Biomedical subjects

Alan R Fersht

Publications and source records attributed to Alan R Fersht.

At least 19 recordsLinked to original sources

PubMed2007

Distinguishing between cooperative and unimodal downhill protein folding.

Conventional cooperative protein folding invokes discrete ensembles of native and denatured state structures in separate free-energy wells. Unimodal noncooperative ("downhill") folding, however, proposes an ensemble of states occupying a single free-energy well for proteins folding at >/=4 x 10(4) s(-1) at 298 K. It is difficult to falsify unimodal mechanisms for such fast folding proteins by standard equilibrium experiments because both cooperative and unimodal mechanisms can present the same time-averaged structural, spectroscopic, and thermodynamic properties when the time scale used for observation is longer than for equilibration. However, kinetics can provide the necessary evidence. Chevron plots with strongly sloping linear refolding arms are very difficult to explain by downhill folding and are a signature for cooperative folding via a transition state ensemble. The folding kinetics of the peripheral subunit binding domain POB and its mutants fit to strongly sloping chevrons at observed rate constants of >6 x 10(4) s(-1) in denaturant solution, extrapolating to 2 x 10(5) s(-1) in water. Protein A, which folds at 10(5) s(-1) at 298 K, also has a well-defined chevron. Single-molecule fluorescence energy transfer experiments on labeled Protein A in the presence of denaturant demonstrated directly bimodal distributions of native and denatured states.

Fluorescence Resonance Energy Transfer↗

PubMed2006

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.

Alanine↗

PubMed2006

Structural basis for understanding oncogenic p53 mutations and designing rescue drugs.

The DNA-binding domain of the tumor suppressor p53 is inactivated by mutation in approximately 50% of human cancers. We have solved high-resolution crystal structures of several oncogenic mutants to investigate the structural basis of inactivation and provide information for designing drugs that may rescue inactivated mutants. We found a variety of structural consequences upon mutation: (i) the removal of an essential contact with DNA, (ii) creation of large, water-accessible crevices or hydrophobic internal cavities with no other structural changes but with a large loss of thermodynamic stability, (iii) distortion of the DNA-binding surface, and (iv) alterations to surfaces not directly involved in DNA binding but involved in domain-domain interactions on binding as a tetramer. These findings explain differences in functional properties and associated phenotypes (e.g., temperature sensitivity). Some mutants have the potential of being rescued by a generic stabilizing drug. In addition, a mutation-induced crevice is a potential target site for a mutant-selective stabilizing drug.

Amino Acid Substitution↗

PubMed2006

Effects of oncogenic mutations and DNA response elements on the binding of p53 to p53-binding protein 2 (53BP2).

The tumor suppressor p53 is frequently mutated in human cancers. Upon activation it can induce cell cycle arrest or apoptosis. ASPP2 can specifically stimulate the apoptotic function of p53 but not cell cycle arrest, but the mechanism of enhancing the activation of pro-apoptotic genes over cell cycle arrest genes remains unknown. In this study, we analyzed the binding of 53BP2 (p53-binding protein 2, the C-terminal domain of ASPP2) to p53 core domain and various mutants using biophysical techniques. We found that several p53 core domain mutations (R181E, G245S, R249S, R273H) have different effects on the binding of DNA response elements and 53BP2. Further, we investigated the existence of a ternary complex consisting of 53BP2, p53, and DNA response elements to gain insight into the specific pro-apoptotic activation of p53. We found that binding of 53BP2 and DNA to p53 is mutually exclusive in the case of GADD45, p21, Bax, and PIG3. Both pro-apoptotic and non-apoptotic response elements were competed off p53 by 53BP2 with no indication of a ternary complex.

Amino Acid Sequence↗

PubMed2006

Characterization of the native and fibrillar conformation of the human Nalpha-acetyltransferase ARD1.

ARD1 (arrest-defective protein 1), together with NAT1 (N-acetyltransferase protein 1), is part of the major N(alpha)-acetyltransferase complex in eukaryotes responsible for alpha-acetylation of proteins and peptides. Protein acetylation has been implicated in gene expression regulation and protein-protein interaction. We characterized the native folded and misfolded conformation of hARD1. Structural characterization of native hARD1 using size exclusion chromatography, circular dichroism, and fluorescence spectroscopy shows the protein consists of a compact globular region comprising two thirds of the protein and a flexible unstructured C terminus. In addition, hARD1 forms protofilaments under physiological conditions of pH and temperature, as judged by electron microscopy and staining with the dyes Congo red and thioflavin T. The process is accelerated by thermal denaturation and high protein concentrations. Limited proteolysis of aggregated hARD1 revealed a resistant fragment whose sequence matched a region contained within the acetyl transferase domain.

