As the functional domain boundaries could not be predicted with certainty, we expressed two constructs of the conserved region, comprising residues 1–83 and 1–135, respectively. 1D-NMR spectra did not show additional signals in the
Molecular cloning
The genes encoding for amino acid residues 1–83 and 1–135, respectively, of human ASPP2 were extracted from a human cDNA library and subcloned into a pRSET derived plasmid containing an N-terminal fusion of 6xHis/lipoamyl domain/TEV protease cleavage site.
Protein expression and purification
N-ASPP2 and N-ASPP2 (1–135) were expressed in Escherichia coli BL21 at 25 °C for 4 h and purified using standard His-tag purification protocols followed by TEV protease digestion, a second Ni-affinity chromatography step to separate the
Acknowledgements
We thank Drs S. Freund, C. Johnson, D. Veprintsev, A. Joerger, M. Bycroft and T. Religa for valuable discussions and help on the experimental setup, and C. Blair for TEV protease. We also thank Drs R. Williams and O. Perisic for the kind gift of H-Ras protein. H.T. was supported by a fellowship from the Boehringer Ingelheim Fonds.
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