Human epidermal growth factor. High resolution solution structure and comparison with human transforming growth factor alpha.

The solution structure of the 53 amino acid peptide hormone, human epidermal growth factor (hEGF), has been determined to high resolution from nuclear magnetic resonance (n.m.r.) data. A large number of internuclear distance and dihedral restraints was obtained, including data from uniformly 15N-labelled hEGF. Dynamical simulated annealing methods using the program XPLOR were used for structure calculation. An improved protocol was developed combining efficient conformational searching at a reduced computational cost. The general fold of the calculated structures compared well with that of a derivative of the carboxy-terminally truncated hEGF determined previously. A group of 44 structures were calculated with no violations greater than 0.3 A and 3 degrees for distance and dihedral restraints, respectively. The average pairwise root mean square (r.m.s.) deviation of all backbone atoms for these structures was 2.25 A for all 53 residues, 0.92 A for the bulk of the protein, and 0.23 A for the functionally important carboxy-terminal domain. Two new helical segments containing highly conserved amino acids have been identified; one between cysteines 6 and 14 and a second at the end of the carboxy-terminal domain. New insight into the molecular architecture of the site of putative receptor binding was provided by comparing the structure of hEGF with its biologically equipotent analogue, human transforming growth factor alpha. This comparison revealed a close structural relationship between the two growth factors and provides an improved understanding of the structure/function relationships in EGF.     

Circular dichroism studies on lipid-protein complexes of a hydrophobic myelin protein

Circular dichroism spectra of lipophilin (a hydrophobic protein purified from human central nervous system myelin) were analyzed by the method of Chen et al. (1974) to obtain information on its secondary structure in aqueous and lipid environments. When introduced into phosphatidylcholine vesicles by dialysis from 2-chloroethanol, the protein possessed about 75% alpha helix. A new water-soluble form of lipophilin also containing over 70% alpha helix was obtained by a similar dialysis in the absence of lipid. This product had a higher helical content than two other water-soluble preparations derived by dialysis from phenol-acetic acid-urea. Interaction of all three aqueous forms of the protein with lysolecithin micelles resulted in increases in total helical content or in the average length of helical segments. The amount of beta sheet was at a minimum for lipophilin incorporated into vesicles, where the presence of lipid also provided some protection against thermal denaturation.     

Primary structure of equine pituitary prolactin

Equine prolactin was determined to be a single chain protein of 199 amino acid containing two tryptophan and six cysteine residues, as found in other mammalian prolactins. The primary sequence of equine prolactin was obtained by automated Edman analyses of S-carboxymethylated protein and proteolytic fragments of modified protein. Of the known prolactin sequences, equine prolactin shows closest homology with porcine (93%) and fin whale (87-91%) prolactins. Genetic mutations have produced changes in 17 of 199 residues of equine prolactin relative to its putative ancestral precursor. Since equine growth hormone has undergone alterations in 4 of 191 residues relative to this putative precursor protein, these results support the theory that prolactins are evolving at a faster rate than growth hormones. Consistent with the previously determined circular dichroic spectrum of equine prolactin, 60% of the protein is predicted to form alpha helices.     

Crystal structure of the FeS cluster-containing nucleotide excision repair helicase XPD

DNA damage recognition by the nucleotide excision repair pathway requires an initial step identifying helical distortions in the DNA and a proofreading step verifying the presence of a lesion. This proofreading step is accomplished in eukaryotes by the TFIIH complex. The critical damage recognition component of TFIIH is the XPD protein, a DNA helicase that unwinds DNA and identifies the damage. Here, we describe the crystal structure of an archaeal XPD protein with high sequence identity to the human XPD protein that reveals how the structural helicase framework is combined with additional elements for strand separation and DNA scanning. Two RecA-like helicase domains are complemented by a 4Fe4S cluster domain, which has been implicated in damage recognition, and an alpha-helical domain. The first helicase domain together with the helical and 4Fe4S-cluster-containing domains form a central hole with a diameter sufficient in size to allow passage of a single stranded DNA. Based on our results, we suggest a model of how DNA is bound to the XPD protein, and can rationalize several of the mutations in the human XPD gene that lead to one of three severe diseases, xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy.     

Immunochemical similarity of the human plasma growth hormone-binding protein and the rabbit liver growth hormone receptor

We probed the (immunochemical) relationship between the recently discovered growth hormone binding protein in human plasma and the growth hormone receptor using monoclonal and polyclonal antibodies raised against rabbit liver growth hormone receptor. The human binding protein was recognized by these antibodies; its immunological crossreactivity compared to the rabbit receptor was 1-2%. These data suggest a) that the binding protein and the receptor are structurally related and b) that rabbit and human growth hormone receptors share some but not all epitopes.     

