Correction: Energy-dependent nucleolar localization of p53 in vitro requires two discrete regions within the p53 carboxyl terminus.

Following the publication of this article the authors noted that two images were duplicated in Figure 2B. The corrected figure 2B is below. The authors wish to apologize for any inconvenience caused.

Molecular basis of S100 proteins interacting with the p53 homologs p63 and p73.

S100 proteins modulate p53 activity by interacting with its tetramerization (p53TET, residues 325-355) and transactivation (residues 1-57) domains. In this study, we characterized biophysically the binding of S100A1, S100A2, S100A4, S100A6 and S100B to homologous domains of p63 and p73 in vitro by fluorescence anisotropy, analytical ultracentrifugation and analytical gel filtration. We found that S100A1, S100A2, S100A4, S100A6 and S100B proteins bound different p63 and p73 tetramerization domain variants and naturally occurring isoforms with varying affinities in a calcium-dependent manner. Additional interactions were observed with peptides derived from the p63 and p73 N-terminal transactivation domains. Importantly, S100 proteins bound p63 and p73 with different affinities in their different oligomeric states, similarly to the differential modes of binding to p53. On the basis of our data, we hypothesize that S100 proteins regulate the oligomerization state of all three p53 family members and their isoforms, with a potential physiological relevance in developmental and disease-related processes. The regulation of the p53 family by S100 is complicated and depends on the target preference of each individual S100 protein, the concentration of the proteins and calcium, as well as the splicing variation of p63 or p73. Our results outlining the complexity of the interaction should be considered when studying the functional effects of S100 proteins in their biological context.

The accessory subunit of mitochondrial DNA polymerase gamma determines the DNA content of mitochondrial nucleoids in human cultured cells.

The accessory subunit of mitochondrial DNA polymerase gamma, POLGbeta, functions as a processivity factor in vitro. Here we show POLGbeta has additional roles in mitochondrial DNA metabolism. Mitochondrial DNA is arranged in nucleoprotein complexes, or nucleoids, which often contain multiple copies of the mitochondrial genome. Gene-silencing of POLGbeta increased nucleoid numbers, whereas over-expression of POLGbeta reduced the number and increased the size of mitochondrial nucleoids. Both increased and decreased expression of POLGbeta altered nucleoid structure and precipitated a marked decrease in 7S DNA molecules, which form short displacement-loops on mitochondrial DNA. Recombinant POLGbeta preferentially bound to plasmids with a short displacement-loop, in contrast to POLGalpha. These findings support the view that the mitochondrial D-loop acts as a protein recruitment centre, and suggest POLGbeta is a key factor in the organization of mitochondrial DNA in multigenomic nucleoprotein complexes.

Regulation by phosphorylation of the relative affinities of the N-terminal transactivation domains of p53 for p300 domains and Mdm2.

The transcriptional activity of the tumour suppressor, p53, requires direct binding between its transactivation domain (TAD, 1-57) and the transcriptional coactivator, p300. We systematically assessed the role of TAD phosphorylation on binding of the p300 domains CH3, Taz1, Kix and IBiD. Thr18 phosphorylation increased the affinity up to sevenfold for CH3 and Taz1, with smaller increases from phosphorylation of Ser20, Ser15, Ser37, Ser33, Ser46 and Thr55. Binding of Kix and IBiD was less sensitive to phosphorylation. Strikingly, hepta-phosphorylation of all Ser and Thr residues increased binding 40- and 80-fold with CH3 and Taz1, respectively, but not with Kix or IBiD. Substitution of all phospho-sites with aspartates partially mimicked the effects of hepta-phosphorylation. Mdm2, the main negative regulator of p53, competes with p300 for binding to TAD. Binding of Mdm2 to TAD was reduced significantly only on phosphorylation of Thr18 (sevenfold) or by hepta-phosphorylation (24-fold). The relative affinities of Mdm2 and p300 for p53 TAD can thus be changed by up to three orders of magnitude by phosphorylation. Accordingly, phosphorylation of Thr18 and hepta-phosphorylation dramatically shifts the balance towards favouring the binding of p300 with p53, and is thus likely to be an important factor in its regulation.

