Cyclic olefin homopolymer-based microfluidics for protein crystallization and in situ X-ray diffraction

A cyclic olefin homopolymer-based microfluidics system has been established for protein crystallization and in situ X-ray diffraction

A conformational switch regulates p53 DNA binding

The p53 tumour suppressor gene encodes for a transcription factor that encompasses a sequence-specific DNA-binding and an oligomerization domains. This gene is the most frequently mutated gene in human cancers. It remains unclear how p53 recognizes its DNA target sites on the genome. We describe here the structures of p53 in absence and in complex with DNA. Structure and kinetic analyses of p53-DNA complexes reveal that p53 recognizes its specific DNA target sites via an induced fit mechanism. The affinities of p53 for specific and non-specific DNA are similar, but the off-rates are much different. The loop L1 of the DNA-binding domain of p53 undergoes a conformational switch. Overall, these results reveal the importance of the conformational switch of loop L1 in the DNA binding properties of p53. We also describe here the first structure of p53 in complex with the natural CDKN1A (p21) p53-response element

Crystal Structure of a Multidomain Human p53 Tetramer Bound to the Natural <i>CDKN1A</i> (<i>p21</i>) p53-Response Element

The p53 tumor suppressor protein is a sequence-specific DNA-binding transcription factor. Structures of p53 bound to DNA have been described, but, so far, no structure has been determined of p53 bound to a natural p53-response element. We describe here the structure of a human p53 homotetramer encompassing both the DNA-binding and homo-oligomerization domains in complex with the natural p53-response element present upstream of the promoter of the CDKN1A (p21) gene. Similar to our previously described structures of human p53 tetramers bound to an artificial consensus DNA site, p53 DNA binding proceeds via an induced fit mechanism with loops L1 of two subunits adopting recessed conformations. Interestingly, the conformational change involving loop L1 is even more extreme than the one previously observed with the artificial consensus DNA site. In fact, the previously determined loop L1 conformation seems to be a transition intermediate between the non–DNA-bound and CDKN1A-bound states. Thus, the new structure further supports our model that recognition of specific DNA by p53 is associated with conformational changes within the DNA-binding domain of p53. Mol Cancer Res; 9(11); 1493–9. ©2011 AACR.</p

Reversal of the DNA-Binding-Induced Loop L1 Conformational Switch in an Engineered Human p53 Protein

The gene encoding the p53 tumor suppressor protein, a sequence-specific DNA binding transcription factor, is the most frequently mutated gene in human cancer. Crystal structures of homo-oligomerizing p53 polypeptides with specific DNA suggest that DNA binding is associated with a conformational switch. Specifically, in the absence of DNA, loop L1 of the p53 DNA binding domain adopts an extended conformation, whereas two p53 subunits switch to a recessed loop L1 conformation when bound to DNA as a tetramer. We previously designed a p53 protein, p53FG, with amino substitutions S121F and V122G targeting loop L1. These two substitutions enhanced the affinity of p53 for specific DNA yet, counterintuitively, decreased the residency time of p53 on DNA. Here, we confirmed these DNA binding properties of p53FG using a different method. We also determined by crystallography the structure of p53FG in its free state and bound to DNA as a tetramer. In the free state, loop L1 adopted a recessed conformation, whereas upon DNA binding, two subunits switched to the extended loop L1 conformation, resulting in a final structure that was very similar to that of wild-type p53 bound to DNA. Thus, altering the apo structure of p53 changed its DNA binding properties, even though the DNA-bound structure was not altered.</p

Expression, crystallization and preliminary X-ray diffraction analysis of the CMM2 region of the Arabidopsis thaliana Morpheus' molecule 1 protein

Of the known epigenetic control regulators found in plants, the Morpheus' molecule 1 (MOM1) protein is atypical in that the deletion of MOM1 does not affect the level of epigenetic marks controlling the transcriptional status of the genome. A short 197-amino-acid fragment of the MOM1 protein sequence can complement MOM1 deletion when coupled to a nuclear localization signal, suggesting that this region contains a functional domain that compensates for the loss of the full-length protein. Numerous constructs centred on the highly conserved MOM1 motif 2 (CMM2) present in these 197 residues have been generated and expressed in Escherichia coli. Following purification and crystallization screening, diamond-shaped single crystals were obtained that diffracted to approximately 3.2 A resolution. They belonged to the trigonal space group P3(1)21 (or P3(2)21), with unit-cell parameters a=85.64, c=292.74 A. Structure determination is ongoing

An induced fit mechanism regulates p53 DNA binding kinetics to confer sequence specificity

This crystal structure of the p53 DNA binding and homo-tetramerization domains identifies a conformational change involved in an induced fit mechanism of binding to DNA and demonstrates that binding specificity is determined not by affinity but by the dissociation rate of the transcription factor