Design of activated serine-containing catalytic triads with atomic-level accuracy.

A challenge in the computational design of enzymes is that multiple properties, including substrate binding, transition state stabilization and product release, must be simultaneously optimized, and this has limited the absolute activity of successful designs. Here, we focus on a single critical property of many enzymes: the nucleophilicity of an active site residue that initiates catalysis. We design proteins with idealized serine-containing catalytic triads and assess their nucleophilicity directly in native biological systems using activity-based organophosphate probes. Crystal structures of the most successful designs show unprecedented agreement with computational models, including extensive hydrogen bonding networks between the catalytic triad (or quartet) residues, and mutagenesis experiments demonstrate that these networks are critical for serine activation and organophosphate reactivity. Following optimization by yeast display, the designs react with organophosphate probes at rates comparable to natural serine hydrolases. Co-crystal structures with diisopropyl fluorophosphate bound to the serine nucleophile suggest that the designs could provide the basis for a new class of organophosphate capture agents.

Amyloid ß peptide cleavage by kallikrein 7 attenuates fibril growth and rescues neurons from Aß-mediated toxicity in vitro.

The gradual accumulation and assembly of ß-amyloid (Aß) peptide into neuritic plaques is a major pathological hallmark of Alzheimer disease (AD). Proteolytic degradation of Aß is an important clearance mechanism under normal circumstances, and it has been found to be compromised in those with AD. Here, the extended substrate specificity and Aß-degrading capacity of kallikrein 7 (KLK7), a serine protease with a unique chymotrypsin-like specificity, was characterized. Preferred peptide substrates of KLK7 identified using a bacterial display substrate library were found to exhibit a consensus motif of RXΦ(Y/F)↓(Y/F)↓(S/A/G/T) or RXΦ(Y/F)↓(S/T/A) (Φ=hydrophobic), which is remarkably similar to the hydrophobic core motif of Aß (K16L17V18F19F20 A21) that is largely responsible for aggregation propensity. KLK7 was found to cleave after both Phe residues within the core of Aß42 in vitro, thereby inhibiting Aß fibril formation and promoting the degradation of preformed fibrils. Finally, the treatment of Aß oligomer preparations with KLK7, but not inactive pro-KLK7, significantly reduced Aß42-mediated toxicity to rat hippocampal neurons to the same extent as the known Aß-degrading protease insulin-degrading enzyme (IDE). Taken together, these results indicate that KLK7 possesses an Aß-degrading capacity that can ameliorate the toxic effects of the aggregated peptide in vitro.

Electron microscopy studies on the quaternary structure of p53 reveal different binding modes for p53 tetramers in complex with DNA.

The multidomain homotetrameric tumor suppressor p53 has two modes of binding dsDNA that are thought to be responsible for scanning and recognizing specific response elements (REs). The C termini bind nonspecifically to dsDNA. The four DNA-binding domains (DBDs) bind REs that have two symmetric 10 base-pair sequences. p53 bound to a 20-bp RE has the DBDs enveloping the DNA, which is in the center of the molecule surrounded by linker sequences to the tetramerization domain (Tet). We investigated by electron microscopy structures of p53 bound to DNA sequences consisting of a 20-bp RE with either 12 or 20 bp nonspecific extensions on either end. We found a variety of structures that give clues to recognition and scanning mechanisms. The 44- and 60-bp sequences gave rise to three and four classes of structures, respectively. One was similar to the known 20-bp structure, but the DBDs in the other classes were loosely arranged and incompatible with specific DNA recognition. Some of the complexes had density consistent with the C termini extending from Tet to the DNA, adjacent to the DBDs. Single-molecule fluorescence resonance energy transfer experiments detected the approach of the C termini towards the DBDs on addition of DNA. The structural data are consistent with p53 sliding along DNA via its C termini and the DNA-binding domains hopping on and off during searches for REs. The loose structures and posttranslational modifications account for the affinity of nonspecific DNA for p53 and point to a mechanism of enhancement of specificity by its binding to effector proteins.

Single-Molecule characterization of oligomerization kinetics and equilibria of the tumor suppressor p53.