Acetyltransferases↗

PubMed2006

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.

Amino Acid Sequence↗

PubMed2006

Phi-analysis of the folding of the B domain of protein A using multiple optical probes.

We examined the co-operativity of ultra-fast folding of a protein and whether the Phi-value analysis of its transition state depended on the location of the optical probe. We incorporated in turn a tryptophan residue into each of the three helices of the B domain of Protein A. Each Trp mutant of the three-helix bundle protein was used as a pseudo-wild-type parent for Phi-analysis in which the intrinsic Trp fluorescence probed the formation of each helix during the transition state. Apart from local effects in the immediate vicinity of the probe, the three separate sets of Phi-values were in excellent agreement, demonstrating the overall co-operativity of folding and the robustness of the Phi-analysis. The transition state of folding of Protein A contains the second helix being well formed with many stabilizing tertiary hydrophobic interactions. In contrast, the first and the third helices are more poorly structured in the transition state. The mechanism of folding thus involves the concurrent formation of secondary and tertiary interactions, and is towards the nucleation-condensation extreme in the nucleation-condensation-framework continuum of mechanism, with helix 2 being the nucleus. We provide an error analysis of Phi-values derived purely from the kinetics of two-state chevron plots.

Circular Dichroism↗

PubMed2006

Effects of common cancer mutations on stability and DNA binding of full-length p53 compared with isolated core domains.

Common cancer mutations of p53 tend either to lower the stability or distort the core domain of the protein or weaken its DNA binding affinity. We have previously analyzed in vitro the effects of mutations on the core domain of p53. Here, we extend those measurements to full-length p53, using either the wild-type protein or a biologically active superstable construct that is more amenable to accurate biophysical measurements to assess the possibilities of rescuing different types of mutations by anticancer drugs. The tetrameric full-length proteins had similar apparent melting temperatures to those of the individual domains, and the structural mutations lowered the melting temperature by similar amounts. The thermodynamic stability of tetrameric p53 is thus dictated by its core domain. We determined that the common contact mutation R273H weakened binding to the gadd45 recognition sequence by approximately 700-1000 times. Many mutants that have lowered melting temperatures should be good drug targets, although the common R273H mutant binds response elements too weakly for simple rescue.

Cell Cycle Proteins↗

PubMed2006

Solution structure of p53 core domain: structural basis for its instability.

The 25-kDa core domain of the tumor suppressor p53 is inherently unstable and melts at just above body temperature, which makes it susceptible to oncogenic mutations that inactivate it by lowering its stability. We determined its structure in solution using state-of-the-art isotopic labeling techniques and NMR spectroscopy to complement its crystal structure. The structure was very similar to that in the crystal but far more mobile than expected. Importantly, we were able to analyze by NMR the structural environment of several buried polar groups, which indicated structural reasons for the instability. NMR spectroscopy, with its ability to detect protons, located buried hydroxyl and sulfhydryl groups that form suboptimal hydrogen-bond networks. We mutated one such buried pair, Tyr-236 and Thr-253 to Phe-236 and Ile-253 (as found in the paralogs p63 and p73), and stabilized p53 by 1.6 kcal/mol. We also detected differences in the conformation of a mobile loop that might reflect the existence of physiologically relevant alternative conformations. The effects of temperature on the dynamics of aromatic residues indicated that the protein also experiences several dynamic processes that might be related to the presence of alternative hydrogen-bond patterns in the protein interior. p53 appears to have evolved to be dynamic and unstable.

Crystallography↗

PubMed2006

Core domain interactions in full-length p53 in solution.

The tumor suppressor p53 consists of four 393-residue chains, each of which has two natively unfolded (N- and C-terminal) and two folded (core and tetramerization) domains. Their structural organization is poorly characterized as the protein tends to aggregate, has defied crystallization, and is at the limits of NMR studies. We first stabilized the protein by mutation to make it more suitable for extended study and then acquired NMR spectra on full-length protein and various combinations of shorter domain constructs. The NMR spectrum (15N,1H transverse relaxation optimized spectroscopy) of full-length p53 was close to that expected from the sum of the spectra of isolated individual domains. However, patterns of changes in chemical shifts revealed unexpected interactions between the core domains. We used the NMR data as constraints in docking algorithms and found a previously uncharacterized self-complementary surface for the association of core domains into dimers within the tetrameric complex. Binding to DNA requires about a 70 degrees rotation to break those subunit interactions and form the known protein:protein interface in the p53-DNA complex. We verified the interactions by the effects of mutation on DNA binding. Spectroscopic, biophysical, and mutational data conspired to give a picture of the p53 tetramer as a dimer of loosely tethered core dimers of appropriate symmetry to be poised to bind target DNA.

