Design, high-level expression, purification and characterization of soluble fragments of the hepatitis C virus NS3 RNA helicase suitable for NMR-based drug discovery methods and mechanistic studies.

RNA helicases represent a family of enzymes that unwind double-stranded (ds) RNA in a nucleoside triphosphate (NTP)-dependent fashion and which are required in all aspects of cellular RNA metabolism and processing. The hepatitis C virus (HCV) non-structural 3 (NS3) protein possesses a serine protease activity in the N-terminal one-third, whereas RNA-stimulated NTPase and helicase activities reside in the C-terminal portion of the 631 amino acid residue bifunctional enzyme. The HCV NS3 RNA helicase is of key importance in the life cycle of HCV, which makes it a target for the development of therapeutics. However, neither the precise mechanism nor the substrate structure has been defined for this enzyme. For nuclear magnetic resonance (NMR)-based drug discovery methods and for mechanistic studies we engineered, prepared and characterized various truncated constructs of the 451-residue HCV NS3 RNA helicase. Our goal was to produce smaller fragments of the enzyme, which would be amenable to solution NMR techniques while retaining their native NTP and/or nucleic acid binding sites. Solution conditions were optimized to obtain high-quality heteronuclear NMR spectra of nitrogen-15 isotope-labeled constructs, which are typical of well-folded monomeric proteins. Moreover, NMR binding studies and functional data directly support the correct folding of these fragments.

Structure of the Ras-binding domain of c-Raf-1 as determined by NMR spectroscopy and identification of the region that interacts with Ras.

The structure of the Ras-binding domain of human c-Raf-1 (residues 55 to 132) as determined in solution by NMR spectroscopy is presented. It consists of a five-stranded beta-sheet, a twelve residue alpha-helix, and an additional one-turn helix. The fold belongs to a known family whose members include ubiquitin and protein G. The surface of Raf55-132 that interacts with Ras has been identified by resonance perturbation mapping. The binding site is a spatially contiguous patch comprised of the two-N-terminal beta-strands, the loop between them, and the C-terminal end of the alpha-helix. A model of the Raf-Ras complex is presented, which was derived by analogy to the complex between protein G and a Fab fragment of IgG. In the model, edge beta-strands of each protein align in an antiparallel orientation, forming a unified beta-sheet, and side chains from both proteins are able to participate in ionic and hydrophobic interactions at the interface.

Peptide mimetics of thyrotropin-releasing hormone based on a cyclohexane framework: design, synthesis, and cognition-enhancing properties.

The design and synthesis of peptide mimetics of thyrotropin-releasing hormone (TRH) in which the peptide backbone is entirely replaced by a cyclohexane framework are described. The cis-1,3,5-trisubstituted ring was expected to permit key pharmacophoric groups to adopt conformations consistent with proposed bioactive conformations of the peptide. Compounds were synthesized by a stereoselective synthesis starting from L-glutamic acid. In a behavioral model of cognition in which TRH is active, the mimetics are potent, active compounds, exhibiting oral activity. One analog (26, (1S,3R,5(2S),5S)-5-[[5-oxo-1-(phenylmethyl)-2-pyrrolidinyl]-methyl]-5- [(1H-imidazol-5-yl)methyl]cyclohexaneacetamide) was radiolabeled for binding studies and evaluated in other binding assays and pharmacological tests. Competition binding of 26 vs [3H]MeTRH to rat brain slices suggests a two-site model for ligand binding with IC50's of 1 microM and 3 mM. Direct binding of [3H]-26 shows a biphasic curve with IC50's of 80 and 49 microM, respectively. Further studies would be needed to establish a link between the novel binding site(s) and the behavioral activity of 26 and TRH analogs.

Solution structure of the Ras-binding domain of c-Raf-1 and identification of its Ras interaction surface.

