Photochemically enhanced binding of small molecules to the tumor necrosis factor receptor-1 inhibits the binding of TNF-alpha.

The binding of tumor necrosis factor alpha (TNF-alpha) to the type-1 TNF receptor (TNFRc1) plays an important role in inflammation. Despite the clinical success of biologics (antibodies, soluble receptors) for treating TNF-based autoimmune conditions, no potent small molecule antagonists have been developed. Our screening of chemical libraries revealed that N-alkyl 5-arylidene-2-thioxo-1,3-thiazolidin-4-ones were antagonists of this protein-protein interaction. After chemical optimization, we discovered IW927, which potently disrupted the binding of TNF-alpha to TNFRc1 (IC(50) = 50 nM) and also blocked TNF-stimulated phosphorylation of Ikappa-B in Ramos cells (IC(50) = 600 nM). This compound did not bind detectably to the related cytokine receptors TNFRc2 or CD40, and did not display any cytotoxicity at concentrations as high as 100 microM. Detailed evaluation of this and related molecules revealed that compounds in this class are "photochemically enhanced" inhibitors, in that they bind reversibly to the TNFRc1 with weak affinity (ca. 40-100 microM) and then covalently modify the receptor via a photochemical reaction. We obtained a crystal structure of IV703 (a close analog of IW927) bound to the TNFRc1. This structure clearly revealed that one of the aromatic rings of the inhibitor was covalently linked to the receptor through the main-chain nitrogen of Ala-62, a residue that has already been implicated in the binding of TNF-alpha to the TNFRc1. When combined with the fact that our inhibitors are reversible binders in light-excluded conditions, the results of the crystallography provide the basis for the rational design of nonphotoreactive inhibitors of the TNF-alpha-TNFRc1 interaction.

CL22 - a novel cationic peptide for efficient transfection of mammalian cells.

Condensing peptide-DNA complexes have great potential as nonviral agents for gene delivery. To date, however, such complexes have given transfection activities greatly inferior to adenovirus and somewhat inferior to cationic lipid-DNA complexes, even for cell lines and primary cells in vitro. We report here the identification of a novel condensing peptide, CL22, which forms DNA complexes that efficiently transfect many cell lines, as well as primary dendritic and endothelial cells. We report studies with sequence and structure variants that define some properties of the peptide that contribute to efficient transfection. We demonstrate that the superior transfection activity of CL22 compared with other DNA condensing peptides is conferred at a step after uptake of the complexes into cells. We show that CL22-DNA complexes have transfection activity that is at least equivalent to the best available nonviral agents.

In vitro uptake of polystyrene microspheres: effect of particle size, cell line and cell density.

Uptake of polycation-DNA particles is the first step in achieving gene delivery with non-viral vehicles. One of the important characteristics determining uptake of DNA particles is their size. Here we have characterized the ability of several cell lines to internalise labelled polystyrene microspheres of different sizes. All the cell lines tested ingested 20-nm microspheres avidly. With larger microspheres (93, 220, 560 and 1010 nm) cell type as well as growth related differences were observed. Whereas some cell lines (HUVEC, ECV 304 and HNX 14C) took up microspheres up to 1010 nm even when the cells were confluent, others did not take up many microspheres larger than 93 nm (Hepa 1-6 and HepG2). In one cell line (KLN 205), uptake of 93-, 220- and 560-nm microspheres was avid in growing cells, but not detectable when they were confluent. In KLN 205 cells, a good correlation was found between the uptake of 560-nm microspheres and the uptake of a peptide-DNA polyplex formulation, when it was prepared under conditions leading to small particle sizes. Little correlation was found when the polyplex formulation was allowed to aggregate.

NMR studies of tandem WW domains of Nedd4 in complex with a PY motif-containing region of the epithelial sodium channel.

