THE N-TERMINUS OF THE MURINE P53 TUMOR-SUPPRESSOR IS AN INDEPENDENT REGULATORY DOMAIN AFFECTING ACTIVATION AND THERMOSTABILITY

The contribution of each of the structural domains of p53 to its funct ion has been discussed widely in the literature. Crystallographic stud ies have revealed much about the structure of the core DNA binding dom ain, but as it has not been possible to use this approach for the inta ct protein, the effect of the domains flanking the core must be invest igated by more indirect techniques. Ln this study a series of truncate d murine p53 proteins has been investigated for DNA binding activity a t 4 degrees C and 37 degrees C, transcriptional activation, and tumour suppression activity. Full-length p53, and truncations lacking the N terminus, purified from a baculovirus expression system all show laten cy for DNA binding; that is, they must be activated to bind by associa tion with a C-terminal antibody such as PAb421. This demonstrates that latency for DNA binding is independent of the N terminus. Truncations lacking the C-terminal oligomerisation domain, and the isolated core domain, can only be activated to bind DNA and PAb1620 (an antibody rec ognising the wild-type conformation of the core domain) in the presenc e of cross-linking antibodies, while murine core only binds to DNA in the presence of PAb1620. An analysis of the thermostability of DNA bin ding revealed that antibodies that bind the N terminus of p53 could pr otect the protein against loss of activity at 37 degrees C. C-terminal antibodies, however, were ineffective unless the N-terminal 37 amino acid residues were absent. The N terminus may retain some secondary st ructure, since it is the main contributor to the anomalous migration i n SDS-polyacrylamide gels. Our results suggest that the N terminus has a destabilising effect that influences conformation of p53 at 37 degr ees C, so cellular proteins binding to the N terminus in viva may modu late p53 conformation and stability. The effects on thermostability ar e also direct evidence showing that antibodies binding to N-terminal d eletions create a conformational change in the rest of the molecule. i n addition, longer deletions of the C terminus reduce the ability of p 53 to transactivate target genes and inactivate tumour suppression act ivity, while truncations of the N terminus retain partial tumour suppr ession activity. Our results clearly show participation of both the N and C termini in the regulation of all the functions of p53 at 37 degr ees C, indicating that distinct, independent domains interact with eac h other within the flexible structure of p53. (C) 1998 Academic Press Limited.