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.

Application of thermophilic enzymes in commercial biotransformation processes.

Biocatalysis is a useful tool in the provision of chiral technology and extremophilic enzymes are just one component in that toolbox. Their role is not always attributable to their extremophilic properties; as with any biocatalyst certain other criteria should be satisfied. Those requirements for a useful biocatalyst will be discussed including issues of selectivity, volume efficiency, security of supply, technology integration, intellectual property and regulatory compliance. Here we discuss the discovery and commercialization of an L-aminoacylase from Thermococcus litoralis, the product of a LINK project between Chirotech Technology and the University of Exeter. The enzyme was cloned into Escherichia coli to aid production via established mesophilic fermentation protocols. A simple downstream process was then developed to assist in the production of the enzyme as a genetically modified-organism-free reagent. The fermentation and downstream processes are operated at the 500 litre scale. Characterization of the enzyme demonstrated a substrate preference for N-benzoyl groups over N-acetyl groups. The operational parameters have been defined in part by substrate-concentration tolerances and also thermostability. Several examples of commercial biotransformations will be discussed including a process that is successful by virtue of the enzyme's thermotolerance.

The ligand-binding loops in the tunicate C-type lectin TC14 are rigid.

C-type lectin-like domains are very common components of extracellular proteins in animals. They bind to a variety of ligands, including carbohydrates, proteins, ice, and CaCO3 crystals. Their structure is characterized by long surface loops in the area of the protein usually involved in ligand binding. The C-type lectin TC14 from Polyandrocarpa misakiensis specifically binds to D-galactose by coordination of the sugar to a bound calcium atom. We have studied the dynamic properties of TC14 by measuring 15N longitudinal and transverse relaxation rates as well as [1H-15N] heteronuclear NOEs. Relaxation rates and heteronuclear NOE data for holo-TC14 show minimal variations, indicating that there is no substantial difference in rigidity between the elements of regular secondary structure and the extended surface loops. Anisotropic tumbling of the elongated TC14 dimer can account for the main fluctuations in relaxation rates. Loss of the bound calcium does not significantly alter the internal dynamics, suggesting that the stability of the loop region is intrinsic and not dependent on the coordination of the calcium ion. Chemical shift differences between the holo and apo form show that main structural changes occur in the calcium-binding site, but smaller structural changes are propagated throughout the molecule without affecting the overall fold. The disappearance of two resonances for residues following the conserved cis-proline 87 (which is located in the calcium-binding site) in the apo form indicates conformational change on an NMR time scale between the cis and trans configurations of this peptide bond in the absence of calcium. Possible implications of these findings for the ligand binding in C-type lectin-like domains are discussed.

Crystallization and preliminary crystallographic studies of a ribosomal protein L30e from the hyperthermophilic archaeon Thermococcus celer.

Ribosomal protein L30e from the hyperthermophilic archaeon Thermococcus celer is a good model for the study of the thermostability of proteins. It has been overexpressed, purified and crystallized using the hanging-drop vapour-diffusion method using PEG 8000 as precipitant at 290 K. The crystal belongs to the hexagonal space group P6(1)/P6(5), with unit-cell parameters a = b = 48.32, c = 86.42 A. The asymmetric unit contains a single molecule of L30e, with a corresponding crystal volume per protein mass (V(M)) of 2.68 A(3) Da(-1) and a solvent content of 54%. A complete data set diffracting to 1.96 A resolution was collected from a single crystal at 100 K.

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.

The UBX domain: a widespread ubiquitin-like module.

The UBX domain is an 80 amino acid residue module that is present typically at the carboxyl terminus of a variety of eukaryotic proteins. In an effort to elucidate the function of UBX domains, we solved the three-dimensional structure of the UBX domain of human Fas-associated factor-1 (FAF1) by NMR spectroscopy. The structure has a beta-Grasp fold characterised by a beta-beta-alpha-beta-beta-alpha-beta secondary-structure organisation. The five beta strands are arranged into a mixed sheet in the order 21534. The longer first helix packs across the first three strands of the sheet, and a second shorter 3(10) helix is located in an extended loop connecting strands 4 and 5. In the absence of significant sequence similarity, the UBX domain can be superimposed with ubiquitin with an r.m.s.d. of 1.9 A, suggesting that the two structures share the same superfold, and an evolutionary relationship. However, the absence of a carboxyl-terminal extension containing a double glycine motif and of suitably positioned lysine side-chains makes it highly unlikely that UBX domains are either conjugated to other proteins or part of mixed UBX-ubiquitin chains. Database searches revealed that most UBX domain-containing proteins belong to one of four evolutionarily conserved families represented by the human FAF1, p47, Y33K, and Rep8 proteins. A role of the UBX domain in ubiquitin-related processes is suggested.