The state of oligomerization of the tumor suppressor p53 is an important factor in its various biological functions. It has a well-defined tetramerization domain, and the protein exists as monomers, dimers and tetramers in equilibrium. The dissociation constants between oligomeric forms are so low that they are at the limits of measurement by conventional methods in vitro. Here, we have used the high sensitivity of single-molecule methods to measure the equilibria and kinetics of oligomerization of full-length p53 and its isolated tetramerization domain, p53tet, at physiological temperature, pH and ionic strength using fluorescence correlation spectroscopy (FCS) in vitro. The dissociation constant at 37 °C for tetramers dissociating into dimers for full-length p53 was 50 ± 7 nM, and the corresponding value for dimers into monomers was 0.55 ± 0.08 nM. The half-lives for the two processes were 20 and 50 min, respectively. The equivalent quantities for p53tet were 150 ± 10 nM, 1.0 ± 0.14 nM, 2.5 ± 0.4 min and 13 ± 2 min. The data suggest that unligated p53 in unstressed cells should be predominantly dimeric. Single-molecule FCS is a useful procedure for measuring dissociation equilibria, kinetics and aggregation at extreme sensitivity.

Mapping the physical and functional interactions between the tumor suppressors p53 and BRCA2.

p53 maintains genome integrity either by regulating the transcription of genes involved in cell cycle, apoptosis, and DNA repair or by interacting with partner proteins. Here we provide evidence for a direct physical interaction between the tumor suppressors p53 and BRCA2. We found that the transactivation domain of p53 made specific interactions with the C-terminal oligonucleotide/oligosaccharide-binding-fold domains of BRCA2 (BRCA2(CTD)). A second distinct site situated on the p53 DNA-binding domain, bound to a region containing BRC repeats of BRCA2 (BRCA2([BRC1-8])) and may contribute synergistically for high affinity association of intact full-length proteins. Overexpression of BRCA2 and BRCA2(CTD) suppressed the transcriptional activity of p53 with a concomitant reduction in the expression of p53-target genes such as Bax and p21. Consequently, p53-mediated apoptosis was significantly attenuated by BRCA2. The observed physical association of p53 and BRCA2 may have important functional implications in the p53 transactivation-independent suppression of homologous recombination and suggests a possible interregulatory role for both proteins in apoptosis and DNA repair.

Mechanistic differences in the transcriptional activation of p53 by 14-3-3 isoforms.

p53 maintains genome integrity by initiating the transcription of genes involved in cell-cycle arrest, senescence, apoptosis and DNA repair. The activity of p53 is regulated by both post-translational modifications and protein-protein interactions. p53 that has been phosphorylated at S366, S378 and T387 binds 14-3-3 proteins in vitro. Here, we show that these sites are potential 14-3-3 binding sites in vivo. Epsilon (epsilon) and gamma (gamma) isoforms required phosphorylation at either of these sites for efficient interaction with p53, while for sigma (sigma) and tau (tau) these sites are dispensable. Further, sigma and tau bound more weakly to p53 C-terminal phosphopeptides than did epsilon and gamma. However, the four isoforms bound tightly to di-phosphorylated p53 C-terminal peptides than did the mono-phosphorylated counterparts. Interestingly, all the isoforms studied transcriptionally activated wild-type p53. sigma and tau stabilized p53 levels in cells, while epsilon and gamma stimulated p53-DNA binding activity in vitro. Overall, the results suggest that structurally and functionally similar 14-3-3 isoforms may exert their regulatory potential on p53 through different mechanisms. We discuss the isoform-specific roles of 14-3-3 in p53 stabilization and activation of specific-DNA binding.

Structural evolution of p53, p63, and p73: implication for heterotetramer formation.

Oligomerization of members of the p53 family of transcription factors (p53, p63, and p73) is essential for their distinct functions in cell-cycle control and development. To elucidate the molecular basis for tetramer formation of the various family members, we solved the crystal structure of the human p73 tetramerization domain (residues 351-399). Similarly to the canonical p53 tetramer, p73 forms a tetramer with D(2) symmetry that can be described as a dimer of dimers. The most striking difference between the p53 and p73 tetramerization domain is the presence of an additional C-terminal helix in p73. This helix, which is conserved in p63, is essential for stabilizing the overall architecture of the tetramer, as evidenced by the different oligomeric structures observed for a shortened variant lacking this helix. The helices act as clamps, wrapping around the neighboring dimer and holding it in place. In addition, we show by mass spectrometry that the tetramerization domains of p63 and p73, but not p53, fully exchange, with different mixed tetramers present at equilibrium, albeit at a relatively slow rate. Taken together, these data provide intriguing insights into the divergent evolution of the oligomerization domain within the p53 family, from the ancestral p63/p73-like protein toward smaller, less promiscuous monomeric building blocks in human p53, allowing functional separation of the p53 pathway from that of its family members.