Primary structure of a putative receptor for a ligand of the insulin family

Nucleotide sequence analysis of human and guinea pig genomic DNA encoding a new member of the insulin receptor (IR) family revealed that the predicted primary structure of this IR-related protein is as similar to the IR and insulin-like growth factor (IGF) I receptor as the IR and IGF-IR are to each other. The conservation of this IR-related sequence among mammals and with the IR and IGF-IR suggests that this IR-related protein is a novel receptor for insulin, IGF-I, IGF-II, or an as yet unidentified peptide hormone or growth factor belonging to the insulin family.     

The crystal structure of the human hepatitis B virus capsid.

Hepatitis B is a small enveloped DNA virus that poses a major hazard to human health. The crystal structure of the T = 4 capsid has been solved at 3.3 A resolution, revealing a largely helical protein fold that is unusual for icosahedral viruses. The monomer fold is stabilized by a hydrophobic core that is highly conserved among human viral variants. Association of two amphipathic alpha-helical hairpins results in formation of a dimer with a four-helix bundle as the major central feature. The capsid is assembled from dimers via interactions involving a highly conserved region near the C terminus of the truncated protein used for crystallization. The major immunodominant region lies at the tips of the alpha-helical hairpins that form spikes on the capsid surface.     

Construction of a bFGF-tethered extracellular matrix using a coiled-coil helical interaction

A novel method for construction of biomaterials for tissue engineering was developed. Noncovalent associations between extracellular matrix (ECM) and growth factors were achieved by engineering recombinant versions of both proteins that included helical peptides that could form a coiled-coil structure. The helix A peptide, which is capable of forming a coiled-coil helical structure, was fused with a matrix protein that contains a cell-adhesive RGD sequence. The helix B peptide, which is also capable of forming a coiled-coil helical structure, was fused with basic fibroblast growth factor (bFGF). Each protein retained its original activity of promoting cell adhesion and cell proliferation, respectively. These recombinant proteins associated noncovalently through coiled-coil helix formation between helix A and helix B. The resulting complex combined the functions of both proteins, and this method of joining proteins with different functionalities could be used to develop biomaterials for tissue engineering.     

A new microbioassay for the measurement of lactogenic hormones in human serum

The standard Nb2 assay for biologically active prolactin has been modified to allow a rapid convenient microbioassay without loss of specificity or accuracy. Lactogenic hormones specifically stimulate the replication of Nb2 node rat lymphoma cells in suspension culture and form the basis of a currently available bioassay to measure prolactin and growth hormone in human serum. A new microbioassay was developed using microtest plates enabling a large number of samples to be assayed simultaneously whilst maintaining the overall sensitivity of the bioassay for lactogenic hormones. Growth of the Nb2 node lymphoma cells, measured by a light scattering technique using optical density on a spectrophotometer, was shown to be closely correlated with the cell number determined on a Coulter counter. Addition of excess anti-human prolactin and anti-human growth hormone completely inhibited the growth stimulatory effects of both human prolactin and human growth hormone. This new microbioassay (BA) and conventional radioimmunoassay (RIA) were used to measure lactogenic hormones in 48 normal subjects. There was a close correlation between the results of both assays for each hormone studied in the control sera. The mean basal BA/RIA ratio was 1.5 (range 0.8-2.0) for prolactin, 0.7 (range 0-4.5) for growth hormone and 1.3 (range 0.5-1.9) for total lactogenic activity.     

Growth hormone-induced alteration of morphology and tubulin expression in 3T3 preadipose cells

Effects of growth hormone on morphology and cytoskeletal protein expression were examined in 3T3-F442A preadipocytes in serum-free medium. Between 2 and 5 days of culture 2 nM methionyl human growth hormone converted 3T3-F442A cells from a flat fibroblastic morphology to a rounded form with numerous membrane convolutions. Growth hormone treated cultures manifested a 30-40% reduction in cell volume. Growth hormone induced changes in morphology and volume preceded and were independent of lipogenesis. In cells treated with growth hormone, expression of alpha and beta-tubulin as determined by Western blotting was found to increase approximately 50% within 72 h as compared to untreated cells. After 7 days, tubulin levels in growth hormone treated cells were approximately 40% of control levels. This indicated that morphological changes and alteration of tubulin expression were signatures of growth hormone action on 3T3-F442A cells.     