Crystallography↗

PubMed2006

The central region of HDM2 provides a second binding site for p53.

HDM2 is a negative regulator of p53 that inhibits its transcriptional activity and subjects it to degradation by an E3 ligase activity. The primary binding site for HDM2 on p53 is located in its N-terminal domain. A second site on the p53 core domain (p53C) binds to an unidentified site in HDM2. We found that this site is in its acidic domain and part of the zinc finger domain by examining the interaction of full-length and domain constructs of p53 with the N-terminal region of HDM2 and peptide arrays derived from the full-length protein. NMR spectroscopy showed that peptides derived from this region of HDM2 bound to residues in the specific DNA-binding site of p53C. The peptides were displaced from the site by gadd45 sequence-specific DNA. Phosphorylation of single amino acids in the central domain of HDM2 did not abolish the interaction between the HDM2-derived peptides and p53C. We speculate that this second binding site helps in stabilizing the interaction between HDM2 and p53 during p53 degradation.

Amino Acid Sequence↗

PubMed2006

Analogues with fluorescent leaving groups for screening and selection of enzymes that efficiently hydrolyze organophosphorus nerve agents.

Enzymes that efficiently hydrolyze highly toxic organophosphorus nerve agents could potentially be used as medical countermeasures. As sufficiently active enzymes are currently unknown, we synthesized twelve fluorogenic analogues of organophosphorus nerve agents with the 3-chloro-7-oxy-4-methylcoumarin leaving group as probes for high-throughput enzyme screening. This set included analogues of the pesticides paraoxon, parathion, and dimefox, and the nerve agents DFP, tabun, sarin, cyclosarin, soman, VX, and Russian-VX. Data from inhibition of acetylcholinesterase, in vivo toxicity tests of a representative analogue (cyclosarin), and kinetic studies with phosphotriesterase (PTE) from Pseudomonas diminuta, and a mammalian serum paraoxonase (PON1), confirmed that the analogues mimic the parent nerve agents effectively. They are suitable tools for high-throughput screens for the directed evolution of efficient nerve agent organophosphatases.

Acetylcholinesterase↗

PubMed2005

Solution structure of the C4 zinc finger domain of HDM2.

HDM2 is a ubiquitin E3 ligase that is a key negative regulator of the tumor suppressor p53. Here, we report the determination of the solution structure of the C4 zinc finger domain of HDM2 using multidimensional NMR. The HDM2 C4 zinc finger domain has a fold consisting of a 3(10) helix followed by four beta-strands, which shares significant structural similarity to the zinc ribbon protein family. Family based sequence analysis identified two putative binding sites, one of which resembles an RNA binding motif.

Amino Acid Sequence↗

PubMed2005

The transition state for folding of a peripheral subunit-binding domain contains robust and ionic-strength dependent characteristics.

The denaturant dependencies of the folding and unfolding kinetics were used to characterize the structure of the transition state for folding of E3BD, a peripheral subunit-binding domain. For the majority of E3BD mutants, the Phi-values calculated at 298 K from the analysis of chevron plots were in good agreement with those previously determined at 325 K using Arrhenius analysis. This agreement further demonstrates the general robustness of Phi-value analyses, since different experiments, methods of denaturation and thermodynamic assumptions were used to determine each set of Phi(F) values. The structure of the transition state for folding was grossly conserved at 298 K and 325 K, with residues in Helix I playing a lesser role in folding than those located in the 3(10) helix, disordered loop and Helix II. However, the energetic contributions of a cluster of basic residues close to the N-terminus and Helix I, which are an integral part of the ligand-binding site, were susceptible to ionic strength effects because of electrostatic strain in native and transition states of E3BD at low ionic strength. We found no evidence of the downhill folding previously proposed for E3BD, even though the conditions employed in this study significantly increased the energetic bias towards the native state.

Bacterial Proteins↗

PubMed2005

Ultra-fast barrier-limited folding in the peripheral subunit-binding domain family.