Time-resolved fluorescence resonance energy transfer study shows a compact denatured state of the B domain of protein A.

The B domain of protein A (BDPA), a three-helix bundle of 60 residues, folds via a nucleation-condensation mechanism in apparent two-state kinetics. We have applied a time-resolved FRET (tr-FRET) approach to characterize the ensembles of BDPA during chemical denaturation. The distribution of the distance between residues 22 and 55, which are close and separated by helices 2 and 3 in the native state, was determined by global analysis of the time-resolved fluorescence decay curves of the probes. Narrow distributions were observed when the protein was equilibrated in guanidinium chloride (GdmCl) concentrations below 1.5 M (native state, N) and above the transition zone at 2.6-3.0 M GdmCl (denatured state, D). Considerably broader distributions were found around the transition point (2.0 M GdmCl) or much higher GdmCl concentrations (>3.0 M). Comparative global analysis of the tr-FRET data showed a compact denatured state of the protein, characterized by narrow distribution and relatively small mean distance between residues 22 and 55 that was observed at mild denaturing conditions (<3 M GdmCl). This experiment supports the two-state folding mechanism of BDPA and indicates the existence of effective nonlocal, probably hydrophobic, intramolecular interactions that stabilize a pretty uniform ensemble of compact denatured molecules at intermediate denaturing conditions.

Structure-function-rescue: the diverse nature of common p53 cancer mutants.

The tumor suppressor protein p53 is inactivated by mutation in about half of all human cancers. Most mutations are located in the DNA-binding domain of the protein. It is, therefore, important to understand the structure of p53 and how it responds to mutation, so as to predict the phenotypic response and cancer prognosis. In this review, we present recent structural and systematic functional data that elucidate the molecular basis of how p53 is inactivated by different types of cancer mutation. Intriguingly, common cancer mutants exhibit a variety of distinct local structural changes, while the overall structural scaffold is largely preserved. The diverse structural and energetic response to mutation determines: (i) the folding state of a particular mutant under physiological conditions; (ii) its affinity for the various p53 target DNA sequences; and (iii) its protein-protein interactions both within the p53 tetramer and with a multitude of regulatory proteins. Further, the structural details of individual mutants provide the basis for the design of specific and generic drugs for cancer therapy purposes. In combination with studies on second-site suppressor mutations, it appears that some mutants are ideal rescue candidates, whereas for others simple pharmacological rescue by small molecule drugs may not be successful.

Energy-dependent nucleolar localization of p53 in vitro requires two discrete regions within the p53 carboxyl terminus.

The p53 tumor suppressor is a nucleocytoplasmic shuttling protein that is found predominantly in the nucleus of cells. In addition to mutation, abnormal p53 cellular localization is one of the mechanisms that inactivate p53 function. To further understand features of p53 that contribute to the regulation of its trafficking within the cell, we analysed the subnuclear localization of wild-type and mutant p53 in human cells that were either permeabilized with detergent or treated with the proteasome inhibitor MG132. We, here, show that either endogenously expressed or exogenously added p53 protein localizes to the nucleolus in detergent-permeabilized cells in a concentration- and ATP hydrolysis-dependent manner. Two discrete regions within the carboxyl terminus of p53 are essential for nucleolar localization in permeabilized cells. Similarly, localization of p53 to the nucleolus after proteasome inhibition in unpermeabilized cells requires sequences within the carboxyl terminus of p53. Interestingly, genotoxic stress markedly decreases the association of p53 with the nucleolus, and phosphorylation of p53 at S392, a site that is modified by such stress, partially impairs its nucleolar localization. The possible significance of these findings is discussed.

Solution structure of a protein denatured state and folding intermediate.