The structure of the Ras-binding domain of human c-Raf-1 (residues 55-132) has been determined in solution by nuclear magnetic resonance (NMR) spectroscopy. Following complete assignment of the backbone and side-chain 1H, 15N, and 13C resonances, the structure was calculated using the program CHARMM. Over 1300 NOE-derived constraints were applied, resulting in a detailed structure. The fold of Raf55-132 consists of a five-stranded beta-sheet, a 12-residue alpha-helix, and an additional one-turn helix. It is similar to those of ubiquitin and the IgG-binding domain of protein G, although the three proteins share very little sequence identity. The surface of Raf55-132 that interacts with Ras has been identified by monitoring perturbation of line widths and chemical shifts of 15N-labeled Raf55-132 resonances during titration with unlabeled Ras-GMPPNP. The Ras-binding site is contained within a spatially contiguous patch comprised of the N-terminal beta-hairpin and the C-terminal end of the alpha-helix.

Structure-function analysis of human IL-1 alpha: identification of residues required for binding to the human type I IL-1 receptor.

Using oligonucleotide-directed mutagenesis, the binding site on human interleukin-1 alpha (IL-1 alpha) for the human type I IL-1 receptor (IL-1R) has been analyzed. Substitution of seven amino acids (Arg12, Ile14, Asp60, Asp61, Ile64, Lys96 and Trp109) resulted in a significant loss of binding to the receptor. Based on crystallographic information, the side chains of these residues are clustered in one region of IL-1 alpha and exposed on the surface of the protein. Five of the residues in the IL-1 alpha binding site align with the binding residues previously determined in human IL-1 beta, demonstrating that the type I IL-1R recognizes homologous regions in both ligands. Unexpectedly, only three of the aligned residues are identical between IL-1 alpha and IL-1 beta. These observations suggest that the composition of contact residues in the binding site is unique for each ligand-receptor complex in the IL-1 system.

Solution structures of cyclic and dicyclic analogues of growth hormone releasing factor as determined by two-dimensional NMR and CD spectroscopies and constrained molecular dynamics.

Solution structures were determined for a linear analogue of growth hormone releasing factor (GRF), and cyclic and dicyclic analogues in which the side chains of aspartyl and lysyl residues spaced at positions i-(i + 4) were joined to form a lactam. The four analogues were [Ala15]-GRF-(1-29)-NH2 and its cyclo8-12, cyclo21-25, and dicyclo8-12;21-25 derivatives. The peptides were studied in two solvent systems: 75% methanol/25% water at pH 6.0; and 100% water at pH 3.0. CD spectroscopy was used to assess the overall alpha-helical content. Nuclear magnetic resonance spectroscopy was used to determine the structures in more detail. Nearly complete proton resonance assignments were made for each of the peptides, in both solvents. Nuclear Overhauser effects were converted into distance constraints and applied in the molecular dynamics program CHARMM to evaluate the range of low-energy structures that satisfied the nmr data. In 75% methanol, all of the peptides are comprised of a single alpha-helical segment with fraying of one to three residues at each end. The linear analogue has a tendency to kink. In water, the analogues have two helical segments with flexible regions between them and at the termini of the peptides. The linear analogue is helical at residues 7-14 and 21-28. In the cyclo8-12 analogue, the N-terminal helical region extends to include residues 7-19, while the other helical region is slightly shortened. In the cyclo21-25 analogue, the C-terminal helical region is extended to include residues 19-28, while the N-terminal helical region is destabilized. The dicyclic analogue has the largest N-terminal helix, spanning residues 7-20, but its helical segment at residues 21-28 is not well ordered. All of the analogues exhibit substantial biological activity. The cyclic and dicyclic analogues show dramatically increased resistance to degradation during incubation with human plasma. The i-(i + 4) lactam, therefore, appears to be a synthetic means of stabilizing a local alpha-helical conformation, which may be of general use in the design of active, stable peptides.

Identification of the discontinuous binding site in human interleukin 1 beta for the type I interleukin 1 receptor.

Human interleukin 1 beta (IL-1 beta) exerts its diverse biological effects by binding to specific receptors on target cells. Two types of IL-1 receptor (IL-1R) have been identified: the type I IL-1R (p80) and the type II IL-1R (p68). Using site-specific mutagenesis, we have identified the binding site on IL-1 beta for the murine type I IL-1R. Analogs of the IL-1 beta protein containing defined amino acid substitutions were produced and tested for competitive binding to the two IL-1Rs. Substitutions of the amino acids at seven positions resulted in analogs that had greater than or equal to 100-fold reductions in competitive binding to the type I IL-1R, while maintaining substantial binding to the type II IL-1R. These seven amino acids (Arg-4, Leu-6, Phe-46, Ile-56, Lys-93, Lys-103, and Glu-105) are clustered in the IL-1 beta molecule, forming a discontinuous binding site. The side chains of all seven residues are exposed on the surface of IL-1 beta. The cumulative binding energies contributed by each of the residues predict a binding affinity that is consistent with the observed Kd of the wild-type protein for the type I IL-1R.

