Search

The activation of leukocytes by chemokines is believed to be mediated via binding of chemokines to glycosaminoglycan chains of the extracellular matrix. The binding site on the chemokine interleukin-8 (IL-8) for the glycosaminoglycan heparin has been characterized using a systematic series of site-directed mutants of IL-8 in which the basic residues of the protein have been replaced by alanine. Mutation of K64 and R68 caused the largest decrease in affinity for a heparin Sepharose matrix, with smaller effects seen with mutations of K20, R60, and K67. Heparin-derived disaccharides that could disrupt the IL-8-heparin Sepharose interaction were identified by a competitive binding assay. Heteronuclear NMR spectroscopic titration of 15N-labeled IL-8 with a trisulfated disaccharide revealed a cluster of residues on IL-8 which were perturbed by disaccharide binding. These data identify a heparin-binding surface on IL-8 that includes the C-terminal alpha-helix and the proximal loop around residues 18-23. The heparin-binding site is spatially distinct from the residues involved in receptor binding.

Advances in protein NMR have opened new doors for the understanding of macromolecular structure and interactions. Isotope-labelling approaches have extended the size limit for structure determination, the mapping of protein-ligand interactions is now widely used and has led to a new drug discovery approach, pathways of protein folding processes can be followed, and the structures of molecules from membranes can be determined.

The chemokine superfamily can be subdivided into two groups based on their amino terminal cysteine spacing. The CXC chemokines are primarily involved in neutrophil-mediated inflammation and, so far, two human receptors have been cloned. The CC chemokines tend to be involved in chronic inflammation, and recently we have cloned a fourth leukocyte receptor for this group of ligands. Understanding what makes one receptor bind its range of agonists is important if we are to develop potent selective antagonist. We have started to investigate the molecular basis of this receptor selectivity by looking at why CC chemokines do not bind to the CXC receptors in several ways. First, we looked at the role of the three-dimensional structure of the ligand, and have solved the three dimensional structure of RANTES using nuclear magnetic resonance spectroscopy. The structure is similar to that already determined for the CC chemokine macrophage inflammatory protein-1 beta, and it has a completely different dimer interface to that of the CXC chemokine interleukin-8 (IL-8). However, the monomer structures of all the chemokines are very similar, and at physiological concentrations the proteins are likely to be monomeric. Second, by examining all the known CC and CXC chemokines, we have found a region that differs between the two subfamilies. Mutations of one of the residues in this region, Leu-25 in IL-8, to tyrosine (which is conserved at this position in CC chemokines) enables the mutant IL-8 to bind CC chemokine receptor-1 (CC-CKR-1) and introduces monocyte chemoattractant activity. Using other mutations in this region, we can show a direct interaction with the N-terminus of CC-CKR-1. Third, we have found that modification of the amino terminus of RANTES by addition of one amino acid makes it into an antagonist with nanomolar potency. Taken together, this data suggests a two-site model for receptor activation and for selectivity between CC and CXC chemokines, with an initial receptor contact provided by the main body of the chemokine, and activation provided by the amino terminal region.

The solution structure of the chemokine RANTES (regulated on activation, normal T-cell expressed and secreted) has been determined using NMR spectroscopy. Backbone and side-chain 1H and 15N assignments have been obtained using a combination of two-dimensional homonuclear and three-dimensional heteronuclear spectra. Regular elements of secondary structure have been identified on the basis of a qualitative interpretation of NOE data, J(NH-H alpha) coupling constants, and amide exchange rates. Three-dimensional structures were calculated from a total of 2146 experimental restraints using a combination of distance geometry and simulated annealing protocols. For the 13 best structures the average backbone (N, C alpha, C) atomic rmsd from the mean coordinates for residues 5-65 is 0.64 A (+/- 0.14 A) for the dimer and 0.50 A (+/- 0.08 A) for the individual monomers. Each monomer consists of a three-stranded antiparallel beta-sheet (residues 26-30, 38-43, 48-51) in a Greek key motif with a C-terminal helix (56-65) packed across the sheet, an arrangement similar to the monomeric structure of other members of this chemokine family (IL-8, PF4, MGSA/Gro alpha, and MIP-1 beta). Overall, the RANTES dimer resembles that previously reported for MIP-1 beta.

