Biological and structural properties of MIP-1 alpha expressed in yeast.

The murine macrophage inflammatory proteins-1 alpha (MIP-1 alpha) and MIP-1 beta are distinct but closely related cytokines. Partially purified mixtures of the two proteins affect neutrophil function and cause local inflammation and fever. The particular properties of MIP-1 alpha have not been well studied, although it has been identified as being identical to an inhibitor of haemopoietic stem cell growth. We have expressed MIP-1 alpha in yeast cells and purified it to sequence homogeneity. Structural analysis of this biologically active material by circular dichroism and fluorescence spectroscopy confirms that MIP-1 alpha has a very similar secondary and tertiary structure to platelet factor 4 and interleukin 8 with which it shares limited sequence homology. The in-vitro stem cell inhibitory properties have been confirmed using a range of murine progenitor cells including purified bone marrow progenitor cells (FACS-1), the FDCP-mix A4 cell line, and spleen colony forming unit (CFU-S) populations. Plateau levels of inhibition of stem cell growth were achieved using concentrations of 0.15 micrograms/ml MIP-1 alpha. We have also demonstrated that MIP-1 alpha is active in vivo: 5 micrograms of MIP-1 alpha per mouse given as a bolus injection, protects stem cells from subsequent in-vitro killing by tritiated thymidine. MIP-1 alpha was also shown to enhance the proliferation of more committed progenitor granulocyte macrophage-colony forming cells (GM-CFC) in response to granulocyte macrophage-colony stimulating factor (GM-CSF).     

Disulfide structures of human interleukin-6 are similar to those of human granulocyte colony stimulating factor

The amino acid sequences of human interleukin-6 and granulocyte colony stimulating factor are approximately 30% homologous in the N-terminal region. The relative positions of four half-cystines in human interleukin-6 (IL-6) match four of the five in human granulocyte colony stimulating factor. Labeling experiments of recombinant interleukin-6 with tritiated iodoacetate confirmed that the molecule forms two intramolecular disulfide bonds and contains no detectable level of free sulfhydryls. By isolation and characterization of tryptic and subtilytic peptides obtained from different proteolytic digestions, the disulfide bonds of the IL-6 molecule were assigned to Cys44-Cys50 and Cys73-Cys83. The two disulfide bridges form two small loops which are separated by 22 amino acids. These structures are similar to those of recombinant granulocyte colony stimulating factor.     

Fibrinogen: a preliminary analysis of the amino acid sequences of the portion of the alpha, beta and gamma-chains postulated to form the interdomainal link between globular regions of the molecule

It has been suggested (Doolittle et al., 1977) that portions of the α-, β- and γ-chains of fibrinogen form a coiled-coil rope of α-helices and that this rope connects globular domains of the molecule. A fast Fourier transform analysis of the relevant amino acid sequences has shown that there is a significant 3.5-residue period in the linear disposition of the apolar residues in all three chains. This periodicity is characteristic of amino acid sequences of α-fibrous proteins, such as α-tropomyosin and α-keratin, where the tertiary structure is closely related to a coiled-coil of α-helices. However, a detailed study of the fibrinogen sequences shows that the structure is likely to contain several regions which do not have a simple secondary structure. The detailed conformation of the postulated rodlike region of fibrinogen is therefore complex and may approximate a coiled-coil only over relatively short lengths.An important question to emerge from this analysis is whether correct positioning of apolar residues in a pseudo-repeating heptad is sufficiently important to override low α-helix-favouring potential of other residues in the heptad.     

Structure of the cDNA encoding transcobalamin I, a neutrophil granule protein

Transcobalamin I (TCI) is a member of the R binder family of vitamin B12 binding proteins. It is a major protein constituent of secondary granules in neutrophils. We have isolated and characterized full length cDNA clones encoding TCI in order to determine whether its expression is coordinately regulated with the appearance of secondary granules and whether it is consequently a useful marker of granulocyte development. Partial amino acid sequences of human R protein were obtained from tryptic digestion fragments. Using the polymerase chain reaction, a partial TCI cDNA probe was isolated by selective amplification of a region of cDNA located between two oligonucleotides deduced from the available partial amino acid sequences. The amplified probe was then used to obtain full length clones from a granulocyte cDNA library. Identity of the clones was confirmed by matching DNA sequence to known peptide amino acid sequence. TCI is transcribed to a single 1.5-kilobase mRNA species. The predicted protein sequence is 433 amino acids long. We have compared the sequence of TCI to that of rat intrinsic factor. The two proteins have areas of extensive homology which implicate regions potentially important for vitamin B12 binding. TCI mRNA was present in late neutrophil precursors but absent from uninduced and induced HL60 cells.     