Biophysical characterization of elongin C from Saccharomyces cerevisiae.

Elongin C (ELC) is an essential component of the mammalian CBC(VHL) E3 ubiquitin ligase complex. As a step toward understanding the role of ELC in assembly and function of CBC-type ubiquitin ligases, we analyzed the quaternary structure and backbone dynamics of the highly homologous Elc1 protein from Saccharomyces cerevisiae. Analytical ultracentrifugation experiments in conjunction with size exclusion chromatography showed that Elc1 is a nonglobular monomer over a wide range of concentrations. Pronounced line broadening in (1)H,(15)N-HSQC NMR spectra and failure to assign peaks corresponding to the carboxy-terminal helix 4 of Elc1 indicated that helix 4 is conformationally labile. Measurement of (15)N NMR relaxation parameters including T(1), T(2), and the (1)H-(15)N nuclear Overhauser effect revealed (i) surprisingly high flexibility of residues 69-77 in loop 5, and (ii) chemical exchange contributions for a large number of residues throughout the protein. Addition of 2,2,2-trifluoroethanol (TFE) stabilized helix 4 and reduced chemical exchange contributions, suggesting that stabilization of helix 4 suppresses the tendency of Elc1 to undergo conformational exchange on a micro- to millisecond time scale. Binding of a peptide representing the major ELC binding site of the von Hippel-Lindau (VHL) tumor suppressor protein almost completely eliminated chemical exchange processes, but induced substantial conformational changes in Elc1 leading to pronounced rotational anisotropy. These results suggest that elongin C interacts with various target proteins including the VHL protein by an induced fit mechanism involving the conformationally flexible carboxy-terminal helix 4.

The structure of a LysM domain from E. coli membrane-bound lytic murein transglycosylase D (MltD).

The LysM domain is a widespread protein module. It was originally identified in enzymes that degrade bacterial cell walls but is also present in many other bacterial proteins. Several proteins that contain the domain, such as Staphylococcal IgG binding proteins and Escherichia coli intimin, are involved in bacterial pathogenesis. LysM domains are also found in some eukaryotic proteins, apparently as a result of horizontal gene transfer from bacteria. The available evidence suggests that the LysM domain is a general peptidoglycan-binding module. We have determined the structure of this domain from E. coli membrane-bound lytic murein transglycosylase D. The LysM domain has a betaalphaalphabeta secondary structure with the two helices packing onto the same side of an anti- parallel beta sheet. The structure shows no similarity to other bacterial cell surface domains. A potential binding site in a shallow groove on surface of the protein has been identified.

Folding of a dimeric beta-barrel: residual structure in the urea denatured state of the human papillomavirus E2 DNA binding domain.

The dimeric beta-barrel is a characteristic topology initially found in the transcriptional regulatory domain of the E2 DNA binding domain from papillomaviruses. We have previously described the kinetic folding mechanism of the human HPV-16 domain, and, as part of these studies, we present a structural characterization of the urea-denatured state of the protein. We have obtained a set of chemical shift assignments for the C-terminal domain in urea using heteronuclear NMR methods and found regions with persistent residual structure. Based on chemical shift deviations from random coil values, 3'J(NHN alpha) coupling constants, heteronuclear single quantum coherence peak intensities, and nuclear Overhauser effect data, we have determined clusters of residual structure in regions corresponding to the DNA binding helix and the second beta-strand in the folded conformation. Most of the structures found are of nonnative nature, including turn-like conformations. Urea denaturation at equilibrium displayed a loss in protein concentration dependence, in absolute parallel to a similar deviation observed in the folding rate constant from kinetic experiments. These results strongly suggest an alternative folding pathway in which a dimeric intermediate is formed and the rate-limiting step becomes first order at high protein concentrations. The structural elements found in the denatured state would collide to yield productive interactions, establishing an intermolecular folding nucleus at high protein concentrations. We discuss our results in terms of the folding mechanism of this particular topology in an attempt to contribute to a better understanding of the folding of dimers in general and intertwined dimeric proteins such as transcription factors in particular.

