Hansen S, Lane D P, Midgley C A
Department of Biochemistry, University of Dundee, Scotland, UK.
J Mol Biol. 1998 Jan 30;275(4):575-88. doi: 10.1006/jmbi.1997.1507.
The contribution of each of the structural domains of p53 to its function has been discussed widely in the literature. Crystallographic studies have revealed much about the structure of the core DNA binding domain, but as it has not been possible to use this approach for the intact protein, the effect of the domains flanking the core must be investigated by more indirect techniques. In this study a series of truncated murine p53 proteins has been investigated for DNA binding activity at 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 latency for DNA binding; that is, they must be activated to bind by association 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 recognising the wild-type conformation of the core domain) in the presence of cross-linking antibodies, while murine core only binds to DNA in the presence of PAb1620. An analysis of the thermostability of DNA binding revealed that antibodies that bind the N terminus of p53 could protect 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 structure, since it is the main contributor to the anomalous migration in SDS-polyacrylamide gels. Our results suggest that the N terminus has a destabilising effect that influences conformation of p53 at 37 degrees C, so cellular proteins binding to the N terminus in vivo may modulate p53 conformation and stability. The effects on thermostability are also direct evidence showing that antibodies binding to N-terminal deletions create a conformational change in the rest of the molecule. In addition, longer deletions of the C terminus reduce the ability of p53 to transactivate target genes and inactivate tumour suppression activity, while truncations of the N terminus retain partial tumour suppression activity. Our results clearly show participation of both the N and C termini in the regulation of all the functions of p53 at 37 degrees C, indicating that distinct, independent domains interact with each other within, the flexible structure of p53.
p53各结构域对其功能的贡献在文献中已有广泛讨论。晶体学研究揭示了许多关于核心DNA结合结构域的结构信息，但由于无法将这种方法用于完整蛋白质，因此必须通过更间接的技术来研究核心结构域两侧结构域的作用。在本研究中，对一系列截短的小鼠p53蛋白进行了研究，检测它们在4℃和37℃时的DNA结合活性、转录激活能力以及肿瘤抑制活性。从杆状病毒表达系统中纯化得到的全长p53以及缺失N端的截短体，在DNA结合方面均表现出潜伏性；也就是说，它们必须通过与C端抗体（如PAb421）结合才能被激活从而进行结合。这表明DNA结合的潜伏性与N端无关。缺失C端寡聚化结构域的截短体以及分离出的核心结构域，只有在存在交联抗体的情况下才能被激活以结合DNA和PAb1620（一种识别核心结构域野生型构象的抗体），而小鼠核心结构域只有在存在PAb1620时才能与DNA结合。对DNA结合热稳定性的分析表明，结合p53 N端的抗体可以保护该蛋白在37℃时不丧失活性。然而，C端抗体则无效，除非缺失N端的37个氨基酸残基。N端可能保留了一些二级结构，因为它是导致在SDS - 聚丙烯酰胺凝胶中出现异常迁移的主要因素。我们的结果表明，N端具有一种去稳定化作用，会影响p53在37℃时的构象，因此体内与N端结合的细胞蛋白可能会调节p53的构象和稳定性。对热稳定性的影响也是直接证据，表明结合N端缺失部分的抗体在分子的其余部分产生了构象变化。此外，C端更长的缺失会降低p53激活靶基因的能力并使肿瘤抑制活性失活，而N端的截短体则保留部分肿瘤抑制活性。我们的结果清楚地表明，N端和C端都参与了p53在37℃时所有功能的调节，这表明在p53的柔性结构内，不同的独立结构域相互作用。
