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The c-MYC transcription factor is a master regulator of cell growth and proliferation and is an established target for cancer therapy. This basic helix–loop–helix Zip protein forms a heterodimer with its obligatory partner MAX, which binds to DNA via the basic region. Considerable research efforts are focused on targeting the heterodimerization interface and the interaction of the complex with DNA. The only available crystal structure is that of a c-MYC:MAX complex artificially tethered by an engineered disulfide linker and prebound to DNA. We have carried out a detailed structural analysis of the apo form of the c-MYC:MAX complex, with no artificial linker, both in solution using nuclear magnetic resonance (NMR) spectroscopy and by X-ray crystallography. We have obtained crystal structures in three different crystal forms, with resolutions between 1.35 and 2.2 Å, that show extensive helical structure in the basic region. Determination of the α-helical propensity using NMR chemical shift analysis shows that the basic region of c-MYC and, to a lesser extent, that of MAX populate helical conformations. We have also assigned the NMR spectra of the c-MYC basic helix–loop–helix Zip motif in the absence of MAX and showed that the basic region has an intrinsic helical propensity even in the absence of its dimerization partner. The presence of helical structure in the basic regions in the absence of DNA suggests that the molecular recognition occurs via a conformational selection rather than an induced fit. Our work provides both insight into the mechanism of DNA binding and structural information to aid in the development of MYC inhibitors.

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 β-Grasp fold characterised by a β-β-α-β-β-α-β secondary-structure organisation. The five β 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 310 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.

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 coliintimin, 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. colimembrane-bound lytic murein transglycosylase D. The LysM domain has a βααβ secondary structure with the two helices packing onto the same side of an anti-parallel β 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.

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 cerevisiaeRNase HI, determined using NMR spectroscopy, is presented. The 46 residue motif comprises a three-stranded antiparallel β-sheet and two short α-helices which pack onto opposite faces of the β-sheet. Conserved residues involved in packing the α-helices onto the β-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 β-β-α-β-α 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.

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 μM, 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 β-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 α-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.

The NMR solution structure of an immunoglobulin superfamily module of twitchin (Ig 18') has been determined and the kinetic and equilibrium folding behaviour characterised. Thirty molecular coordinates were calculated using a hybrid distance geometry-simulated annealing protocol based on 1207 distance and 48 dihedral restraints. The atomic rms distributions about the mean coordinate for the ensemble of structures is 0.55(±0.09) Å for backbone atoms and 1.10(±0.08) Å for all heavy atoms. The protein has a topology very similar to that of telokin and the titin Ig domains and thus it falls into the I set of the immunoglobulin superfamily. The close agreement between the predicted and observed structures of Ig 18' demonstrates clearly that the I set profile can be applied in the structure prediction of immunoglobulin-like domains of diverse modular proteins. Folding studies reveal that the protein has relatively low thermodynamic stability, ΔG^H₂O_U–F=4.0 kcal mol⁻¹at physiological pH. Unfolding studies suggest that the protein has considerable kinetic stability, the half life of the unfolding is greater than 40 minutes in the absence of denaturant.

Cytosolic phospholipase A2 (cPLA2) is a calcium-sensitive 85-kDa enzyme that hydrolyzes arachidonic acid-containing membrane phospholipids to initiate the biosynthesis of eicosanoids and platelet-activating factor, potent inflammatory mediators. The calcium-dependent activation of the enzyme is mediated by an N-terminal C2 domain, which is responsible for calcium-dependent translocation of the enzyme to membranes and that enables the intact enzyme to hydrolyze membrane-resident substrates. The 2.4-Å x-ray crystal structure of this C2 domain was solved by multiple isomorphous replacement and reveals a β-sandwich with the same topology as the C2 domain from phosphoinositide-specific phospholipase Cδ1. Two clusters of exposed hydrophobic residues surround two adjacent calcium binding sites. This region, along with an adjoining strip of basic residues, appear to constitute the membrane binding motif. The structure provides a striking insight into the relative importance of hydrophobic and electrostatic components of membrane binding for cPLA2. Although hydrophobic interactions predominate for cPLA2, for other C2 domains such as in “conventional” protein kinase C and synaptotagmins, electrostatic forces prevail.

Barstar, a polypeptide inhibitor of ribonucleases, has been studied by 2D and 3D NMR techniques using uniformly 15N‐labeled protein. Backbone (15NH‐CαH‐CβH) resonances were assigned for all but 5 of the 89 residues. Dihedral angle and deuterium exchange studies were used in conjunction with medium range inter‐proton NOEs to characterize the secondary structure of barstar. The protein is composed of four α‐helices and three short stretches of extended strand. By further analysis of the NOE data three of the helices were found to be parallel to each other with the single disulphide bond linking the second and fourth helices at their C‐terminal ends.