Crystallization and preliminary crystallographic studies of a SAM domain at the C-terminus of human p73alpha.

p73 is a recently discovered homologue of the tumour suppressor p53 and contains all three functional domains of p53. The alpha-splice variant of p73 (p73alpha) contains an additional structural domain near its C--terminus that has sequence homology with the sterile alpha-motif (SAM) domain. This domain is considered to be responsible for mediating protein-protein interactions. Pyramidal crystals of human p73alpha SAM domain were obtained by the hanging-drop vapour-diffusion method with ammonium dihydrogen orthophosphate as the precipitant. The crystals diffract to 2.54 A resolution and belong to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 32.02, c = 133.84 A. The structure was solved by molecular replacement using the NMR structure of the same protein as the search model.

RNA recognition by a Staufen double-stranded RNA-binding domain.

The double-stranded RNA-binding domain (dsRBD) is a common RNA-binding motif found in many proteins involved in RNA maturation and localization. To determine how this domain recognizes RNA, we have studied the third dsRBD from Drosophila Staufen. The domain binds optimally to RNA stem-loops containing 12 uninterrupted base pairs, and we have identified the amino acids required for this interaction. By mutating these residues in a staufen transgene, we show that the RNA-binding activity of dsRBD3 is required in vivo for Staufen-dependent localization of bicoid and oskar mRNAs. Using high-resolution NMR, we have determined the structure of the complex between dsRBD3 and an RNA stem-loop. The dsRBD recognizes the shape of A-form dsRNA through interactions between conserved residues within loop 2 and the minor groove, and between loop 4 and the phosphodiester backbone across the adjacent major groove. In addition, helix alpha1 interacts with the single-stranded loop that caps the RNA helix. Interactions between helix alpha1 and single-stranded RNA may be important determinants of the specificity of dsRBD proteins.

Efficient purification and kinetic characterization of a bimodular derivative of the erythromycin polyketide synthase.

Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), are giant multienzymes that biosynthesize a number of clinically important natural products. The modular nature of PKSs suggests the possibility of a combinatorial approach to the synthesis of novel bioactive polyketides, but the efficacy of such a strategy depends critically on gaining fundamental insight into PKS structure and function, most directly through experiments with purified PKS proteins. Several recent investigations into important aspects of the activity of these enzymes have used only partially purified proteins (often 3-4% of total protein), reflecting how difficult it is to purify these multienzymes in amounts adequate for kinetic and structural analysis. We report here the steady-state kinetic analysis of a typical bimodular PKS, 6-deoxyerythronolide B synthase 1-thioesterase (DEBS 1-TE), purified from recombinant Saccharopolyspora erythraea JCB101 by a new, high-yielding procedure consisting of three steps: ammonium sulfate precipitation, hydrophobic interaction chromatography and size-exclusion chromatography. The method provides 13-fold purification with a recovery of 11% of the applied PKS activity. The essentially homogeneous synthase exhibits an intrinsic methylmalonyl-CoA hydrolase activity, which competes with polyketide chain extension. The most reliable value for the kcat for synthesis of (3S,5R)-dihydroxy-(2R,4R)-dimethyl-n-heptanoic acid-delta-lactone is 0.84 min-1, and the apparent Km for (2RS)-methylmalonyl-CoA is 17 microM. This kcat is approximately 10-fold lower than the value reported previously for a differently engineered version of the truncated PKS, DEBS 1+TE. The difference likely reflects the fact that the DEBS 1-TE contains a hybrid acyl carrier protein (ACP) domain in its second module, which lowers its catalytic efficiency.

Knowledge-based design of bimodular and trimodular polyketide synthases based on domain and module swaps: a route to simple statin analogues.