Nedd4 (neuronal precursor cell-expressed developmentally down-regulated 4) is a ubiquitin-protein ligase containing multiple WW domains. We have previously demonstrated the association between the WW domains of Nedd4 and PPxY (PY) motifs of the epithelial sodium channel (ENaC). In this paper, we report the assignment of backbone 1H alpha, 1HN, 15N, 13C', 13C alpha, and aliphatic 13C resonances of a fragment of rat Nedd4 (rNedd4) containing the two C-terminal WW domains, WW(II+III), complexed to a PY motif-containing peptide derived from the beta subunit of rat ENaC, the betaP2 peptide. The secondary structures of these two WW domains, determined from chemical shifts of 13C alpha and 13C beta resonances, are virtually identical to those of the WW domains of the Yes-associated protein YAP65 and the peptidyl-prolyl isomerase Pin1. Triple resonance experiments that detect the 1H alpha chemical shift were necessary to complete the chemical shift assignment, owing to the large number of proline residues in this fragment of rNedd4. A new experiment, which correlates sequential residues via their 15N nuclei and also detects 1H alpha chemical shifts, is introduced and its utility for the chemical shift assignment of sequential proline residues is discussed. Data collected on the WW(II+III)-betaP2 complex indicate that these WW domains have different affinities for the betaP2 peptide.

Contributions to protein entropy and heat capacity from bond vector motions measured by NMR spin relaxation.

The backbone dynamics of both folded and unfolded states of staphylococcal nuclease (SNase) and the N-terminal SH3 domain from drk (drkN SH3) are studied at two different temperatures. A simple method for obtaining order parameters, describing the amplitudes of motion of bond vectors, from NMR relaxation measurements of both folded and unfolded proteins is presented and the data obtained for 15N-NH bond vectors in both the SNase and drkN SH3 systems analyzed with this approach. Using a recently developed theory relating the amplitude of bond vector motions to conformational entropy, the entropy change between the folded and unfolded forms of SNase is calculated on a per residue basis. It is noteworthy that the region of the molecule with the smallest entropy change includes those residues showing native-like structure in the unfolded form of the molecule, as established by NOE-based experiments. Order parameters of backbone 15N-NH bond vectors show significantly larger changes with temperature in the unfolded states of both proteins relative to the corresponding folded forms. The differential temperature dependence is interpreted in terms of differences in the heat capacities of folded and unfolded polypeptide chains. The contribution to the heat capacity of the unfolded chain from rapid 15N-NH bond vector motions is calculated and compared with estimates of the heat capacity of the backbone unit, -CHCONH-, obtained from calorimetric data. Methyl dynamics measured at 14 and 30 degrees C establish that the amplitudes of side-chain motions in the folded SH3 domain are more sensitive to changes in temperature than the backbone dynamics, suggesting that over this temperature range side-chain ps to ns time-scale motions contribute more to the heat capacity than backbone motions for this protein.

Pulse schemes for the measurement of 3JC'C gamma and 3JNC gamma scalar couplings in 15N,13C uniformly labeled proteins.

Pulse sequences are presented for the measurement of 3JC'C gamma and 3JNC gamma scalar couplings for all C gamma containing residues in 15N,13C uniformly labeled proteins. The methods described are based on quantitative J correlation spectroscopy pioneered by Bax and co-workers [Bax et. al. (1994) Methods Enzymol., 239, 79-105]. The combination of 3JC'C gamma and 3JNC gamma scalar coupling constants allows the assignment of discrete rotameric states about the chi 1 torsion angle in cases where such states exist or, alternatively, facilitates the establishment of noncanonical chi 1 conformations or the presence of rotameric averaging. The methods are applied to a 1.5 mM sample of staphylococcal nuclease.

Characterization of the backbone dynamics of folded and denatured states of an SH3 domain.