Conformational homologies among cytokines: Interleukins and colony stimulating factors

Some 30 cytokine amino acid sequences (mainly interleukins, colony stimulating factors and tumor necrosis factors) have been examined for evidence of secondary structure as well as longer-range interactions of a type likely to lead to stable α-helical bundles. Most, though not all, of the cytokines examined have a high predicted α-helical content (40–60%) and quasi-repeating heptads containing i/i+3 apolar periodicities. This major subset of the cytokines is predicted to be characterized by molecules in which 4-α-helical bundles with an average length of 25Å are the most marked conformational features. Based on these conclusions, we suggest structures for huG-CSF, huGM-CSF and muIL-5 in which defined loop segments at the ends of helical bundles are the most likely sites for binding and recognition by specific cell receptors. As such, they provide a means for testing or refining the three working models we have defined, using currently available methods of site-directed substitution and deletion mutagenesis, as well as synthetic peptides corresponding to the proposed loop sequences and the use of monoclonal antibodies of defined epitopic specificity. The structure arrived at for huGM-CSF is consistent with the limited data currently available concerning the residues which are important for binding and activity.     

Protein plasticity to the extreme: changing the topology of a 4-alpha-helical bundle with a single amino acid substitution.

BACKGROUND: Conventional wisdom has it that two proteins sharing 98.4% sequence identity have nearly identical three-dimensional structures. Here we provide a counter-example to this statement by showing that a single amino acid substitution can change the topology of a homodimeric 4-alpha-helical bundle protein. RESULTS: We have determined the high-resolution crystal structure of a 4-alpha-helical protein with a single alanine to proline mutation in the turn region, and show that this single amino acid substitution leads to a complete reorganisation of the whole molecule. The protein is converted from the canonical left-handed all-antiparallel form, to a right-handed mixed parallel and antiparallel bundle, which to the best of our knowledge and belief represents a novel topological motif for this class of proteins. CONCLUSIONS: The results suggest a possible new mechanism for the creation and evolution of topological motifs, show the importance of loop regions in determining the allowable folding pathways, and illustrate the malleability of protein structures.     

Differential fMet-Leu-Phe- and platelet-activating factor-induced signaling toward Ral activation in primary human neutrophils.

We have measured the activation of the small GTPase Ral in human neutrophils after stimulation with fMet-Leu-Phe (fMLP), platelet activating factor (PAF), and granulocyte macrophage-colony stimulating factor and compared it with the activation of two other small GTPases, Ras and Rap1. We found that fMLP and PAF, but not granulocyte macrophage-colony stimulating factor, induce Ral activation. All three stimuli induce the activation of both Ras and Rap1. Utilizing specific inhibitors we demonstrate that fMLP-induced Ral activation is mediated by pertussis toxin-sensitive G-proteins and partially by Src-like kinases, whereas fMLP-induced Ras activation is independent of Src-like kinases. PAF-induced Ral activation is mediated by pertussis toxin-insensitive proteins, Src-like kinases and phosphatidylinositol 3-kinase. Phosphatidylinositol 3-kinase is not involved in PAF-induced Ras activation. The calcium ionophore ionomycin activates Ral, but calcium depletion partially inhibits fMLP- and PAF-induced Ral activation, whereas Ras activation was not affected. In addition, 12-O-tetradecanoylphorbol-13-acetate-induced activation of Ral is completely abolished by inhibitors of protein kinase C, whereas 12-O-tetradecanoylphorbol-13-acetate-induced Ras activation is largely insensitive. We conclude that in neutrophils Ral activation is mediated by multiple pathways, and that fMLP and PAF induce Ral activation differently.     

Packed protein bilayers in the 0.90 A resolution structure of a designed alpha helical bundle.

A 12-residue peptide designed to form an alpha-helix and self-associate into an antiparallel 4-alpha-helical bundle yields a 0.9 A crystal structure revealing unanticipated features. The structure was determined by direct phasing with the "Shake-and-Bake" program, and contains four crystallographically distinct 12-mer peptide molecules plus solvent for a total of 479 atoms. The crystal is formed from nearly ideal alpha-helices hydrogen bonded head-to-tail into columns, which in turn pack side-by-side into sheets spanning the width of the crystal. Within each sheet, the alpha-helices run antiparallel and are closely spaced (9-10 A center-to-center). The sheets are more loosely packed against each other (13-14 A between helix centers). Each sheet is amphiphilic: apolar leucine side chains project from one face, charged lysine and glutamate side chains from the other face. The sheets are stacked with two polar faces opposing and two apolar faces opposing. The result is a periodic biomaterial composed of packed protein bilayers, with alternating polar and apolar interfaces. All of the 30 water molecules in the unit cell lie in the polar interface or between the stacked termini of helices. A section through the sheet reveals that the helices packed at the apolar interface resemble the four-alpha-helical bundle of the design, but the helices overhang parts of the adjacent bundles, and the helix crossing angles are less steep than intended (7-11 degrees rather than 18 degrees).     