BACKGROUND: Polyketides are structurally diverse natural products that have a range of medically useful activities. Nonaromatic bacterial polyketides are synthesised on modular polyketide synthase (PKS) multienzymes, in which each cycle of chain extension requires a different 'module' of enzymatic activities. Attempts to design and construct modular PKSs that synthesise specified novel polyketides provide a particularly stringent test of our understanding of PKS structure and function. RESULTS: We have constructed bimodular and trimodular PKSs based on DEBS1-TE, a derivative of the erythromycin PKS that contains only modules 1 and 2 and a thioesterase (TE), by substituting multiple domains with appropriate counterparts derived from the rapamycin PKS. Hybrid PKSs were obtained that synthesised the predicted target triketide lactones, which are simple analogues of cholesterol-lowering statins. In constructing intermodular fusions, whether between modules in the same or in different proteins, it was found advantageous to preserve intact the acyl carrier protein-ketosynthase (ACP-KS) didomain that spans the junction between successive modules. CONCLUSIONS: Relatively simple considerations govern the construction of functional hybrid PKSs. Fusion sites should be chosen either in the surface-accessible linker regions between enzymatic domains, as previously revealed, or just inside the conserved margins of domains. The interaction of an ACP domain with the adjacent KS domain, whether on the same polyketide or not, is of particular importance, both through conservation of appropriate protein-protein interactions, and through optimising molecular recognition of the altered polyketide chain in the key transfer of the acyl chain from the ACP of one module to the KS of the downstream module.

NMR structure of the N-terminal domain of Saccharomyces cerevisiae RNase HI reveals a fold with a strong resemblance to the N-terminal domain of ribosomal protein L9.

In addition to the conserved and well-defined RNase H domain, eukaryotic RNases HI possess either one or two copies of a small N-terminal domain. The solution structure of one of the N-terminal domains from Saccharomyces cerevisiae RNase HI, determined using NMR spectroscopy, is presented. The 46 residue motif comprises a three-stranded antiparallel beta-sheet and two short alpha-helices which pack onto opposite faces of the beta-sheet. Conserved residues involved in packing the alpha-helices onto the beta-sheet form the hydrophobic core of the domain. Three highly conserved and solvent exposed residues are implicated in RNA binding, W22, K38 and K39. The beta-beta-alpha-beta-alpha topology of the domain differs from the structures of known RNA binding domains such as the double-stranded RNA binding domain (dsRBD), the hnRNP K homology (KH) domain and the RNP motif. However, structural similarities exist between this domain and the N-terminal domain of ribosomal protein L9 which binds to 23 S ribosomal RNA.

Hot-spot mutants of p53 core domain evince characteristic local structural changes.

Most of the oncogenic mutations in the tumor suppressor p53 map to its DNA-binding (core) domain. It is thus a potential target in cancer therapy for rescue by drugs. To begin to understand how mutation inactivates p53 and hence to provide a structural basis for drug design, we have compared structures of wild-type and mutant p53 core domains in solution by NMR spectroscopy. Structural changes introduced by five hot-spot mutations (V143A, G245S, R248Q, R249S, and R273H) were monitored by chemical-shift changes. Only localized changes are observed for G245S, R248Q, R249S, and R273H, suggesting that the overall tertiary folds of these mutant proteins are similar to that of wild type. Structural changes in R273H are found mainly in the loop-sheet-helix motif and the loop L3 of the core domain. Mutations in L3 (G245S, R248Q, and R249S) introduce structural changes in the loop L2 and L3 as well as terminal residues of strands 4, 9, and 10. It is noteworthy that R248Q, which is often regarded as a contact mutant that affects only interactions with DNA, introduces structural changes as extensive as the other loop L3 mutations (G245S and R249S). These changes suggest that R248Q is also a structural mutant that perturbs the structure of loop L2-L3 regions of the p53 core domain. In contrast to other mutants, replacement of the core residue valine 143 to alanine causes chemical-shift changes in almost all residues in the beta-sandwich and the DNA-binding surface. Long-range effects of V143A mutation may affect the specificity of DNA binding.

The structure of a tunicate C-type lectin from Polyandrocarpa misakiensis complexed with D -galactose.

