MDMX contains an autoinhibitory sequence element.

MDM2 and MDMX are homologous proteins that bind to p53 and regulate its activity. Both contain three folded domains and ~70% intrinsically disordered regions. Previous detailed structural and biophysical studies have concentrated on the isolated folded domains. The N-terminal domains of both exhibit high affinity for the disordered N-terminal of p53 (p53TAD) and inhibit its transactivation function. Here, we have studied full-length MDMX and found a ~100-fold weaker affinity for p53TAD than does its isolated N-terminal domain. We found from NMR spectroscopy and binding studies that MDMX (but not MDM2) contains a conserved, disordered self-inhibitory element that competes intramolecularly for binding with p53TAD. This motif, which we call the WWW element, is centered around residues Trp200 and Trp201. Deletion or mutation of the element increased binding affinity of MDMX to that of the isolated N-terminal domain level. The self-inhibition of MDMX implies a regulatory, allosteric mechanism of its activity. MDMX rests in a latent state in which its binding activity with p53TAD is masked by autoinhibition. Activation of MDMX would require binding to a regulatory protein. The inhibitory function of the WWW element may explain the oncogenic effects of an alternative splicing variant of MDMX that does not contain the WWW element and is found in some aggressive cancers.

Structures of SAS-6 suggest its organization in centrioles.

Centrioles are cylindrical, ninefold symmetrical structures with peripheral triplet microtubules strictly required to template cilia and flagella. The highly conserved protein SAS-6 constitutes the center of the cartwheel assembly that scaffolds centrioles early in their biogenesis. We determined the x-ray structure of the amino-terminal domain of SAS-6 from zebrafish, and we show that recombinant SAS-6 self-associates in vitro into assemblies that resemble cartwheel centers. Point mutations are consistent with the notion that centriole formation in vivo depends on the interactions that define the self-assemblies observed here. Thus, these interactions are probably essential to the structural organization of cartwheel centers.

Conservation of DNA-binding specificity and oligomerisation properties within the p53 family.

BACKGROUND: Transcription factors activate their target genes by binding to specific response elements. Many transcription factor families evolved from a common ancestor by gene duplication and subsequent divergent evolution. Members of the p53 family, which play key roles in cell-cycle control and development, share conserved DNA binding and oligomerisation domains but exhibit distinct functions. In this study, the molecular basis of the functional divergence of related transcription factors was investigated. RESULTS: We characterised the DNA-binding specificity and oligomerisation properties of human p53, p63 and p73, as well as p53 from other organisms using novel biophysical approaches. All p53 family members bound DNA cooperatively as tetramers with high affinity. Despite structural differences in the oligomerisation domain, the dissociation constants of the tetramers was in the low nanomolar range for all family members, indicating that the strength of tetramerisation was evolutionarily conserved. However, small differences in the oligomerisation properties were observed, which may play a regulatory role. Intriguingly, the DNA-binding specificity of p53 family members was highly conserved even for evolutionarily distant species. Additionally, DNA recognition was only weakly affected by CpG methylation. Prediction of p53/p63/p73 binding sites in the genome showed almost complete overlap between the different homologs. CONCLUSION: Diversity of biological function of p53 family members is not reflected in differences in sequence-specific DNA binding. Hence, additional specificity factors must exist, which allowed the acquisition of novel functions during evolution while preserving original roles.

Biophysical characterizations of human mitochondrial transcription factor A and its binding to tumor suppressor p53.

Human mitochondrial transcription factor A (TFAM) is a multi-functional protein, involved in different aspects of maintaining mitochondrial genome integrity. In this report, we characterized TFAM and its interaction with tumor suppressor p53 using various biophysical methods. DNA-free TFAM is a thermally unstable protein that is in equilibrium between monomers and dimers. Self-association of TFAM is modulated by its basic C-terminal tail. The DNA-binding ability of TFAM is mainly contributed by its first HMG-box, while the second HMG-box has low-DNA-binding capability. We also obtained backbone resonance assignments from the NMR spectra of both HMG-boxes of TFAM. TFAM binds primarily to the N-terminal transactivation domain of p53, with a K(d) of 1.95 +/- 0.19 microM. The C-terminal regulatory domain of p53 provides a secondary binding site for TFAM. The TFAM-p53-binding interface involves both TAD1 and TAD2 sub-domains of p53. Helices alpha1 and alpha2 of the HMG-box constitute the main p53-binding region. Since both TFAM and p53 binds preferentially to distorted DNA, the TFAM-p53 interaction is implicated in DNA damage and repair. In addition, the DNA-binding mechanism of TFAM and biological relevance of the TFAM-p53 interaction are discussed.

Physical and functional interactions between human mitochondrial single-stranded DNA-binding protein and tumour suppressor p53.

Single-stranded DNA-binding proteins (SSB) form a class of proteins that bind preferentially single-stranded DNA with high affinity. They are involved in DNA metabolism in all organisms and serve a vital role in replication, recombination and repair of DNA. In this report, we identify human mitochondrial SSB (HmtSSB) as a novel protein-binding partner of tumour suppressor p53, in mitochondria. It binds to the transactivation domain (residues 1-61) of p53 via an extended binding interface, with dissociation constant of 12.7 (+/- 0.7) microM. Unlike most binding partners reported to date, HmtSSB interacts with both TAD1 (residues 1-40) and TAD2 (residues 41-61) subdomains of p53. HmtSSB enhances intrinsic 3'-5' exonuclease activity of p53, particularly in hydrolysing 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) present at 3'-end of DNA. Taken together, our data suggest that p53 is involved in DNA repair within mitochondria during oxidative stress. In addition, we characterize HmtSSB binding to ssDNA and p53 N-terminal domain using various biophysical measurements and we propose binding models for both.