The most controversial area in protein folding concerns its earliest stages. Questions such as whether there are genuine folding intermediates, and whether the events at the earliest stages are just rearrangements of the denatured state or progress from populated transition states, remain unresolved. The problem is that there is a lack of experimental high-resolution structural information about early folding intermediates and denatured states under conditions that favour folding because competent states spontaneously fold rapidly. Here we have solved directly the solution structure of a true denatured state by nuclear magnetic resonance under conditions that would normally favour folding, and directly studied its equilibrium and kinetic behaviour. We engineered a mutant of Drosophila melanogaster Engrailed homeodomain that folds and unfolds reversibly just by changing ionic strength. At high ionic strength, the mutant L16A is an ultra-fast folding native protein, just like the wild-type protein; however, at physiological ionic strength it is denatured. The denatured state is a well-ordered folding intermediate, poised to fold by docking helices and breaking some non-native interactions. It unfolds relatively progressively with increasingly denaturing conditions, and so superficially resembles a denatured state with properties that vary with conditions. Such ill-defined unfolding is a common feature of early folding intermediate states and accounts for why there are so many controversies about intermediates versus compact denatured states in protein folding.

Designing a metal-binding site in the scaffold of Escherichia coli KDO8PS.

KDO8PS (3-deoxy-d-manno-octulosonate-8-phosphate synthase) and DAH7PS (3-deoxy-d-arabino-heptulosonic acid-7-phosphate synthase) enzymes catalyse analogous condensation reactions between phosphoenolpyruvate and arabinose 5-phosphate or erythrose 4-phosphate, respectively. All known DAH7PS and some of KDO8PS enzymes (Aquifex aeolicus KDO8PS) require a metal ion for activity whereas another class of KDO8PS (including Escherichia coli KDO8PS) does not. Based on sequence alignment of all known KDO8PS and DAH7PS enzymes, we identified a single amino acid residue that might define the metal dependence of KDO8PS activity. One of the four metal-binding residues, a cysteine, is conserved only among metal-binding KDO8PS and DAH7PS enzymes and is replaced by an asparagine residue in other KDO8PS enzymes. We introduced a metal binding site into E.coli KDO8PS by a single N26C and a double M25P N26C mutation, which led to an increased k(cat) of the enzymes in the presence of activating Mn(2+) ions. The M25P N26C mutant of E.coli KDO8PS had a value of k(cat)/K(M) in the presence of Mn(2+) ions four times higher than A.aeolicus KDO8PS. KDO8PS and DAH7PS may have evolved from a common ancestor protein that required a divalent metal ion for activity. A non-metal-binding KDO8PSs may have evolved from an ancestor protein that was able to bind Mn(2+) but no longer required Mn(2+) to function and eventually lost one of metal-binding residues.

Ultrafast folding of WW domains without structured aromatic clusters in the denatured state.

Ultrafast-folding proteins are important for combining experiment and simulation to give complete descriptions of folding pathways. The WW domain family comprises small proteins with a three-stranded antiparallel beta-sheet topology. Previous studies on the 57-residue YAP 65 WW domain indicate the presence of residual structure in the chemically denatured state. Here we analyze three minimal core WW domains of 38-44 residues. There was little spectroscopic or thermodynamic evidence for residual structure in either their chemically or thermally denatured states. Folding and unfolding kinetics, studied by using rapid temperature-jump and continuous-flow techniques, show that each domain folds and unfolds very rapidly in a two-state transition through a highly compact transition state. Folding half-times were as short as 17 micros at 25 degrees C, within an order of magnitude of the predicted maximal rate of loop formation. The small size and topological simplicity of these domains, in conjunction with their very rapid two-state folding, may allow us to reduce the difference in time scale between experiment and theoretical simulation.

Using flexible loop mimetics to extend phi-value analysis to secondary structure interactions.