Measurements of 15N NMR relaxation parameters have been used to characterize the backbone dynamics of folded and denatured states of the N-terminal SH3 domain from the adapter protein drk, in high salt or guanidinium chloride, respectively. Values of the spectral density function evaluated at a number of frequencies are compared. The levels of backbone dynamics in the folded protein show little variation across the molecule and are of similar magnitude to those determined previously for the folded state of the protein in exchange with an unfolded state at low salt concentrations [Farrow et al. (1995) Biochemistry 34, 868-878]. The denatured state of the domain exhibits both more extensive and more heterogeneous dynamics than the folded state. In particular the profile of the spectral density function evaluated at zero-frequency for the unfolded state of the domain indicates that residues in the middle of the protein sequence are considerably less mobile than those at the termini. These data suggest that the molecule is not behaving as an extended polymer and that concerted motions of the central portions of the molecule are occurring, consistent with a reasonably compact conformation in this region. The backbone dynamics of the folded and unfolded states were studied at two temperatures. The level of high-frequency motions in the folded molecule is largely unaffected by changes in temperature, whereas an increase in temperature results in increased high-frequency motion in the unfolded state.

Structure and dynamics of bacteriophage IKe major coat protein in MPG micelles by solution NMR.

The structure and dynamics of the 53-residue filamentous bacteriophage IKe major coat protein in fully protonated myristoyllysophosphatidylglycerol (MPG) micelles were characterized using multinuclear solution NMR spectroscopy. Detergent-solubilized coat protein [sequence: see text] mimics the membrane-bound "assembly intermediate" form of the coat protein which occurs during part of the phage life cycle. NMR studies of the IKe coat protein show that the coat protein is largely alpha-helical, exhibiting a long amphipathic surface. helix (Asn 4 to Ser 26) and a shorter "micelle-spanning" C-terminal helix which begins at TRP 29 and continues at least to Phe 48. Pro 30 likely occurs in the first turn of the C-terminal helix, where it is ideally situated given the hydrogen bonding and steric restrictions imposed by this residue. The similarity of 15N relaxation values (T1, T2, and NOE and 500 MHz and T2 at 600 MHz) among much of the N-terminal helix and all of the TM helix indicates that the N-terminal helix is as closely associated with the micelle as the TM helix. The description of the protein in the micelle is supported by the observation of NOEs between lysolipid protons and protein amide protons between asn 8 and Ser 50. The N-terminal and TM helices exhibit substantial mobility on the microsecond to second time scale, which likely reflects changes in the orientation between the two helices. The overall findings serve to clarify the role of individual residues in the context of a TM alpha-helix and provide an understanding of the secondary structure, dynamics, and aqueous and micellar environments of the coat protein.

Correlation between dynamics and high affinity binding in an SH2 domain interaction.

Protein-protein interfaces can consist of interactions between large numbers of residues of each molecule; some of these interactions are critical in determining binding affinity and conferring specificity, while others appear to play only a marginal role. Src-homology-2 (SH2) domains bind to proteins containing phosphorylated tyrosines, with additional specificity provided by interactions with residues C-terminal to the phosphotyrosine (pTyr) residue. While the C-terminal SH2 domain of phospholipase C-gamma 1 (PLCC SH2) interacts with eight residues of a pTyr-containing peptide from its high affinity binding site on the beta-platelet-derived growth factor receptor, it can still bind tightly to a phosphopeptide containing only three residues. Novel deuterium (2H) based nuclear magnetic resonance (NMR) spin relaxation experiments which probe the nanosecond-picosecond time scale dynamics of methyl containing side chain residues have established that certain regions of the PLCC SH2 domain contacting the residues C-terminal to the pTyr have a high degree of mobility in both the free and peptide complexed states. In contrast, there is significant restriction of motion in the pTyr binding site. These results suggest a correlation between the dynamic behavior of certain groups in the PLCC SH2 complex and their contribution to high affinity binding and binding specificity.

Spectral density function mapping using 15N relaxation data exclusively.

