The solution structure of the N-terminal domain of hepatocyte growth factor reveals a potential heparin-binding site.

BACKGROUND: Hepatocyte growth factor (HGF) is a multipotent growth factor that transduces a wide range of biological signals, including mitogenesis, motogenesis, and morphogenesis. The N-terminal (N) domain of HGF, containing a hairpin-loop region, is important for receptor binding and the potent biological activities of HGF. The N domain is also the primary binding site for heparin or heparan sulfate, which enhances, receptor/ligand oligomerization and modulates receptor-dependent mitogenesis. The rational design of artificial modulators of HGF signaling requires a detailed understanding of the structures of HGF and its receptor, as well as the role of heparin proteoglycan; this study represents the first step towards that goal. RESULTS: We report here a high-resolution structure of the N domain of HGF. This first structure of HGF reveals a novel folding topology with a distinct pattern of charge distribution and indicates a possible heparin-binding site. CONCLUSIONS: The hairpin-loop region of the N domain plays a major role in stabilizing the structure and contributes to a putative heparin-binding site, which explains why it is required for biological functions. These results suggest several basic and/or polar residues that may be important for use in further mutational studies of heparin binding.

A 2H-NMR study of Acholeplasma laidlawii membranes highly enriched in myristic acid.

Myristic acid specifically deuterated at several positions along the acyl chain was biosynthetically incorporated into the membrane lipids of Acholeplasma laidlawii B to the level of greater than or equal to 90%. 2H-NMR was used to study the molecular order and lipid phase composition of the membranes as a function of temperature. Isolated membranes and intact cells give rise to similar 2H spectra. Below 25 degrees C the spectra exhibit a broad gel phase component which at 0 degrees C reaches the rigid limit value expected for an immobilized methylene group. Spectral moments were used to determine the relative amounts of gel and liquid crystalline phase lipids throughout the gel-liquid crystal phase transition. The results indicate that at the growth temperature (37 or 30 degrees C) the A. laidlawii B membrane lipids are approximately 85-90% in the gel state, and that protein has little effect on lipid order of the liquid crystalline lipid, but leads to an increase in the linewidth by approx. 20%.

A 31P-NMR study of structural and functional aspects of phosphate and phosphonate distribution in Tetrahymena.

31P-NMR has been used to study the chemical nature of cytoplasmic components of live Tetrahymena in a non-invasive manner. The technique has further been used to characterized the physical behaviour of lipids extracted from this organism. In particular, we have shown the presence of large quantities of pyrophosphate and of tripolyphosphate in acid extracts of the organism. These are not detectable in the live cells due to the motionally rigid nature of the storage granules. We have characterized the distribution of phosphonic acids in the organism and followed the phase behavior of the extracted cell lipids. Aqueous dispersions of extracted lipid show both bilayer and non-bilayer behaviour in the range of the growth temperature. The phosphonolipid in Tetrahymena appears to play a role similar to that of phosphatidylethanolamine in regulating the phase behaviour of the membrane. The high degree of unsaturation in the fatty acids of Tetrahymena is most likely responsible for the polymorphic phase behaviour observed near the growth temperature.

Solution structure, domain features, and structural implications of mutants of the chromo domain from the fission yeast histone methyltransferase Clr4.

The encapsulation of otherwise transcribable loci within transcriptionally inactive heterochromatin is rapidly gaining recognition as an important mechanism of epigenetic gene regulation. In the fission yeast Schizosaccharomyces pombe, heterochromatinization of the mat2/mat3 loci silences the mating-type information encoded within these loci. Here, we present the solution structure of the chromo domain from the cryptic loci regulator protein Clr4. Clr4 is known to regulate silencing and switching at the mating-type loci and to affect chromatin structure at centromeres. Clr4 and its human and Drosophila homologs have been identified as histone H3-specific methyltransferases, further implicating this family of proteins in chromatin remodeling. Our structure highlights a conserved surface that may be involved in chromo domain-ligand interactions. We have also analyzed two chromo domain mutants (W31G and W41G) that previously were shown to affect silencing and switching in full-length Clr4. Both mutants are significantly destabilized relative to wild-type.

Solution structure of interleukin-13 and insights into receptor engagement.

The complex and interrelated function of the interleukin cytokines relies on a range of pro-inflammatory and anti-inflammatory immune responses mediated by an array of receptors, and there is considerable cross-reactivity for related cytokines. Recent findings continue to elucidate the expression patterns of interleukin receptors associated with a range of diseases, including cancer. We report here the first experimentally determined high-resolution structure of human interleukin-13 (IL-13). The experimental structure is significantly different from an earlier homology model, which could have led to improper estimation of receptor interaction surfaces and design of mutational experiments. Similarities between the presented IL-13 structure and the homologous interleukin-4 (IL-4) are discussed. Additionally, mutation data for IL-4 and IL-13 are analyzed and combined with a detailed structural analysis of the IL-4/IL4Ralpha interface that leads us to postulate interactions at the IL-13/receptor interface. The structural comparison is used to interpret the different affinities for various receptors and establishes the basis for further mutational experiments and antagonist design.