Three-dimensional structure of porcine C5adesArg from 1H nuclear magnetic resonance data.

Two-dimensional nuclear magnetic resonance spectra of porcine C5adesArg (73 residues) have been used to construct a list of 34 hydrogen bonds, 27 dihedral angle constraints, and 151 distance constraints, derived from nuclear Overhauser effect data. These constraints were used in restrained molecular dynamics calculations on residues 1-65 of C5a, starting from a folded structure modeled on the crystal structure of a homologous protein, C3a. Forty-one structures have been calculated, which fall into three similar families with few violations of the imposed constraints. Structures in the most populated family have a root-mean-square deviation from the average structure of 1.02 A for the C alpha atoms, with good definition of the internal residues. There is good agreement between the calculated structures and other nuclear magnetic resonance data. The structure is very similar to that recently reported for human C5a [Zuiderweg et al. (1989) Biochemistry 28, 172-185]. Some biological implications of these structures are discussed.

Structure of interleukin 1 alpha at 2.7-A resolution.

The interleukin 1 (IL-1) family of proteins has a central role in modulating immune and inflammatory responses. Two major IL-1 proteins, designated alpha (IL-1 alpha) and beta (IL-1 beta), are produced by activated macrophages and other cell types. In an effort to understand the similarities and differences in the physicochemical and functional properties of these two proteins, a program was initiated to determine their structures. Crystals of IL-1 alpha were grown, and the three-dimensional structure at 2.7-A resolution was solved. The technique of multiple-wavelength anomalous dispersion (MAD) with the selenomethionine form of IL-1 alpha was utilized in combination with a single mercury derivative to provide the starting phases. Partial refinement of the IL-1 alpha model has been performed as well. The overall structure is composed of 14 beta-strands and a 3(10) helix. The core of this structure is a capped beta-barrell that possesses 3-fold symmetry and displays a topology similar to that observed for IL-1 beta [Priestle, J. P., et al. (1988) EMBO J. 7, 339-343] and soybean trypsin inhibitor (STI) [McLachlan, A. D. (1979) J. Mol. Biol. 133, 557-563]. In this paper, the overall structure of IL-1 alpha and the nature and fidelity of the internal 3-fold symmetry are discussed. Comparisons with IL-1 beta and STI are made within these contexts.

Solution structure of an analogue of vasoactive intestinal peptide as determined by two-dimensional NMR and circular dichroism spectroscopies and constrained molecular dynamics.

Structures have been determined for a potent analogue of vasoactive intestinal peptide (VIP), Ac-[Lys12, Lys14, Nle17, Val26, Thr28]VIP (VIP'), in methanol/water solutions. In CD studies, both VIP and VIP' were helical in methanol/water, with the percentage of alpha-helix increasing with percentage methanol. The pH had little effect on the structure. Complete 1H NMR assignments were made for VIP' in 25% methanol at pH 4 and 6 and in 50% methanol at pH 6, using two-dimensional COSY, NOESY, and relay-COSY experiments. There were no widespread changes in chemical shifts between the samples at pH 4 and 6; however, widespread changes were observed between the samples in 25% and 50% methanol. Complete sets of NOEs were obtained for VIP' in 25% methanol, pH 4, and in 50% methanol, pH 6. These NOEs were converted into distance constraints and applied in molecular dynamics and energy minimization calculations using the program CHARMM. A set of low-energy structures was obtained for VIP' in each solvent system. In 25% methanol, VIP' has two helical segments at residues 9-17 and 23-28. The remainder of the structure is not well determined. In 50% methanol, residues 8-26 form a regular, well-defined alpha-helix and residues 5-8 form a type III beta-turn. The remaining residues are not ordered. These structural assessments agree with the CD data. In the lowest energy structure in 50% methanol, the side chains of Asp3, Phe6, Thr7, and Tyr10 are clustered together--these residues are conserved throughout the family of peptide hormones homologous to VIP.