The tertiary structure and backbone dynamics of human prolactin

Human prolactin is a 199-residue (23 kDa) protein closely related to growth hormone and placental lactogen with properties and functions resembling both a hormone and a cytokine. As a traditional hormone, prolactin is produced by lactotrophic cells in the pituitary and secreted into the bloodstream where it acts distally to regulate reproduction and promote lactation. Pituitary cells store prolactin in secretory granules organized around large prolactin aggregates, which are produced within the trans layer of the Golgi complex. Extrapituitary prolactin is synthesized by a wide variety of cells but is not stored in secretory granules. Extrapituitary prolactin displays immunomodulatory activities and acts as a growth factor for cancers of the breast, prostate and tissues of the female reproductive system. We have determined the tertiary structure of human prolactin using three-dimensional (3D) and four-dimensional (4D) heteronuclear NMR spectroscopy. As expected, prolactin adopts an "up-up-down-down" four-helical bundle topology and resembles other members of the family of hematopoietic cytokines. Prolactin displays three discrete structural differences from growth hormone: (1) a structured N-terminal loop in contact with the first helix, (2) a missing mini-helix in the loop between the first and second helices, and (3) a shorter loop between the second and third helices lacking the perpendicular mini-helix observed in growth hormone. Residues necessary for functional binding to the prolactin receptor are clustered on the prolactin surface in a position similar to growth hormone. The backbone dynamics of prolactin were investigated by analysis of 15N NMR relaxation phenomena and demonstrated a rigid four-helical bundle with relatively mobile interconnecting loops. Comparison of global macromolecular tumbling at 0.1mM and 1.0mM prolactin revealed reversible oligomerization, which was correlated to dynamic light scattering experiments. The existence of a reversible oligomerization reaction in solution provides insight into previous results describing the in vitro and in vivo aggregation properties of human prolactin.     

Transgenic rabbit producing human growth hormone in milk

The gene construct WAP(6xHisThr):hGH containing the entire human growth hormone gene (hGH) under the rat whey acidic protein (WAP) promoter regulating the expression in mammary glands of mammals was prepared. The 5' end of the gene was modified by the addition of a sequence encoding six histidine residues and a sequence recognized by thrombin. The gene construct was introduced by microinjection into the male pronucleus of a fertilized oocyte. The founder male rabbit was obtained with the transgene mapping to chromosome 7. The presence of the growth hormone was confirmed in samples of milk collected during the lactation of F1 generation females. The growth hormone can be easily purified by affinity chromatography and cleavage by thrombin to an active form.     

Apolipoprotein structure and dynamics

PURPOSE OF REVIEW: This review highlights recent advances in structural studies of exchangeable human apolipoproteins and the insights they provide into lipoprotein action in cardiovascular and amyloid diseases. RECENT FINDINGS: The high-resolution X-ray crystal structure of free apoA-II reveals a parallel helical array that may represent other lipid-poor apolipoproteins, and the structure in complex with detergent substantiates the belt model for the protein arrangement on lipoproteins. Nuclear magnetic resonance structures of apolipoprotein-detergent complexes show a repertoire of curved helical conformations, suggesting multiple helical arrangements on the lipid. Low-resolution spectroscopic analyses, interface studies and molecular modeling provide new insights into the 'hinge-domain' mechanism of apolipoprotein adaptation at variable lipoprotein surfaces. A kinetic mechanism for lipoprotein stabilization is proposed. SUMMARY: Cumulative evidence supports the belt model that provides a general structural basis for understanding the molecular mechanisms of functional apolipoprotein reactions, such as binding to lipoprotein receptors, lipid transporters, and the activation of lipophilic enzymes. However, the detailed protein and lipid conformations on lipoproteins and the underlying molecular interactions are unclear. New insights will hopefully emerge once the first detailed lipoprotein structure is solved.     

Regulation of ghrelin structure and membrane binding by phosphorylation

The peptide hormone ghrelin requires Ser-3 acylation for receptor binding, orexigenic and anti-inflammatory effects. Functions of desacylghrelin are less well understood. In vitro kinase assays reveal that the evolutionarily conserved Ser-18 in the basic C-terminus is an excellent substrate for protein kinase C. Circular dichroism reveals that desacylghrelin is 12% helical in aqueous solution and 50% helical in trifluoroethanol. Ser-18-phosphorylation, Ser-18-Ala substitution, or Ser-3-acylation reduces the helical character in trifluoroethanol to 24%. Both ghrelin and desacylghrelin bind to phosphatidylcholine:phosphatidylserine sucrose-loaded vesicles in a phosphatidylserine-dependent manner. Phosphoghrelin and phosphodesacylghrelin show greatly diminished phosphatidylserine-dependent binding. These results are consistent with binding of ghrelin and desacylghrelin to acidic lipids via the basic face of an amphipathic helix with Ser-18 phosphorylation disrupting both helical character and membrane binding.     