Solution structure of the human Hck SH3 domain and identification of its ligand binding site.

SH3 domains are protein binding domains that occur widely among signal transduction proteins. Here, we present the NMR-determined solution structure of the SH3 domain from the cytoplasmic protein tyrosine kinase, Hck. Hck is involved in a number of cell signal transduction pathways, frequently in pathways associated with immune response. SH3 domains bind proteins via a left-handed polyproline type II helix on the target protein. We have assessed the structural impact of binding to a ligand through addition of a peptide corresponding to a proline-rich region of a Hck target, the GTPase activating protein of the Ras pathway. Ligand binding effects small structural changes and stabilizes the SH3 domain structure. Also, we have compared the solution structure of the Hck SH3 domain to the crystal structure of Hck, in which the SH3 domain exhibits an intramolecular binding to an interdomain linker region. These structures are interpreted as the apo- and holo- forms of the Hck SH3 domain.

Dynamics of the Hck-SH3 domain: comparison of experiment with multiple molecular dynamics simulations.

Molecular dynamics calculations provide a method by which the dynamic properties of molecules can be explored over timescales and at a level of detail that cannot be obtained experimentally from NMR or X-ray analyses. Recent work (Philippopoulos M, Mandel AM, Palmer AG III, Lim C, 1997, Proteins 28:481-493) has indicated that the accuracy of these simulations is high, as measured by the correspondence of parameters extracted from these calculations to those determined through experimental means. Here, we investigate the dynamic behavior of the Src homology 3 (SH3) domain of hematopoietic cell kinase (Hck) via 5N backbone relaxation NMR studies and a set of four independent 4 ns solvated molecular dynamics calculations. We also find that molecular dynamics simulations accurately reproduce fast motion dynamics as estimated from generalized order parameter (S2) analysis for regions of the protein that have experimentally well-defined coordinates (i.e., stable secondary structural elements). However, for regions where the coordinates are not well defined, as indicated by high local root-mean-square deviations among NMR-determined structural family members or high B-factors/low electron density in X-ray crystallography determined structures, the parameters calculated from a short to moderate length (less than 5-10 ns) molecular dynamics trajectory are dependent on the particular coordinates chosen as a starting point for the simulation.

Sequential assignments and secondary structure of the RNA-binding transcriptional regulator NusB.

The NusB protein is involved in transcriptional regulation in bacteriophage lambda. NusB binds to the RNA form of the nut site and along with N, NusA, NusE and NusG, stabilizes the RNA polymerase transcription complex and allows stable, persistent antitermination. NusB contains a 10 residue Arg-rich RNA-binding motif (ARM) at the N-terminus but is not sequentially homologous to any other proteins. In contrast to other known ARM-containing proteins, NusB forms a stable structure in solution in the absence of RNA. NMR spectroscopy was used to determine that NusB contains six alpha-helices: R10-Q21, 127-F34, V45-L65, Q79-S93, Y100-F114 and D118-L127. The structure of NusB makes it a member of a newly emerging class of alpha-helical RNA-binding proteins.

Insights into the structure of hepatocyte growth factor/scatter factor (HGF/SF) and implications for receptor activation.

The modular structure of HGF/SF offers a reductionist or 'divide and rule' approach to the analysis of structure and function. Domain deletion experiments have established that the N domain, kringle 1 and kringle 2 are essential for HGF/SF activity and that truncated variants containing the N domain and kringle 1 (NK1) or kringles 1 and 2 (NK2) can exhibit partial agonistic or antagonistic activity depending on target cells. Comparative modelling has been used to predict the 3D structures of the six domains of HGF/SF. More recently, NMR methods have shown that the N domain has a novel fold, the charge distribution of which suggests a heparin binding site. Crystals of NK1 indicate the relationship of this domain to the kringle 1, offering further insights into the mechanism of domain interactions and receptor activation.

Intramolecular binding of a proximal PPII helix to an SH3 domain in the fusion protein SH3Hck : PPIIhGAP.