Interleukin 2 (IL-2) is one of the major cytokines produced by T lymphocytes in response to antigen. It is a potent growth and differentiation factor for several cell-types and is structurally related to the four-helix bundle family of cytokines. Mutation of residue Phe42 to Ala abolishes binding to the alpha chain of the tri-partite IL-2 receptor. The three-dimensional structure of the F42A mutant IL-2 has been calculated by two dimensional NMR methods and compared to a structure of wild-type IL-2 determined by X-ray crystallography. The overall topology of the two structures is the same. The main differences between the structures are within the ill-defined loops connecting the helices and the region of the protein that is believed to interact with the alpha-chain of the receptor. Thus, the mutation of Phe42 to Ala does not perturb the overall three-dimensional structure of IL-2, and does not appear to change the putative binding sites for the beta and gamma chains of the receptor. The structural differences observed in this mutant suggest that the replacement of Phe42 with Ala causes the re-orientation of neighbouring side-chains that are also involved in binding the alpha-chain of the receptor.

High-field NMR spectroscopy has been used to study the complex formed by the tetrasaccharide sialyl Lewis X and its receptor, E-selectin. Transferred NOEs demonstrate a specific interaction between the protein and ligand and enable measurement of the dissociation constant for the complex to be between approximately 1.1 and 2.0 mM. Differences between Overhauser spectra for free and bound sialyl Lewis X highlight a conformational change upon binding. This can be pinpointed to a change in the torsion angle of the glycosidic link between the sialyl and galactosyl residues and used to select a likely "bound" conformation from four low-energy species. Docking the bound form of sialyl Lewis X onto a model of the lectin domain of E-selectin suggests that the conformational change upon binding results primarily from steric interactions.

In chronic autoimmune thrombocytopenic purpura (AITP), an indirect monoclonal antibody immobilised platelet antigen (MAIPA) assay detected serum glycoprotein (GP) IIb/IIIa antibodies in 16/39 (41%) cases. In patients with clinically active AITP, a direct MAIPA assay detected platelet-associated GP IIb/IIIa kantibodies in 8/13 (62%) cases. Platelet bound and serum antibody concentrations suggested a high antibody affinity for platelet membrane glycoprotein IIb/IIIa. Five AITP patients with platelet associated glycoprotein IIb/IIIa antibodies were treated with intravenous anti D immunoglobulin. All showed an increase in platelet counts and a decrease in platelet associated autoantibody. These responses could be due to immunosuppressive anti-idiotype antibodies in anti D immunoglobulin.

The solution structure of the fibrinogen antagonist, echistatin, has been determined by a combination of NMR and simulated annealing methods. While the structure of the disulphide-linked core is well-defined by the NMR data, the N- and C-termini and the loop bearing the RGD sequence (which is responsible for the fibrinogen antagonist properties) are poorly defined. The pattern of disulphide bridges, which could not be determined by classical methods, was predicted by a statistical analysis of the simulated annealing structures. This pattern is distinct from that for the homologous protein kistrin, leading to the novel suggestion that homologous proteins possess non-conserved patterns of disulphide bridges.

Recombinant 15N-labeled human interleukin 2 (IL-2) has been studied by 2D and 3D NMR using uniformly 15N-labeled protein. Assignment of the backbone resonances has enabled the secondary structure of the protein to be defined. The secondary structure was found to consist of four alpha-helical regions and a short section of antiparallel beta-sheet. This structure is more similar to recent published structures of interleukin 4 and granulocyte-macrophage colony-stimulating factor than to a structure of IL-2 previously obtained from low-resolution X-ray diffraction data.

The 1H-NMR spectrum of the snake toxin echistatin has been assigned using homonuclear two-dimensional methods. Consideration of the NOE patterns, coupling constants and putative hydrogen bonds enabled two regular features of secondary structure to be deduced: a beta-sheet/turn between residues 8 and 13 and a small anti-parallel beta-sheet and bulge linking residues 16-20 with residues 30-33. The recognition region of the protein containing the residues RGD lies in a loop joining the two strands of the beta-sheet. The beta-bulge and the loop containing the RGD sequence undergo pH-dependent conformational interconversion, modulated by the side chain of Asp29.

The solution structure of transforming growth factor alpha has been determined by a combination of high-resolution 1H-nuclear magnetic resonance and distance geometry and restrained molecular dynamics. The 382 restraints derived from the NMR experiments were used to calculate many distance geometry structures, which were then refined by restrained molecular mechanics. Five of these structures were further refined using a variety of methods. Comparison of independently measured parameters, such as calculated hydrogen bonding patterns and experimental amide exchange rates, have been used to evaluate the accuracy of the structures. Also, possible mechanisms to explain the pH-dependent conformational interconversion observed are suggested. Finally comparisons between this work and others on this topic have been made.