Chemical synthesis allows the incorporation of nonnatural amino acids into proteins that may provide previously untried probes of their folding pathway and thermodynamic stability. We have used a flexible thioether linker as a loop mimetic in the human yes kinase-associated protein (YAP 65) WW domain, a three-stranded, 44-residue, beta-sheet protein. This linkage avoids problems of incorporating sequences that constrain loops to the extent that they significantly change the nature of the denatured state with concomitant effects on the folding kinetics. An NMR solution structure shows that the thioether linker had little effect on the global fold of the domain, although the loop is apparently more dynamic. The thioether variants are destabilized by up to 1.4 kcal/mol (1 cal = 4.18 J). Preliminary Phi-value analysis showed that the first loop is highly structured in the folding transition state, and the second loop is essentially unstructured. These data are consistent with results from simulated unfolding and detailed protein-engineering studies of structurally homologous WW domains. Previously, Phi-value analysis was limited to studying side-chain interactions. The linkers used here extend the protein engineering method directly to secondary-structure interactions.

The binding of bis-ANS to the isolated GroEL apical domain fragment induces the formation of a folding intermediate with increased hydrophobic surface not observed in tetradecameric GroEL.

The extent of hydrophobic exposure upon bis-ANS binding to the functional apical domain fragment of GroEL, or minichaperone (residues 191-345), was investigated and compared with that of the GroEL tetradecamer. Although a total of seven molecules of bis-ANS bind cooperatively to this minichaperone, most of the hydrophobic sites were induced following initial binding of one to two molecules of probe. From the equilibrium and kinetics studies at low bis-ANS concentrations, it is evident that the native apical domain is converted to an intermediate conformation with increased hydrophobic surfaces. This intermediate binds additional bis-ANS molecules. Tyrosine fluorescence detected denaturation demonstrated that bis-ANS can destabilize the apical domain. The results from (i) bis-ANS titrations, (ii) urea denaturation studies in the presence and absence of bis-ANS, and (iii) intrinsic tyrosine fluorescence studies of the apical domain are consistent with a model in which bis-ANS binds tightly to the intermediate state, relatively weakly to the native state, and little to the denatured state. The results suggest that the conformational changes seen in apical domain fragments are not seen in the intact GroEL oligomer due to restrictions imposed by connections of the apical domain to the intermediate domain and suppression of movement due to quaternary structure.

Experimental assignment of the structure of the transition state for the association of barnase and barstar.

Association of a protein complex follows a two step reaction mechanism, with the first step being the formation of an encounter complex which evolves into the final complex. Here we present new experimental data for the association of the bacterial ribonuclease barnase and its polypeptide inhibitor barstar which shed light on the thermodynamics and structure of the transition state and preceding encounter complex of association at diminishing electrostatic attraction. We show that the activation entropy at the transition state is close to zero, with the activation enthalpy being equal to the free energy of binding. This observation was independent of the magnitude of the mutual electrostatic attraction, which were altered by mutagenesis or by addition of salt. The low activation entropy implies that the transition state is mostly solvated at all ionic strengths. The structure of the transition state was probed by measuring pairwise interaction energies using double-mutant-cycles. While at low ionic strength all proximal charge-pairs form contacts, at high salt only a subset of these interactions are maintained. More specifically, charge-charge interactions between partially buried residues are lost, while exposed charged residues maintain their ability to form specific interactions even at the highest salt concentration. Uncharged residues do not interact at any ionic strength. The results presented here suggest that the barnase-barstar binding sites are correctly aligned during the transition state even at diminishing electrostatic attraction, although specific short range interactions of uncharged residues are not yet formed. Furthermore, most of the interface desolvation (which contributes to the entropy of the system) has not yet occurred. This picture seems to be valid at low and high salt. However, at high salt, interactions of the activated complex are limited to a more restricted set of residues which are easier approached during diffusion, prior to final docking. This suggest that the steering region at high salt is more limited, albeit maintaining its specificity.