A method is presented for the determination of values of the spectral density function, J(omega), describing the dynamics of amide bond vectors from 15N relaxation parameters alone. Assuming that the spectral density is given by the sum of Lorentzian functions, the approach allows values of J(omega) to be obtained at omega = 0, omega N and 0.870 omega H, where omega N and omega H are Larmor frequencies of nitrogen and proton nuclei, respectively, from measurements of 15N T1, T2 and 1H-15N steady-state NOE values at a single spectrometer frequency. Alternatively, when measurements are performed at two different spectrometer frequencies of i and j MHz, J(omega) can be mapped at omega = 0, omega iN, omega jN, 0.870 omega iH and 0.870 omega iH, where omega iN, for example, is the 15N Larmor frequency for a spectrometer operating at 1 MHz. Additionally, measurements made at two different spectrometer frequencies enable contributions to transverse relaxation from motions on millisecond-microsecond time scales to be evaluated and permit assessment of whether a description of the internal dynamics is consistent with a correlation function consisting of a sum of exponentials. No assumptions about the specific form of the spectral density function describing the dynamics of the 15N-NH bond vector are necessary, provided that dJ(omega)/d omega is relatively constant between omega = omega H + omega N to omega = omega H - omega N. Simulations demonstrate that the method is accurate for a wide range of protein motions and correlation times, and experimental data establish the validity of the methodology. Results are presented for a folded and an unfolded form of the N-terminal SH3 domain of the protein drk.

Rapid corepressor exchange from the trp-repressor/operator complex: an NMR study of [ul-13C/15N]-L-tryptophan.

[ul-13C/15N]-L-tryptophan was prepared biosynthetically and its dynamic properties and intermolecular interaction with a complex of Escherichia coli trp-repressor and a 20 base-pair operator DNA were studied by heteronuclear isotope-edited NMR experiments. The resonances of the free and bound corepressor (L-Trp) were unambiguously identified from gradient-enhanced 15N-1H HSQC, 13C-1H HSQC, 13C- and 15N-edited 2D NOESY spectra. The exchange off-rate of the corepressor between the bound and free states was determined to be 3.4 +/- 0.52 s-1 at 45 degrees C, almost three orders of magnitude faster than the dissociation of the protein-DNA complex. Examination of the experimental NOE buildup curves indicates that it may be desirable to use longer mixing times than would normally be used for a large molecule, in order to detect weak intermolecular NOEs in the presence of exchange. Intermolecular NOEs from bound corepressor to trp-repressor and DNA were analyzed with respect to the mechanism of ligand exchange. This analysis suggests that, in order for the ligand to diffuse out of the complex, there must be significant movement or 'breathing' of the protein and/or DNA.

Comparison of the backbone dynamics of a folded and an unfolded SH3 domain existing in equilibrium in aqueous buffer.

Two-dimensional NMR 15N relaxation studies have been used to characterize the backbone dynamics and folding transition of the N-terminal SH3 domain of the protein drk (drkN SH3). The isolated drkN SH3 domain exists in equilibrium between a folded and an unfolded state in aqueous buffer and near neutral pH [Zhang et al. (1994) J. Biomol. NMR 4, 845]. The backbone dynamics of both the folded and unfolded states in this exchanging system have been determined and the rates of the folding transition estimated at 14 degrees C. For 12 residues, the values of the spectral density functions of the backbone amide bond vectors at a number of frequencies have been established. Results show that while the unfolded state has considerably greater dynamic behavior, the overall motional properties are consistent with it having a reasonably compact structure in solution. In contrast to the folded state, considerable variations are seen in the values of the spectral densities of the unfolded state as a function of residue number; these variations do not appear to be strongly correlated with structural elements in the folded state. The mean value of the exchange rate associated with the folding transition was determined to be 0.89 s-1, similar to previous measurements of the rate of formation of beta-structure.

A heteronuclear correlation experiment for simultaneous determination of 15N longitudinal decay and chemical exchange rates of systems in slow equilibrium.

A heteronuclear correlation experiment is described which permits simultaneous characterization of both 15N longitudinal decay rates and slow conformational exchange rates. Data pertaining to the exchange between folded and unfolded forms of an SH3 domain is used to illustrate the technique. Because the unfolded form of the molecule, on average, shows significantly higher NH exchange rates than the folded form, an approach which minimizes the degree of water saturation is employed, enabling the extraction of accurate rate constants.

A structure for the signal sequence binding protein SRP54: 3D reconstruction from STEM images of single molecules.