Macrophage-colony stimulating factor enhances MHC-restricted presentation of exogenous antigen in dendritic cells

Previous studies have shown that dendritic cells (DCs) can phagocytize, process and present a microencapsulated form of ovalbumin (OVA) in the context of class I MHC as well as class II MHC. In the present study, we examined the effects of recombinant human macrophage-colony stimulating factor (M-CSF) on the MHC-restricted presentation of microencapsulated OVA by DCs. Two types of DCs were generated from mouse bone marrow (BM) cells, one type with granulocyte/macrophage-colony stimulating factor (GM-CSF) alone, the other type with GM-CSF and interleukin (IL)-4. Pretreatment with M-CSF significantly enhanced both class I MHC and class II MHC-restricted presentation of exogenous OVA by both types of DCs. The enhancing activity of M-CSF on antigen presentation was more potent in DCs generated with GM-CSF alone compared to DCs generated with both GM-CSF and IL-4. Pretreatment of the DCs with M-CSF did not increase phagocytic activity or total level of expression of class I MHC (H-2K(b)) molecules, but increased expression of OVA peptide-H-2K(b) complexes upon phagocytosis of microencapsulated OVA. These results demonstrate that M-CSF increases intracellular processing events of phagocytized antigen in DCs.     

Secondary structure and stability of the selenocysteine insertion sequences (SECIS) for human thioredoxin reductase and glutathione peroxidase

We have used high resolution NMR and thermodynamics to characterize the secondary structure and stability of the selenocysteine insertion sequences (SECIS) of human glutathione peroxidase (58 nt) and thioredoxin reductase (51 nt). These sequences are members of the two classes of SECIS recently identified with two distinct structures capable of directing selenocysteine incorporation into proteins in eukaryotes. UV melting experiments showed a single cooperative and reversible transition for each RNA, which indicates the presence of stable secondary structures. Despite their large size, the RNAs gave well resolved NMR spectra for the exchangeable protons. Using NOESY, the imino protons as well as the cytosine amino protons of all of the Watson-Crick base pairs were assigned. In addition, a number of non-canonical base pairs including the wobble G.U pairs were identified. The interbase-pair NOEs allowed definition of the hydrogen-bonded structure of the oligonucleotides, providing an experimental model of the secondary structure of these elements. The derived secondary structures are consistent with several features of the predicted models, but with some important differences, especially regarding the conserved sequence motifs.     

A critique of the utility of the prediction of protein secondary structure

The Chou-Fasman predictive algorithm for determining the secondary structure of proteins from the primary sequence is reviewed. Many examples of its use are presented which illustrate its wide applicability, such as predicting (a) regions with the potential for conformational change, (b) sequences which are capable of assuming several conformations in different environments, (c) effects of single amino acid mutations, (d) amino acid replacements in synthesis of peptides to bring about a change in conformation, (e) guide to the synthesis of polypeptides with definitive secondary structure,e.g. signal sequences, (f) conformational homologues from varying sequences and (g) the amino acid requirements for amphiphilicα-helical peptides.     

Side-chain conformations in 4-alpha-helical bundles.

The distribution of the chi(1), chi(2) dihedral angles in a dataset consisting of 12 unrelated 4-alpha-helical bundle proteins was determined and qualitatively compared with that observed in globular proteins. The analysis suggests that the 4-alpha-helical bundle motif could occasionally impose steric constraints on side chains: (i) the side-chain conformations are limited to only a subset of the conformations observed in globular proteins and for some amino acids they are sterically more constrained than those in helical regions of globular proteins; (ii) aspartic acid and asparagine occasionally adopt rotamers that have not been previously reported for globular or helical proteins; (iii) some rotamers of tyrosine and isoleucine are predominantly or exclusively associated with hydrophobic core positions (a, d); (iv) mutations in the hydrophobic core occur preferentially between residue types which among other physicochemical properties also share a predominant rotamer.     

CD14 is a member of the family of leucine-rich proteins and is encoded by a gene syntenic with multiple receptor genes.