Effects of CpG methylation on recognition of DNA by the tumour suppressor p53.

Methylation of DNA is one of the mechanisms controlling the expression landscape of the genome. Its pattern is altered in cancer and often results in the hypermethylation of the promoter regions and abnormal expression of tumour suppressor genes. Methylation of CpG dinucleotides located in the binding sites of transcription factors may contribute to the development of cancers by preventing their binding or altering their specificity. We studied the effects of CpG methylation on DNA recognition by the tumour suppressor p53, a transcription factor involved in the response to carcinogenic stress. p53 recognises a large number of DNA sequences, many of which contain CpG dinucleotides. We systematically substituted a CpG dinucleotide at each position in the consensus p53 DNA binding sequence and identified substitutions tolerated by p53. We compared the binding affinities of methylated versus non-methylated sequences by fluorescence anisotropy titration. We found that binding of p53 was not affected by cytosine methylation in a majority of cases. However, for a few sequences containing multiple CpG dinucleotides, such as sites in the RB and Met genes, methylation resulted in a four- to sixfold increase in binding of p53. This approach can be used to quantify the effects of CpG methylation on the DNA recognition by other DNA-binding proteins.

General structural motifs of amyloid protofilaments.

Human CA150, a transcriptional activator, binds to and is co-deposited with huntingtin during Huntington's disease. The second WW domain of CA150 is a three-stranded beta-sheet that folds in vitro in microseconds and forms amyloid fibers under physiological conditions. We found from exhaustive alanine scanning studies that fibrillation of this WW domain begins from its denatured conformations, and we identified a subset of residues critical for fibril formation. We used high-resolution magic-angle-spinning NMR studies on site-specific isotopically labeled fibrils to identify abundant long-range interactions between side chains. The distribution of critical residues identified by the alanine scanning and NMR spectroscopy, along with the electron microscopy data, revealed the protofilament repeat unit: a 26-residue non-native beta-hairpin. The structure we report has similarities to the hairpin formed by the A(beta)((1-40)) protofilament, yet also contains closely packed side-chains in a "steric zipper" arrangement found in the cross-beta spine formed from small peptides from the Sup35 prion protein. Fibrillation of unrelated amyloidogenic sequences shows the common feature of zippered repeat units that act as templates for fiber elongation.

Phi-analysis at the experimental limits: mechanism of beta-hairpin formation.

The 37-residue Formin-binding protein, FBP28, is a canonical three-stranded beta-sheet WW domain. Because of its small size, it is so insensitive to chemical denaturation that it is barely possible to determine accurately a denaturation curve, as the transition spans 0-7 M guanidinium hydrochloride (GdmCl). It is also only marginally stable, with a free energy of denaturation of just 2.3 kcal/mol at 10 degrees Celsius so only small changes in energy upon mutation can be tolerated. But these properties and relaxation times for folding of 25 micros-400 micros conspire to allow the rapid acquisition of accurate and reproducible kinetic data for Phi-analysis using classical temperature-jump methods. The transition state for folding is highly polarized with some regions having Phi-values of 0 and others 1, as readily seen in chevron plots, with Phi-values of 0 having the refolding arms overlaying and those of 1 the unfolding arms superimposable. Good agreement is seen with transition state structures identified from independent molecular dynamics (MD) simulations at 60, 75, and 100 degrees Celsius, which allows us to explore further the details of the folding and unfolding pathway of FBP28. The first beta-turn is near native-like in the transition state for folding (experimental) and unfolding (MD and experiment). The simulations show that there are transient contacts between the aromatic side-chains of the beta-strands in the denatured state and that these interactions provide the driving force for folding of the first beta-hairpin of this three-stranded sheet. Only after the backbone hydrogen bonds are formed between beta1 and beta2 does a hydrogen bond form to stabilize the intervening turn, or the first beta-turn.

Rapid amyloid fiber formation from the fast-folding WW domain FBP28.

The WW domains are small proteins that contain a three-stranded, antiparallel beta-sheet. The 40-residue murine FBP28 WW domain rapidly formed twirling ribbon-like fibrils at physiological temperature and pH, with morphology typical of amyloid fibrils. These ribbons were unusually wide and well ordered, making them highly suitable for structural studies. Their x-ray and electron-diffraction patterns displayed the characteristic amyloid fiber 0.47-nm reflection of the cross-beta diffraction signature. Both conventional and electron cryomicroscopy showed clearly that the ribbons were composed of many 2.5-nm-wide subfilaments that ran parallel to the long axis of the fiber. There was a region of lower density along the center of each filament. Lateral association of these filaments generated twisted, often interlinked, sheets up to 40 nm wide and many microns in length. The pitch of the helix varied from 60 to 320 nm, depending on the width of the ribbon. The wild-type FBP28 fibers were formed under conditions in which multiexponential folding kinetics is observed in other studies and which was attributed to a change in the mechanism of folding. It is more likely that those phases result from initial events in the off-pathway aggregation observed here.