The 54-kDa subunit SRP54 of the signal recognition particle in eukaryotic cells is responsible for the recognition of nascent proteins destined for secretion or membrane integration. The three-dimensional structure of this protein was determined using computational techniques applied to images of the molecule obtained via high-resolution, low-dose, scanning transmission electron microscopy at low temperature. The reconstructions at spatial resolutions between 12 and 15 A feature two unequal domains joined by a slender linker. The two-domain structure is in agreement with genetic and biochemical data indicating organization of SRP54 into a larger N-terminal GTP-binding region and a smaller C-terminal peptide-binding region. The structure has similarities to other protein domains with related functions and similar amino acid sequences. The larger domain of the 3D reconstruction is consistent in shape and size with the GTP-binding domains of EF-Tu and p21-RAS, while the smaller domain is compatible in structure with part of the peptide-binding protein calmodulin. The overall shape of SRP54 and the deduced location of critical functional regions of the molecule provide a structural framework for its known biochemical properties in the targeting cycle of the signal recognition particle.

Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation.

The backbone dynamics of the C-terminal SH2 domain of phospholipase C gamma 1 have been investigated. Two forms of the domain were studied, one in complex with a high-affinity binding peptide derived from the platelet-derived growth factor receptor and the other in the absence of this peptide. 2-D 1H-15N NMR methods, employing pulsed field gradients, were used to determine steady-state 1H-15N NOE values and T1 and T2 15N relaxation times. Backbone dynamics were characterized by the overall correlation time (tau m), order parameters (S2), effective correlation times for internal motions (tau e), and, if required, terms to account for motions on a microsecond-to-millisecond-time scale. An extended two-time-scale formalism was used for residues having relaxation data and that could not be fit adequately using a single-time-scale formalism. The overall correlation times of the uncomplexed and complexed forms of SH2 were found to be 9.2 and 6.5 ns, respectively, suggesting that the uncomplexed form is in a monomer-dimer equilibrium. This was subsequently confirmed by hydrodynamic measurements. Analysis of order parameters reveals that residues in the so-called phosphotyrosine-binding loop exhibited higher than average disorder in both forms of SH2. Although localized differences in order parameters were observed between the uncomplexed and complexed forms of SH2, overall, higher order parameters were not found in the peptide-bound form, indicating that on average, picosecond-time-scale disorder is not reduced upon binding peptide. The relaxation data of the SH2-phosphopeptide complex were fit with fewer exchange terms than the uncomplexed form. This may reflect the monomer-dimer equilibrium that exists in the uncomplexed form or may indicate that the complexed form has lower conformational flexibility on a microsecond-to-millisecond-time scale.

Effect of deletions 5' to the translation initiation sequence on the expression of an mRNA in animal cells.

To learn if an mRNA.18S rRNA interaction or a special secondary structure in the mRNA start region is essential for translation in eukaryotic cells, we constructed recombinant plasmids with the SV40 early promoter 5' to part of the Escherichia coli tufB-lacZ gene. Deletion of bases potentially complementary to the 18S rRNA highly increased the transient beta-galactosidase expressed in transfected CHO cells. Deletion of bases that fostered formation of potential hairpins with the mRNA 5'-terminus or altered the structure of the coding region reduced beta-galactosidase activity suggesting that these features of the mRNA secondary structure may be essential for initiation of translation. Computer aided analysis of the potential structure of 290 mRNAs suggests these are conserved features of the initiation region.

Metabolic disorders of the brain in chronic hepatic encephalopathy detected with H-1 MR spectroscopy.

Proton magnetic resonance (MR) spectroscopy of the brain was performed in 11 patients with chronic hepatic encephalopathy (CHE), and the results were compared with those of patients with liver disease but without CHE; clinical control subjects with diabetes, uremia, or cortical atrophy; and healthy subjects. The technique of water-suppressed stimulated-echo hydrogen-1 MR spectroscopy for detection of cerebral glutamate, glutamine, glucose, N-acetylaspartate, choline metabolites, (phospho)creatine, and myo-inositol is described. Specific changes in the brain of CHE patients included the anticipated elevation in cerebral glutamine levels (P less than or equal to .0001), a 23% reduction in choline metabolite levels (P less than or equal to .0001), and a more than 50% reduction in cerebral myo-inositol levels (P less than or equal to .0001). In four of the 15 patients with liver disease but without clinical CHE, a significant reduction in the myo-inositol level was detected, and in two of these patients an elevation in the glutamine concentration was also observed. These findings indicate a role for image-guided H-1 MR spectroscopy in the diagnosis and monitoring of both overt and preclinical CHE.