The solution structure of human insulin-like growth factor 1 has been investigated with a combination of nuclear magnetic resonance and restrained molecular dynamics methods. The results show that the solution structure is similar to that of insulin, but minor differences exist. The regions homologous to insulin are well-defined, while the remainder of the molecule exhibits greater disorder. The resultant structures have been used to visualize the sites for interaction with a number of physiologically important proteins.

The 1H-NMR spectra of native human epidermal growth factor (EGF) and a derivative lacking the final five residues have been assigned by two-dimensional methods, enabling their structures to be compared. The same structural features are observed for each protein, although the final five residues of native human EGF interact with residues earlier in the sequence. Comparison of the resonance shifts of human, rat and mouse EGF and human transforming growth factor alpha (TGF alpha) enables shifts characteristic of the EGF conformation to be identified, providing standards by which the structures of related proteins may be assessed.

The solution structures of the homologous growth factors human epidermal growth factor (hEGF) and human transforming growth factor-alpha (hTGF-alpha), as determined by high resolution NMR and various computational methods, are described. Knowledge of these structures and the sequences of other homologous proteins leads to predictions about growth factor residues which may be involved in the receptor/ligand interface. Recent experiments designed to check these predictions are described briefly. These involve site-specific mutagenesis, receptor binding assays and high resolution NMR studies.

The role of leucine-47 in determining the structure and activity of human epidermal growth factor was examined using site-directed mutagenesis. Wild type protein and four variants in which Leu47 was replaced by valine, glutamate, aspartate and alanine were produced from yeast. 1H NMR experiments demonstrated that substitution of Leu47 had little effect on the protein structure. The observed reduction in receptor binding affinity caused by the substitutions could thus be attributed to perturbation of a residue directly involved in receptor interactions.

The conformations of the neuropeptide galanin in water and trifluoroethanol solutions have been examined by 1H NMR spectroscopy. Analysis of two-dimensional NMR experiments enabled the assignment of virtually all the 1H resonances of galanin in trifluoroethanol solution and many of the 1H resonances in aqueous solution. Interpretation of the NMR data in structural terms suggests that in trifluoroethanol galanin is predominantly helical while in water it does not adopt a fixed conformation.

The solution structures of the homologous growth factors hEGF and hTGF-alpha, have been determined independently from high resolution nuclear magnetic resonance (NMR) data. A model of the insulin-like growth factor structure based on insulin coordinates (Blundell et al. (1978) Proc natn. Acad. Sci. U.S.A. 75, 180-184), has also been refined using molecular dynamics simulations with NMR-determined restraints. Knowledge of these structures, together with known sequences of other homologous proteins and experiments with site-specific residue changes, allows predictions to be made about growth factor residues which might be involved in the receptor-ligand interfaces.

A peptide (S4) of the rat brain sodium channel has been synthesized, studied by high-resolution NMR and its secondary structure modelled by distance geometry and restrained molecular dynamics techniques.

A general discussion of knowledge dependence in risk calculations shows that the assumption of independence underlying standard Monte Carlo simulation in uncertainty analysis is frequently violated. A model is presented for performing Monte Carlo simulation when the variabilities of the component failure probabilities are either negatively or positively coupled. The model is applied to examples in human reliability analysis and the results are compared to the results of Sandia Laboratories as published in the Peer Review Study and to recalculations using more recent methods of uncertainty analysis.

The 500-MHz and 600-MHz 1H-NMR spectra of recombinant human transforming growth factor alpha have been recorded at pH values of 3.8, 6.5 and 9.4. Analysis of various two-dimensional spectra has enabled sequence-specific assignments to be made and the secondary structure to be identified. Information on the tertiary fold has also been obtained from observed nuclear Overhauser effects and titration of histidine residues. The overall fold of the protein is very similar to that of epidermal growth factor, as might be expected from the sequence similarity. However, the structure of transforming growth factor alpha at pH 3.8 is found to show interesting differences from those at the two higher pHs and from that of epidermal growth factor.

The structures of human epidermal growth factor (EGF) and human transforming growth factor alpha (TGF alpha) have been determined in solution using nuclear magnetic resonance techniques. The features of each structure are described and similarities and differences between them are discussed. The structures are combined with information from sequence homologies to produce a model of the receptor-recognition sites of EGF and TGF alpha, which can be tested in a site-directed mutagenesis programme. The model assists in explaining previous observations of sequence-activity relationships. The TGF alpha and EGF structures also serve as models for homologous modules in other extracellular proteins.