The structural and dynamic properties of an immunoglobulin superfamily domain (IgSF), Ig 18′, have been characterised by NMR at 285 K, in the presence of 4.2 M and 6.0 M urea, respectively. Analysis of chemical shift deviations, 3JHNHαcoupling constants, sequential NOE pattern, and 15N relaxation data reveals that although the two urea-denatured states are highly disordered, some local turn-like residual structures do exist. Moreover, some distinct differences between the properties of the two denatured states are observed. In 4.2 M urea-denatured Ig 18′, regions 80–83 and 86–92 adopt turn-like conformations, furthermore, region 84–93 is involved in slow exchange processes that occur on a micro- to millisecond time-scale. In the 6.0 M urea-denatured state, these turn-like conformations are less occupied, and chemical exchange processes in region 84–93 are largely reduced. In contrast, region 32–36 has persistent turn-like structures in both urea-denatured states. Some correlation between the spectral density function at 0 frequency, Jeff(0), for the urea-denatured states and the secondary structure elements of the folded state have been observed. Except for the terminal regions, residues corresponding to β-strands have higher Jeff(0) values compared to residues corresponding to loops. The characterisation and comparison of the two urea-denatured states highlight residues that possess properties that may be crucial for the initiation of folding of this domain.

NMR spectroscopy has been used to determine the secondary structure of one of the double‐stranded RNA binding domains from the Drosophilaprotein staufen. The domain has an αβββα arrangement of secondary structure, with the β strands forming an antiparallel β sheet. The secondary structure differs from that found in the RNP RNA binding domain.

The α-helix in proteins has a dipole moment resulting from the alignment of dipoles of the peptide bond which can perturb the pKas of ionizing groups. One of the two histidine residues (His18) in barnase, the small ribonuclease from Bacillus amyloliquefaciens, is located at the negatively charged end (C-terminal) of an α-helix. From NMR titrations of wild-type and engineered mutants we find that the pKaof His18 is 7.9 in wild-type enzyme, 1.6 units above the value in the urea-denatured enzyme and in model peptides. This implies that there is a favourable interaction between the protonated form of His18 and the α-helix that should stabilize the native structure at neutral pH by 2.1 kcal mol−1. Denaturation at various values of pH of wild-type and muant enzymes engineered at position 18 shows that this is so. The increase in stability of the enzyme as the pH changes from 8.5 to 6.3 is attributable to this interaction, and the pH-stability curve fits pKavalues for Hisl8 in native and urea-denatured enzymes that are consistent with the NMR data.

The dimeric DNA binding domain of the human papillomavirus E2 protein displays a two-state concerted unfolding and dissociation, with no detectable monomeric intermediate species accumulated at equilibrium. We investigated the kinetic folding mechanism of the dimeric domain using stopped-flow spectroscopic techniques and observed a fast forming monomeric intermediate, followed by a slower bimolecular reaction. Both phases involve secondary structure rearrangements of similar magnitude. Our results support a folding pathway in which the formation of an early monomeric intermediate, with characteristics of hydrophobic collapse, is followed by a bimolecular step encompassing association and folding. The interwoven folding topology of this particular type of dimeric β-barrel found in the E2 DNA binding domain strongly suggests that any monomeric species formed could not be native-like.

The extracellular ribonuclease from Bacillus amyloliquifaciens, barnase, forms a tightly‐bound one‐to‐one complex with its intracellular inhibitor barstar. The barstar binding site on barnase was characterised by comparing the differences in the chemical shift and hydrogen‐deuterium exchange rates between free and bound barnase. Chemical shift assignments of barnase in the complex with barstar were determined from 3D NOESY‐HMQC and TOCSY‐HMQC spectra of a complex that had been prepared with uniformly 15N‐labelled barnase and unlabelled barstar. Hydrogen exchange rates were obtained from an analysis of a series of [15N]HMQC spectra of a sample prepared in the same manner exchanged into D2O. The largest changes in either chemical shift or hydrogen‐deuterium exchange rate are observed for residues located in the active‐site and substrate binding loops indicating that barstar inhibits barnase activity by sterically blocking the active site.

The denatured states of barnase that are induced by urea, acid, and high temperature and acid have been assigned and characterised by high resolution heteronuclear NMR. The assignment was completed using a combination of triple-resonance and magnetisation-transfer methods. The latter was facilitated by selecting a suitable mutant of barnase (Ile→Val51) which has an appropriate rate of interconversion between native and denatured states in urea.³JNH-C^αH coupling constants were determined for pH and urea-denatured barnase and intrinsic "random coil" coupling constants are shown to be different for different residue types. All the denatured states are highly unfolded. But, a consistent series of weak correlations in chemical shift, NOESY and coupling constant data provides evidence that the acid-denatured state has some residual structure in regions that form the first and second helices and the central strands of β-sheet in the native protein. The acid/temperature-denatured states has less structure in these regions, and the urea-denatured state, less still. These observations may be combined with detailed analyses of the folding pathway of barnase from kinetic studies to illuminate the relevance of residual structure in the denatured states of proteins to the mechanism of protein folding. First, the folding of barnase is known to proceed in its later stages through structures in which the first helix and centre of the β-sheet are extensively formed. Thus, embryonic initiation sites for these do exist in the denatured states and so could well develop into true nuclei. Second, it has been clearly established that the second helix is unfolded in these later states, and so residual structure in this region of the protein is non-productive. These data fit a model of protein folding in which local nucleation sites are latent in the denatured state and develop only when they make interactions elsewhere in the protein that stabilise them during the folding process. Thus, studies of the structure of denatured states pinpoint where nucleation sites may be, and the kinetic and protein engineering studies show which ones are productive.