We have determined the solution structures, equilibrium properties and ultra-fast folding kinetics for three bacterial homologues of the peripheral subunit-binding domain (PSBD) family. The mesophilic homologue, BBL, was less stable than the thermophilic and hyper-thermophilic variants (E3BD and POB, respectively). The broad unfolding transitions of each PSBD, when probed by different techniques, were essentially superimposable, consistent with co-operative denaturation. Temperature-jump and continuous-flow fluorescence methods were used to measure the folding kinetics for E3BD, POB and BBL. E3BD folded fairly rapidly at 298K (folding half-time approximately 25 micros) and BBL and POB folded even faster (folding half-times approximately 3-5 micros). The variations in equilibrium and kinetic behaviour observed for the PSBD family resembles that of the homeodomain family, where the folding pattern changes from apparent two-state transitions to multi-state kinetics as the denatured state becomes more structured. The faster folding of POB may be a consequence of its higher propensity to form helical structure in the region corresponding to the folding nucleus of E3BD. The ultra-fast folding of BBL appears to be a consequence of residual structure in the denatured ensemble, as with engrailed homeodomain. We discuss issues concerning "one-state", downhill folding, and find no evidence for, and strong evidence against, it occurring in these PSBDs. The shorter construct used previously for BBL was destabilized significantly and the stability further perturbed by the introduction of fluorescent probes. Thermal titrations for 11 side-chains scattered around the protein, when probed by (13)C-NMR experiments, could be fit globally to a common co-operative transition.

Amino Acid Sequence↗

PubMed2005

Determination of the folding transition states of barnase by using PhiI-value-restrained simulations validated by double mutant PhiIJ-values.

The protein barnase folds from the denatured state into its native conformation via a high-energy intermediate. Using PhiI-values determined experimentally from single-point mutations as restraints in all-atom molecular dynamics simulations, we have determined ensembles of structures corresponding to the transition states for the formation of the folding intermediate and its conversion into the native state. We have also introduced a stringent validation of the approach used to calculate such structures by predicting interaction PhiIJ-values determined experimentally from a series of double-mutant cycles. The ensembles that we have obtained illustrate the heterogeneity in the nucleation-condensation process by which barnase folds. Obligatory steps of this process include the sequential formation of two folding nuclei, which correspond to the two main hydrophobic cores of the protein. Nonobligatory steps include the elongation of the strand beta1 and the formation of the helix alpha2. The results confirm that the use of experimental observables such as PhiI-values as restraints in molecular dynamics simulations is a powerful general strategy to characterize the relatively heterogeneous structural ensembles that populate nonnative regions of the energy landscapes of proteins.

Bacterial Proteins↗

PubMed2005

Simulation and experiment conspire to reveal cryptic intermediates and a slide from the nucleation-condensation to framework mechanism of folding.

There is a change from three-state to two-state kinetics of folding across the homeodomain superfamily of proteins as the mechanism slides from framework to nucleation-condensation. The tendency for framework folding in this family correlates with inherent helical propensity. The cellular myeloblastis protein (c-Myb) falls in the mechanistic transition region. An earlier, preliminary report of protein engineering experiments and molecular dynamics simulations (MD) showed that the folding mechanism for this protein has aspects of both the nucleation-condensation and framework models. In the more in-depth analysis of the MD trajectories presented here, we find that folding may be attributed to both of these mechanisms in different regions of the protein. The folding of the loop, middle helix, and turn is best described by nucleation-condensation, whereas folding of the N and C-terminal helices may be described by the framework model. Experimentally, c-Myb folds by apparent two-state kinetics, but the MD simulations predict that the kinetics hide a high-energy intermediate. We stabilized this hypothetical folding intermediate by deleting a residue (P174) in the loop between its second and third helices, and the mutant intermediate is long-lived in the simulations. Equilibrium and kinetic experiments demonstrate that folding of the DeltaP174 mutant is indeed three-state. The presence and shape of the intermediate observed in the simulations were confirmed by small angle X-ray scattering experiments.

Computer Simulation↗

PubMed2005

The crystal structure of human CD1d with and without alpha-galactosylceramide.

The glycolipid alpha-galactosylceramide binds with high affinity to CD1d and stimulates natural killer T cells. Here we report the crystal structure of human CD1d in complex with synthetic alpha-galactosylceramide at a resolution of 3.0 A. The structure shows a tightly fit lipid in the CD1d binding groove, with the sphingosine chain bound in the C' pocket and the longer acyl chain anchored in the A' pocket. We also present the CD1d structure without lipid, which has a more open conformation of the binding groove, suggesting a dual conformation of CD1d in which the 'open' conformation is more able to load lipids. These structures provide clues as to how CD1 molecules load glycolipids as well as data to guide the design of new therapeutic agents.

Animals↗