Multiple conformations of full-length p53 detected with single-molecule fluorescence resonance energy transfer.

The tumor suppressor p53 is a member of the emerging class of proteins that have both folded and intrinsically disordered domains, which are a challenge to structural biology. Its N-terminal domain (NTD) is linked to a folded core domain, which has a disordered link to the folded tetramerization domain, which is followed by a disordered C-terminal domain. The quaternary structure of human p53 has been solved by a combination of NMR spectroscopy, electron microscopy, and small-angle X-ray scattering (SAXS), and the NTD ensemble structure has been solved by NMR and SAXS. The murine p53 is reported to have a different quaternary structure, with the N and C termini interacting. Here, we used single-molecule FRET (SM-FRET) and ensemble FRET to investigate the conformational dynamics of the NTD of p53 in isolation and in the context of tetrameric full-length p53 (flp53). Our results showed that the isolated NTD was extended in solution with a strong preference for residues 66-86 forming a polyproline II conformation. The NTD associated weakly with the DNA binding domain of p53, but not the C termini. We detected multiple conformations in flp53 that were likely to result from the interactions of NTD with the DNA binding domain of each monomeric p53. Overall, the SM-FRET results, in addition to corroborating the previous ensemble findings, enabled the identification of the existence of multiple conformations of p53, which are often averaged and neglected in conventional ensemble techniques. Our study exemplifies the usefulness of SM-FRET in exploring the dynamic landscape of multimeric proteins that contain regions of unstructured domains.

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.

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.

14-3-3 activation of DNA binding of p53 by enhancing its association into tetramers.

Activation of the tumour suppressor p53 on DNA damage involves post-translational modification by phosphorylation and acetylation. Phosphorylation of certain residues is critical for p53 stabilization and plays an important role in DNA-binding activity. The 14-3-3 family of proteins activates the DNA-binding affinity of p53 upon stress by binding to a site in its intrinsically disordered C-terminal domain containing a phosphorylated serine at 378. We have screened various p53 C-terminal phosphorylated peptides for binding to two different isoforms of 14-3-3, epsilon and gamma. We found that phosphorylation at either S366 or T387 caused even tighter binding to 14-3-3. We made by semi-synthesis a tetrameric construct comprised of the tetramerization plus C-terminal domains of p53 that was phosphorylated on S366, S378 and T387. It bound 10 times tighter than did the monomeric counterpart to dimeric 14-3-3. We showed indirectly from binding curves and directly from fluorescence-detection analytical ultracentrifugation that 14-3-3 enhanced the binding of sequence-specific DNA to p53 by causing p53 dimers to form tetramers at lower concentrations. If the in vitro data extrapolate to in vivo, then it is an attractive hypothesis that p53 activity may be subject to control by accessory proteins lowering its tetramer-dimer dissociation constant from its normal value of 120-150 nM.

Interaction between the transactivation domain of p53 and PC4 exemplifies acidic activation domains as single-stranded DNA mimics.

The tumor suppressor p53 regulates cell cycle arrest and apoptosis by transactivating several genes that are critical for these processes. The transcriptional activity of p53 is often regulated by post-translational modifications and its interactions with various transcriptional coactivators. Here we report a physical interaction between the N-terminal transactivation domain (TAD) of p53 and the C-terminal DNA-binding domain of positive cofactor 4 (PC4(CTD)). Using NMR spectroscopy, we showed that residues 35-57 (TAD2) interact with PC4. (15)N,(1)H HSQC and fluorescence competition experiments indicated that TAD binds to the DNA-binding site of PC4. Hepta-phosphorylation of the TAD peptide increased its binding affinity. Computer modeling of the p53N-PC4 complex revealed several important interactions that are reminiscent of those in the single-stranded DNA-PC4 complex. The ubiquitous nature of the acidic transactivation domain of p53 in mediating interactions with several transcription cofactors is also manifested as a DNA mimetic.