Results obtained in the past few years in the study of the reaction mechanism of plant RNA polymerases are reviewed and discussed. They suggest that valuable information can be obtained using a highly simplified transcription system composed of purified plant enzymes and cloned genes. This type of approach may provide a starting point for the development of an in vitro transcription system. The detailed study of the fundamental enzymatic properties of the plant RNA polymerases allows a comparison with the well documented corresponding bacterial enzyme.

Transcription of a cloned cauliflower mosaic virus (CaMV) DNA fragment (plasmid pCa 8) was studied at a low enzyme: DNA ratio. Preincubation with purine nucleoside triphosphates leads to essentially random transcription, while in the presence of a dinucleoside monophosphate and a purine nucleoside triphosphate in the preincubation medium certain combinations prime preferential transcription of the eucaryotic moiety of the chimeric plasmid. Characterisation of transcription primed by the most efficient combination, ApG + ATP, shows that a low enzyme: DNA ratio is absolutely essential for selective initiation. Interestingly the presence of the eucaryotic insertion is essential for the transcription of vector sequences. Analysis of RNA primed by ApG + ATP and of short chains synthesised in the presence of the GTP analogue 3'-OMeGTP shows a high degree of selectivity of transcription initiation sites. Hybridisation of primed RNA to restriction fragments of pCa8 shows that initiation occurs within a limited region of the inserted CaMV fragment.

500 MHz 1H NMR studies of human epidermal growth factor are described. The backbone resonances of the 1-48 derivative of hEGF have been assigned using two-dimensional techniques. Analysis of the type and magnitude of the observed sequential nuclear Overhauser effects and the NH-alpha CH spin-spin coupling constants allowed prediction of the secondary structure. Aspects of the tertiary structure are also identified. A pair of antiparallel beta-sheets involving residues 18-23 and 28-34 is a dominant feature of the solution structure.

Two-dimensional 1H-NMR methods have been used to assign heme and amino acid proton resonances in both isomeric states of the carbon monoxide complexes of two Glycera dibranchiata monomeric hemoglobins, HbA and HbB. For each hemoglobin, there are small differences in heme pocket structure in the two isomeric forms. The largest structural perturbations associated with heme isomerism involve residues close to pyrrole rings I and II. The positions relative to the heme of phenylalanine CD1 and the proximal histidine ligand are almost unaffected by heme isomerism. These residues probably play a key role in determining the location of the heme within the heme pocket.

The epidermal growth factors (EGFs) are powerful mitogens for a wide variety of cells in culture; human EGF (hEGF), known as urogastrone, also inhibits gastric acid secretion in vivo. The transforming growth factors (TGF-alpha) are related to the EGF family both in sequence and activity and EGF-like sequences are often observed in a wide range of functionally unrelated proteins. Attempts to examine the structure of EGF by diffraction methods have not yet succeeded because of difficulties with crystallization. We report here a three-dimensional structure of a biologically active derivative (residues 1-48) of the 53-residue human EGF. An analysis of high resolution 1H nuclear magnetic resonance (NMR) spectra was used together with a combination of distance geometry, restrained energy minimization and restrained molecular dynamics methods. The three-dimensional structure provides a basis for understanding the properties of EGFs and for predicting the structures of homologous sequences in other proteins.

Two-dimensional 1H-NMR methods have been used to assign side-chain resonances for the tryptophan residues and for several amino acids located in the heme pockets of the carbon monoxide complexes of the major monomeric hemoglobins from Glycera dibranchiata. The NMR spectra reveal a high degree of conservation of the heme pocket structure in the different hemoglobins. However some conformational differences are evident and residues at positions B10 and G8 on the distal side of the heme pocket are not conserved. From the present NMR studies it appears that the monomeric G. dibranchiata hemoglobin examined by X-ray crystallography [Padlan, E. A. & Love, W. (1974) J. Biol. Chem. 249, 4067-4078] corresponds to HbC. Except that the orientation of the heme in solution is the reverse of that reported in the crystal structure, there is a close correspondence between the heme pocket structure in the crystal and in solution. The proximal histidine coordination geometry is almost identical in the CO complexes of the three monomeric hemoglobins studied. Distal residues are strongly implicated in determining the observed kinetic differences in ligand binding reactions. In particular, steric crowding of the ligand binding site in hemoglobin A is probably a major factor in the slower kinetics of this component.

1H NMR studies of the carbon monoxide complexes of the major monomeric hemoglobins from Glycera dibranchiata show that distal leucine is conserved at position E7. The observed ring current shifts and nuclear Overhauser enhancements indicate conformational disorder of the leucine E7 side chain. The conformational substates interconvert rapidly on the NMR time scale. The rapid conformational fluctuations of leucine E7 may play a fundamental role in governing diffusion of ligands to the heme.