SH3 domains are a conserved feature of many nonreceptor protein tyrosine kinases, such as Hck, and often function in substrate recruitment and regulation of kinase activity. SH3 domains modulate kinase activity by binding to polyproline helices (PPII helix) either intramolecularly or in target proteins. The preponderance of bimolecular and distal interactions between SH3 domains and PPII helices led us to investigate whether proximal placement of a PPII helix relative to an SH3 domain would result in tight, intramolecular binding. We have fused the PPII helix region of human GAP to the C-terminus of Hck SH3 and expressed the recombinant fusion protein in Escherichia coli. The fusion protein, SH3Hck : PPIIhGAP, folded spontaneously into a structure in which the PPII helix was bound intramolecularly to the hydrophobic crevice of the SH3 domain. The SH3Hck : PPIIhGAP fusion protein is useful for investigating SH3: PPII helix interactions, for studying concepts in protein folding and design, and may represent a protein structural motif that is widely distributed in nature.

The selective estrogen receptor modulator, raloxifene: segment II studies in rats and rabbits.

Raloxifene is a nonsteroidal, selective estrogen receptor modulator developed by Eli Lilly and Company primarily as a therapeutic agent for postmenopausal osteoporosis. Two Segment II studies were conducted that examined maternal reproductive parameters and fetal outcome following gestational exposure to raloxifene. Pregnant CD rats (25/group) and New Zealand white rabbits (20/group) were dosed once daily by oral gavage with 0, 0.1, 1, or 10 mg/kg on Gestation Days (GD) 6 through 17 and 7 through 19, respectively. Maternal body weight and food consumption were monitored throughout pregnancy. Caesarean sections were performed on GD 20 and GD 28 for rats and rabbits, respectively, to evaluate fetal viability, weight, and morphology. In rats, maternal body weight, body weight gain, and food consumption were reduced in all raloxifene treatment groups. Fetal viability was depressed in the 10-mg/kg group and was often associated with signs of hemorrhaging from the vagina. Fetal growth retardation was indicated in the 1- and/or 10-mg/kg groups by increased incidences of fetal runts and the developmental deviations, wavy ribs and kidney cavitation. There was no evidence of treatment-related malformations in rat fetuses. In rabbits, depressions in body weight gain and food consumption occurred in the 10-mg/kg group, and a single abortion occurred in the 1-mg/kg group. Fetal viability and weights were not affected in any of the raloxifene treatment groups. The overall proportions of fetuses with malformations, deviations, or variations were not affected by treatment with raloxifene; however, one fetus each from the 0.1-, 1-, and 10-mg/kg groups had incomplete closure of the interventricular septum. Therefore, maternal and fetal no-effect levels were not obtained in this study of raloxifene.

The selective estrogen receptor modulator, raloxifene: reproductive assessments in adult male rats.

Raloxifene HCl is a nonsteroidal, selective estrogen receptor modulator developed for postmenopausal osteoporosis. Reproductive toxicity of raloxifene was examined in adult male CD rats after the oral administration of doses of 0, 10, 30, or 100 mg/kg/d. In the first study, males (12/group) were treated for 2 weeks followed by 2 weeks without treatment. After dose administration on Day 13, 6 males/group were cohabited with untreated females (1:2) for up to 7 d. Males were killed on Day 14 or 28 (6/group each day). Sperm were collected from the right cauda epididymis and evaluated for relative concentration, motion characteristics, and breakage. The kinetics of spermatogenesis were examined by DNA flow cytometry. The left testis and epididymis were preserved for histopathologic evaluation. Females were examined for reproductive status on Gestation Day 13. In a second study, males (20/group) were treated for 7 weeks (4 weeks prior to cohabitation during a 2-week cohabitation period, and for 1 additional week). Treated males were cohabited with untreated females (1:1). On Gestation Day 20, untreated females were examined for reproductive status and fetuses were examined for viability, weight, gender, and morphology. At necropsy, male reproductive tissues were collected, weighed, and preserved for histopathologic evaluation. In both studies, male body weight gain and food consumption were depressed at all dose levels. There was no indication in either study that raloxifene caused important changes in sperm production, sperm quality, or male reproductive performance at doses as high as 100 mg/kg/d.

Developmental toxicity of the dopamine agonist pergolide mesylate in CD-1 mice. I: Gestational exposure.

Pergolide was given by oral gavage to mated CD-1 female mice at doses of 0, 1, 20, or 60 mg/kg/day on gestation days (GD) 6-15. Animals assigned to the teratology segment were killed on GD 18 for evaluation of maternal organ weights, and fetal viability, weights and morphology. Animals assigned to the postnatal segment were allowed to deliver and physical development and behavioral performance of the progeny were monitored until weaning. Maternal organ weights were collected at termination after weaning. One F1 offspring per sex per litter was maintained for postweaning physical, behavioral and reproductive assessments and for terminal examinations and organ weight evaluations. No adverse effects of pergolide treatment were found in the 1 mg/kg/day group. Dose-related hyperactivity, chewing and squinting that were consistent with dopaminergic stimulation occurred following dosing in the 20 and 60 mg/kg/day groups; F0 body weights and food consumption were reduced during the initial phase of treatment in the 60 mg/kg/day group. Gravid uterine weights and fetal weights were decreased in the 60 mg/kg/day group of the teratology segment, but there was no indication of teratogenicity in any group. Mammary inflammation, attributed to increased progeny suckling, occurred during the second week postpartum in a few postnatal segment females of the 20 and 60 mg/kg/day groups. Mean negative geotaxis performance was delayed slightly, but mean progeny survival and body weights were not affected. Although after weaning the F1 offspring from the treatment-derived groups tended to weigh more than controls and to perform more effectively in the active avoidance task, these findings were attributed to unusually low values obtained in the control group. Startle amplitudes were increased significantly in the males from the 60 mg/kg/day treatment-derived group. These dose-related maternal and developmental findings were all consistent with the mixed D1/D2 agonist properties of pergolide mesylate, and suggest that only very high doses may result in persistent effects on the developing central dopaminergic systems.