A 15N-NMR study of isolated brain in portacaval-shunted rats after acute hyperammonemia.

Acute hyperammonemia was induced by 15NH4+ infusion in portacaval-shunted (PCS) and control rats to investigate its effects on cerebral metabolism of glutamine, glutamate and gamma-aminobutyrate. Cerebral 15N-metabolites were observed by 15N-NMR spectroscopy in the ex vivo brain, removed in toto at the end of infusion. Key 15N-metabolites in the brain and liver were quantitated and their specific activities measured by NMR and biochemical assays in perchloric acid extracts of the freeze-clamped organs. In the ex vivo brain, [gamma-15N]glutamine, present at tissue concentrations of 3-5 mumol/g with 15N enrichment of 36-48%, was observable within 6-13 min of data acquisition. [alpha-15N]glutamine/glutamate, each present at 0.5-1 mumol/g (approx. 10% enrichment), were observed in 27 min. The results demonstrate the feasibility of observing these cerebral metabolites by 15N-NMR within a physiological time scale. In a rat pretreated with glutamine synthetase inhibitor, L-methionine DL-sulfoximine, cerebral [15N]gamma-aminobutyrate was observed after 910 min. In PCS rats, decreased 15NH4+ removal in the liver was accompanied by formation of approx. 2-fold higher concentration of cerebral [gamma-15N]glutamine relative to that in weight-matched controls. The result suggests that increased diffusion of blood-borne 15NH3 into the brain led to increased [gamma-15N]glutamine synthesis in astrocytes as well as ammonia-mediated inhibition of glutaminase.

A 15N-n.m.r. study of cerebral, hepatic and renal nitrogen metabolism in hyperammonaemic rats.

1. Rats were infused with 15NH4+ or L-[15N]alanine to induce hyperammonaemia, a potential cause of hepatic encephalopathy. HClO4 extracts of freeze-clamped brain, liver and kidney were analysed by 15N-n.m.r. spectroscopy in combination with biochemical assays to investigate the effects of hyperammonaemia on tissue concentrations of ammonia, glutamine, glutamate and urea. 2. 15NH4+ infusion resulted in a 36-fold increase in the concentration of blood ammonia. Cerebral glutamine concentration increased, with 15NH4+ incorporated predominantly into the gamma-nitrogen atom of glutamine. Incorporation into glutamate was very low. Cerebral ammonia concentration increased 5-10-fold. The results suggest that the capacity of glutamine synthetase for ammonia detoxification was saturated. 3. Pretreatment with the glutamine synthetase inhibitor L-methionine DL-sulphoximine resulted in 84% inhibition of [gamma-15N]glutamine synthesis, but incorporation of 15N into other metabolites was not observed. The result suggests that no major alternative pathway for ammonia detoxification, other than glutamine synthetase, exists in rat brain. 4. In the liver 15NH4+ was incorporated into urea, glutamine, glutamate and alanine. The specific activity of 15N was higher in the gamma-nitrogen atom of glutamine than in urea. A similar pattern was observed when [15N]alanine was infused. The results are discussed in terms of the near-equilibrium states of the reactions involved in glutamate and alanine formation, heterogeneous distribution in the liver lobules of the enzymes involved in ammonia removal and their different affinities for ammonia. 5. Synthesis of glutamine, glutamate and hippurate de novo was observed in kidney. Hippurate, as well as 15NH4+, was contributed by co-extracted urine. 6. The potential utility and limitations of 15N n.m.r. for studies of mammalian metabolism in vivo are discussed.