We have isolated and characterized genomic and cDNA clones encoding the murine homolog of the human monocyte/granulocyte cell surface glycoprotein, CD14. As in man, the expression of murine CD14 is limited to the myeloid lineage. The murine and human CD14 genes are highly conserved in their intron-exon organization and nucleotide sequence. Their deduced protein sequences show 66% amino acid identity. In both mouse and man, the CD14 protein contains a repeating (10 times) leucine-rich motif (LXXLXLX) that is also found in a group of heterogeneous proteins from phylogenetically distant species. The CD14 gene has been mapped to mouse chromosome 18 which also contains at least five genes encoding receptors (Pdgfr, Adrb2r, li, Grl-1, Fms). Thus CD14 and the receptor genes form a conserved syntenic group localized on mouse chromosome 18 and human chromosome 5. The inclusion of CD14 in the family of leucine-rich proteins, its expression profile and the murine chromosomal localization support the hypothesis that CD14 may function as a receptor.     

人、猴、兔BPI活性部位基因的克隆和序列分析

为比较不同动物来源杀菌／通透性增加蛋白（BPI）的活性部位在基因组成上的差异,为今后对人BPI进行分子改构打下基础,分别从人、恒河猴和家兔外周血白细胞中克隆出BPI全长或活性部位基因,进行序列分析和比较,并进行蛋白二级结构预测。结果发现：猴、兔BPI活性部位序列与人序列在核苷酸水平的同源性分别为94％和77％,在氨基酸水平的同源性分别为88％和62％;人氨基酸序列中包含一个糖基化位点,而猴、兔序列没有;兔序列比人、猴序列少一个氨基酸;3种BPI活性部位有近似的二级结构。     

Membrane insertion and lateral mobility of synthetic amphiphilic signal peptides in lipid model membranes

Amphiphilic signal sequences with the potential to form alpha-helices with a polar, charged face and an apolar face are common in proteins which are imported into mitochondria, in the PTS permeases of bacteria, and in bacterial rhodopsins. Synthetic peptides of such sequences partition into the surface region of lipid membranes where they can adopt different secondary structures. A finely controlled balance of electrostatic and hydrophobic interactions determines the 'affinity' of amphiphilic signal peptides for lipid membranes, as well as the structure, orientation and depth of penetration of these peptides in lipid bilayer membranes. The ability of an individual peptide to associate with lipid bilayer membranes in several different modes is, most likely, a general feature of amphiphilic signal peptides and is reflected in several common physical properties of their amino acid sequences.     

Molecular basis of co-operativity in protein folding. III. Structural identification of cooperative folding units and folding intermediates.

The hierarchical partition function formalism for protein folding developed earlier has been extended through the use of three-dimensional polar and apolar contact plots. For each amino acid residue in the protein, these plots indicate the apolar and polar surfaces that are buried from the solvent, the identity of all amino acid residues that contribute to this shielding, and the magnitude of their contributions. These contact plots are then used to examine the distribution of the free energy of stabilization throughout the protein molecule. Analysis of these data allows identification of co-operative folding units and their hierarchical levels, and the identification of partially folded intermediates with a significant probability of being populated. The overall folding/unfolding thermodynamics of 12 globular proteins, for which crystallographic and experimental thermodynamics are available, is predicted within error. An energetic classification of partially folded intermediates is presented and the results compared to those cases for which structural and thermodynamic experimental information is available. Four different types of partially folded states and their structural energies are considered. (1) Local intermediates, in which only a local region of the protein loses secondary and tertiary interactions, while the rest of the protein remains intact. (2) Global intermediates, corresponding to the standard molten globule definition, in which significant secondary structure is maintained but native-like tertiary structure contacts are disrupted. (3) Extended intermediates characterized by the existence of secondary structure elements (e.g. alpha-helices) exposed to solvent. (4) Folding intermediates in proteins with two structural domains. The structure and energetics of folding intermediates of apo-myoglobin, alpha-lactalbumin, phosphoglycerate kinase and arabinose-binding protein are considered in detail.     

An Integrated Sequence-Structure Database incorporating matching mRNA sequence, amino acid sequence and protein three-dimensional structure data.

We have constructed a non-homologous database, termed the Integrated Sequence-Structure Database (ISSD) which comprises the coding sequences of genes, amino acid sequences of the corresponding proteins, their secondary structure and straight phi,psi angles assignments, and polypeptide backbone coordinates. Each protein entry in the database holds the alignment of nucleotide sequence, amino acid sequence and the PDB three-dimensional structure data. The nucleotide and amino acid sequences for each entry are selected on the basis of exact matches of the source organism and cell environment. The current version 1.0 of ISSD is available on the WWW at http://www.protein.bio.msu.su/issd/ and includes 107 non-homologous mammalian proteins, of which 80 are human proteins. The database has been used by us for the analysis of synonymous codon usage patterns in mRNA sequences showing their correlation with the three-dimensional structure features in the encoded proteins. Possible ISSD applications include optimisation of protein expression, improvement of the protein structure prediction accuracy, and analysis of evolutionary aspects of the nucleotide sequence-protein structure relationship.