Effects of Parkinson's disease-linked mutations on the structure of lipid-associated alpha-synuclein

Alpha-synuclein (alphaS) is a lipid-binding synaptic protein of unknown function that is found in an aggregated amyloid fibril form in the intraneuronal Lewy body deposits that are a defining characteristic of Parkinson's disease (PD). Although intrinsically unstructured when free in solution, alphaS adopts a highly helical conformation in association with lipid membranes or membrane mimetic detergent micelles. Two mutations in the alphaS gene have been linked to early onset autosomal dominant hereditary forms of PD, and have been shown to affect the aggregation kinetics of the protein in vitro. We have used high-resolution NMR spectroscopy, circular dichroism, and limited proteolysis to investigate the effects of these PD-linked mutations on the helical structure adopted by alphaS in the lipid or detergent micelle-bound form. We show that neither the A53T nor the A30P mutation has a significant effect on the structure of the folded protein, although the A30P mutation may cause a minor perturbation in the helical structure around the site of the mutation. The A30P, but not the A53T, mutation also appears to decrease the affinity of the protein for lipid surfaces, possibly by perturbing the nascent helical structure of the free protein. The potential implications of these results for the role of alphaS in PD are discussed.     

Monoclonal antibodies to the pituitary growth-hormone receptor by the anti-idiotypic approach. Production and initial characterization.

We obtained 10/192 and 3/384 antibody-secreting hybrids after immunization of Balb/c mice with either human growth hormone or affinity-purified rabbit anti-(human growth hormone) respectively. Radiolabelled rabbit anti-(human growth hormone) antibodies, but not human growth hormone, were specifically bound by supernatants from the 13 hybrids. The binding was completely inhibited by human-growth-hormone serum binding protein. However, anti-(human growth hormone antibodies) were detected in the sera of all the mice immunized with human growth hormone. In an independent fusion, which was carried out after immunization with fewer doses of human growth hormone, anti-(human growth hormone) antibodies were also obtained. Five hybrids, where the starting antigen was human growth hormone, were selected for ascites production, and the corresponding monoclonal antibodies were partially purified and characterized with respect to their immunoglobulin isotype and their interaction with human-growth-hormone receptors. These antibodies were found to enhance the binding of radioiodinated human growth hormone to human-growth-hormone serum binding protein from human and rabbit plasma by 40%. Scatchard analysis of the effect of one of the monoclonal antibodies showed that this enhancement was due to an increased number of binding sites. All of the partially purified antibodies but one (F12) inhibited the binding of human growth hormone to rat but not rabbit, liver microsomes to various extents, as well as to H-4-II-E rat hepatoma cells. Monoclonal antibody F12 enhanced the binding of radiolabelled human growth hormone to rat liver microsomes and H-4-II-E hepatoma cells. This enhancement was found to be due to an increase in the number of binding sites.     

Flt3 ligand structure and unexpected commonalities of helical bundles and cystine knots

Human Flt3 ligand (Flt3L) stimulates early hematopoiesis by activating a type III tyrosine kinase receptor on primitive bone marrow stem cells. The crystal structure of soluble Flt3L reveals that it is a homodimer of two short chain alpha-helical bundles. Comparisons of structure-function relationships of Flt3L with the homologous hematopoietic cytokines macrophage colony stimulating factor (MCSF) and stem cell factor (SCF) suggest that they have a common receptor binding mode that is distinct from the paradigm derived from the complex of growth hormone with its receptor. Furthermore, we identify recognition features common to all helical and cystine-knot protein ligands that activate type III tyrosine kinase receptors, and the closely related type V tyrosine kinase receptors.     

Time-resolved solution X-ray scattering of tobacco mosaic virus coat protein: kinetics and structure of intermediates

The kinetics of assembly and disassembly of tobacco mosaic virus coat protein (TMVP) following temperature jumps have been studied by small-angle X-ray scattering and turbidimetry. The structures of the principal aggregates of TMVP oligomers (A protein), intermediate size (helix I) and large size helical rods (helix II), have been characterized by their average radii of gyration of thickness, cross section, and shape obtained from the corresponding regimes of the small-angle scattering pattern. This structural information was obtained within seconds after the temperature-induced initiation of either polymerization or depolymerization and allowed us to detect transient intermediates. This methodology made it possible to observe and characterize the structure of a principal intermediate. Taken together with other kinetic information, these data suggest that polymerization of TMVP under virus self-assembly conditions may proceed via a single-layered helical nucleus that contains about 20 subunits. Previous studies have shown that overshoot polymerization of TMVP can occur and results in metastable long helical viruslike rods which subsequently depolymerize and then form short helical rods, depending on the conditions of the final equilibrium state. The longer rods (helix II) are overshoot polymers which form within seconds and contain 17 1/3 subunits per turn (helix IIB), in contrast to the subunit packing arrangement of 16 1/3 subunits per turn found in the shorter helical rods (helix IA). The latter packing arrangement is the one found in TMV. An overall polymerization scheme is proposed for the formation of these two helical forms of TMVP.