C-type lectins are calcium-dependent carbohydrate-recognising proteins. Isothermal titration calorimetry of the C-type Polyandrocarpa lectin (TC14) from the tunicate Polyandrocarpa misakiensis revealed the presence of a single calcium atom per monomer with a dissociation constant of 2.6 microM, and confirmed the specificity of TC14 for D -galactose and related monosaccharides. We have determined the 2.2 A X-ray crystal structure of Polyandrocarpa lectin complexed with D -galactose. Analytical ultracentrifugation revealed that TC14 behaves as a dimer in solution. This is reflected by the presence of two molecules in the asymmetric unit with the dimeric interface formed by antiparallel pairing of the two N-terminal beta-strands and hydrophobic interactions. TC14 adopts a typical C-type lectin fold with differences in structure from other C-type lectins mainly in the diverse loop regions and in the second alpha-helix, which is involved in the formation of the dimeric interface. The D -galactose is bound through coordination of the 3 and 4-hydroxyl oxygen atoms with a bound calcium atom. Additional hydrogen bonds are formed directly between serine, aspartate and glutamate side-chains of the protein and the sugar 3 and 4-hydroxyl groups. Comparison of the galactose binding by TC14 with the mannose binding by rat mannose-binding protein reveals how monosaccharide specificity is achieved in this lectin. A tryptophan side-chain close to the binding site and the distribution of hydrogen-bond acceptors and donors around the 3 and 4-hydroxyl groups of the sugar are essential determinants of specificity. These elements are, however, arranged in a very different way than in an engineered galactose-specific mutant of MBPA. Possible biological functions can more easily be understood from the fact that TC14 is a dimer under physiological conditions.

Molecular basis of Celmer's rules: the role of two ketoreductase domains in the control of chirality by the erythromycin modular polyketide synthase.

BACKGROUND: Polyketides are compounds that possess medically significant activities. The modular nature of the polyketide synthase (PKS) multienzymes has generated interest in bioengineering new PKSs. Rational design of novel PKSs, however, requires a greater understanding of the stereocontrol mechanisms that operate in natural PKS modules. RESULTS: The N-acetyl cysteamine (NAC) thioester derivative of the natural beta-keto diketide intermediate was incubated with DEBS1-TE, a derivative of the erythromycin PKS that contains only modules 1 and 2. The reduction products of the two ketoreductase (KR) domains of DEBS1-TE were a mixture of the (2S, 3R) and (2R,3S) isomers of the corresponding beta-hydroxy diketide NAC thioesters. Repeating the incubation using a DEBS1-TE mutant that only contains KR1 produced only the (2S,3R) isomer. CONCLUSIONS: In contrast with earlier results, KR1 selects only the (2S) isomer and reduces it stereospecifically to the (2S, 3R)-3-hydroxy-2-methyl acyl product. The KR domain of module 1 controls the stereochemical outcome at both methyl-and hydroxyl-bearing chiral centres in the hydroxy diketide intermediate. Earlier work showed that the normal enzyme-bound ketoester generated in module 2 is not epimerised, however. The stereochemistry at C-2 is therefore established by a condensation reaction that exclusively gives the (2R)-ketoester, and the stereo-chemistry at C-3 by reduction of the keto group. Two different mechanisms of stereochemical control, therefore, operate in modules 1 and 2 of the erythromycin PKS. These results should provide a more rational basis for designing hybrid PKSs to generate altered stereochemistry in polyketide products.

The structure of a PKD domain from polycystin-1: implications for polycystic kidney disease.

Most cases of autosomal dominant polycystic kidney disease (ADPKD) are the result of mutations in the PKD1 gene. The PKD1 gene codes for a large cell-surface glycoprotein, polycystin-1, of unknown function, which, based on its predicted domain structure, may be involved in protein-protein and protein-carbohydrate interactions. Approximately 30% of polycystin-1 consists of 16 copies of a novel protein module called the PKD domain. Here we show that this domain has a beta-sandwich fold. Although this fold is common to a number of cell-surface modules, the PKD domain represents a distinct protein family. The tenth PKD domain of human and Fugu polycystin-1 show extensive conservation of surface residues suggesting that this region could be a ligand-binding site. This structure will allow the likely effects of missense mutations in a large part of the PKD1 gene to be determined.