Structure of the C-terminal sterile alpha-motif (SAM) domain of human p73 alpha.

p73 is a homologue of the tumour suppressor p53 and contains all three functional domains of p53. The alpha-splice variant of p73 (p73 alpha) contains near its C-terminus an additional structural domain known as the sterile alpha-motif (SAM) that is probably responsible for regulating p53-like functions of p73. Here, the 2.54 A resolution crystal structure of this protein domain is reported. The crystal structure and the published solution structure have the same five-helix bundle fold that is characteristic of all SAM-domain structures, with an overall r.m.s.d. of 1.5 A for main-chain atoms. The hydrophobic core residues are well conserved, yet some large local differences are observed. The crystal structure reveals a dimeric organization, with the interface residues forming a mini four-helix bundle. However, analysis of solvation free energies and the surface area buried upon dimer formation indicated that this arrangement is more likely to be an effect of crystal packing rather than reflecting a physiological state. This is consistent with the solution structure being a monomer. The p73 alpha SAM domain also contains several interesting structural features: a Cys-X-X-Cys motif, a 3(10)-helix and a loop that have elevated B factors, and short tight inter-helical loops including two beta-turns; these elements are probably important in the normal function of this domain.

Protein folding from a highly disordered denatured state: the folding pathway of chymotrypsin inhibitor 2 at atomic resolution.

Previous experimental and theoretical studies have produced high-resolution descriptions of the native and folding transition states of chymotrypsin inhibitor 2 (CI2). In similar fashion, here we use a combination of NMR experiments and molecular dynamics simulations to examine the conformations populated by CI2 in the denatured state. The denatured state is highly unfolded, but there is some residual native helical structure along with hydrophobic clustering in the center of the chain. The lack of persistent nonnative structure in the denatured state reduces barriers that must be overcome, leading to fast folding through a nucleation-condensation mechanism. With the characterization of the denatured state, we have now completed our description of the folding/unfolding pathway of CI2 at atomic resolution.

Ligand-independent assembly of recombinant human CD1 by using oxidative refolding chromatography.

CD1 is an MHC class I-like antigen-presenting molecule consisting of a heavy chain and beta(2)-microglobulin light chain. The in vitro refolding of synthetic MHC class I molecules has always required the presence of ligand. We report here the use of a folding method using an immobilized chaperone fragment, a protein disulphide isomerase, and a peptidyl-prolyl cis-trans isomerase (oxidative refolding chromatography) for the fast and efficient assembly of ligand-free and ligand-associated CD1a and CD1b, starting with material synthesized in Escherichia coli. The results suggest that "empty" MHC class I-like molecules can assemble and remain stable at physiological temperatures in the absence of ligand. The use of oxidative refolding chromatography thus is extended to encompass complex multisubunit proteins and specifically to members of the extensive, functionally diverse and important immunoglobulin supergene family of proteins, including those for which a ligand has yet to be identified.

Human CD1d-glycolipid tetramers generated by in vitro oxidative refolding chromatography.

CD1 molecules are specialized in presenting lipids to T lymphocytes, but identification and isolation of CD1-restricted lipid specific T cells has been hampered by the lack of reliable and sensitive techniques. We here report the construction of CD1d-glycolipid tetramers from fully denatured human CD1d molecules by using the technique of oxidative refolding chromatography. We demonstrate that chaperone- and foldase-assisted refolding of denatured CD1d molecules and beta(2)-microglobulin in the presence of synthetic lipids is a rapid method for the generation of functional and specific CD1d tetramers, which unlike previously published protocols ensures isolation of CD1d tetramers loaded with a single lipid species. The use of human CD1d-alpha-galactosylceramide tetramers for ex vivo staining of peripheral blood lymphocytes and intrahepatic T cells from patients with viral liver cirrhosis allowed for the first time simultaneous analysis of frequency and specificity of natural killer T cells in human clinical samples. Application of this protocol to other members of the CD1 family will provide powerful tools to investigate lipid-specific T cell immune responses in health and in disease.

Rescuing the function of mutant p53.

One protein--p53--plays nemesis to most cancers by condemning damaged cells to death or quarantining them for repair. But the activity of p53 relies on its intact native conformation, which can be lost following mutation of a single nucleotide. With thousands of such mutations identified in patients, how can a future cancer drug buttress this fragile protein structure and restore the cell's natural defence?