A conformational study of alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----OMe) by NMR nuclear Overhauser effect spectroscopy (NOESY) and molecular dynamics calculations.

The conformational preference of the disaccharide alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----OMe) (1) about the glycosidic torsion angles, phi and psi, was studied by NMR NOESY spectroscopy and molecular mechanics calculations. The NOE data were consistent with either of two distinct conformations close to minima on a calculated phi/psi potential energy surface. Starting from the lowest energy conformation, a 1-ns molecular dynamics (MD) trajectory was computed in vacuo, from which the NOE curves were simulated and compared to the experimentally observed NOESY data.

New n.m.r.-spectroscopic approaches for structural studies of polysaccharides: application to the Haemophilus influenzae type a capsular polysaccharide.

The extension of several modern nuclear magnetic resonance (n.m.r.) spectroscopic techniques to polysaccharides is discussed and illustrated, using the native Haemophilus influenzae type a capsular polysaccharide. These techniques provide for the unambiguous assignment of all n.m.r. resonances (1H, 13C, and 31P) via high-sensitivity homonuclear and 1H-detected heteronuclear correlations, and they are capable of locating the intersaccharide linkages (both O-linked and phosphoric diester-linked) and appended groups (e.g. O-acetyl groups). To illustrate the power and sensitivity of these methods, a 10-mg sample of the H. Influenzae type a polysaccharide (repeat unit mol. wt. = 376) was studied. The combined acquisition time for the two-dimensional 1H-13C correlation data (one-bond and multiple-bond), the 1H-31P correlation data, and the 1H-1H (homonuclear Hartmann-Hahn) data was approximately 18 h.

Determination of the structure of the Escherichia coli K100 capsular polysaccharide, cross-reactive with the capsule from type b Haemophilus influenzae.

The structure of the Escherichia coli K100 capsular polysaccharide, cross-reactive with that from type b Haemophilus influenzae, was determined by using a combination of chemical and spectroscopic techniques. The structure of the K100 repeating unit was found to be----3)-beta-D-Ribf-(1----2)-D-ribitol-5-(PO4----. The K100 polysaccharide is thus identical in composition to, but different in linkage from, the H. influenzae type b capsular polysaccharide, which has beta-D-Ribf-(1----1)-D-ribitol linkages.

Structure of the type 5 capsular polysaccharide of Staphylococcus aureus.

The Staphylococcus aureus type 5 capsular polysaccharide is composed of 2-acetamido-2-deoxy-L-fucose (1 part), 2-acetamido-2-deoxy-D-fucose (1 part), and 2-acetamido-2-deoxy-D-mannuronic acid (1 part). On the basis of methylation analysis, optical rotation, high-field one- and two-dimensional 1H- and 13C-n.m.r. experiments, and selective cleavage with 70% aqueous hydrogen fluoride, the polysaccharide was found to be a partially O-acetylated (50%) polymer of the repeating trisaccharide unit, [----4)-3-O-Ac-beta-D-ManpNAcA-(1----4)-a-L-FucpNAc-(1----3) -beta-D-FucpNAc-(1----]n.

Simultaneous differential staining of cartilage and bone in rodent fetuses: an alcian blue and alizarin red S procedure without glacial acetic acid.

Differential staining of cartilage and bone has several applications including developmental toxicology studies of new chemical candidates for pharmaceutical, industrial, and environmental use. It has been more common to stain fetal bone only using the dye alizarin red S; however, failure to evaluate the cartilaginous portion of the skeleton may result in the failure to identify toxicologically important alterations in skeletal morphology. Previously, differential staining of fetal cartilage and bone was best achieved by combining alizarin red S for staining bone with alcian blue to stain cartilage in glacial acetic acid solution; however, occupational hazards posed by the use of glacial acetic acid make these methods undesirable. Replacement of the glacial acetic acid with potassium hydrogen phthalate eliminates these hazards without compromising the quality of the stained specimen.