Secondary structure determines protein topology

Using a test set of 13 small, compact proteins, we demonstrate that a remarkably simple protocol can capture native topology from secondary structure information alone, in the absence of long-range interactions. It has been a long-standing open question whether such information is sufficient to determine a protein's fold. Indeed, even the far simpler problem of reconstructing the three-dimensional structure of a protein from its exact backbone torsion angles has remained a difficult challenge owing to the small, but cumulative, deviations from ideality in backbone planarity, which, if ignored, cause large errors in structure. As a familiar example, a small change in an elbow angle causes a large displacement at the end of your arm; the longer the arm, the larger the displacement. Here, correct secondary structure assignments (alpha-helix, beta-strand, beta-turn, polyproline II, coil) were used to constrain polypeptide backbone chains devoid of side chains, and the most stable folded conformations were determined, using Monte Carlo simulation. Just three terms were used to assess stability: molecular compaction, steric exclusion, and hydrogen bonding. For nine of the 13 proteins, this protocol restricts the main chain to a surprisingly small number of energetically favorable topologies, with the native one prominent among them.     

Secondary structure and membrane topology of dengue virus NS4A protein in micelles

Dengue virus (DENV) non-structural (NS) 4A is a membrane protein essential for viral replication. The N-terminal region of NS4A contains several helices interacting with the cell membrane and the C-terminal region consists of three potential transmembrane regions. The secondary structure of the intact NS4A is not known as the previous structural studies were carried out on its fragments. In this study, we purified the full-length NS4A of DENV serotype 4 into dodecylphosphocholine (DPC) micelles. Solution NMR studies reveal that NS4A contains six helices in DPC micelles. The N-terminal three helices are amphipathic and interact with the membrane. The C-terminal three helices are embedded in micelles. Our results suggest that NS4A contains three transmembrane helices. Our studies provide for the first time structural information of the intact NS4A of DENV and will be useful for further understanding its role in viral replication.     

Three-dimensional structure of interleukin 8 in solution

The solution structure of the interleukin 8 (IL-8) dimer has been solved by nuclear magnetic resonance (NMR) spectroscopy and hybrid distance geometry-dynamical simulated annealing calculations. The structure determination is based on a total of 1880 experimental distance restraints (of which 82 are intersubunit) and 362 torsion angle restraints (comprising phi, psi, and chi 1 torsion angles). A total of 30 simulated annealing structures were calculated, and the atomic rms distribution about the mean coordinate positions (excluding residues 1-5 of each subunit) is 0.41 +/- 0.08 A for the backbone atoms and 0.90 +/- 0.08 A for all atoms. The three-dimensional solution structure of the IL-8 dimer reveals a structural motif in which two symmetry-related antiparallel alpha-helices, approximately 24 A long and separated by about 14 A, lie on top of a six-stranded antiparallel beta-sheet platform derived from two three-stranded Greek keys, one from each monomer unit. The general architecture is similar to that of the alpha 1/alpha 2 domains of the human class I histocompatibility antigen HLA-A2. It is suggested that the two alpha-helices form the binding site for the cellular receptor and that the specificity of IL-8, as well as that of a number of related proteins involved in cell-specific chemotaxis, mediation of cell growth, and the inflammatory response, is achieved by the distinct distribution of charged and polar residues at the surface of the helices.     

Secondary structure of histones in solution

The secondary structure of histones H2B and H3 from calf thymus has been quantitatively studied in heavy water solutions in a wide range of histone concentrations, pD, and concentrations of sodium chloride by an infrared spectroscopy method. Also, the interactions between molecules of different histones in equimolar mixtures H2A-H2B, H2A-H3, H2A-H4, H2B-H3, H2B-H4, H3-H4, and H2A-H2B-H3-H4 have been investigated using the same method. For H2B and H3 conditions favourable for aggregation have been shown to induce the formation of pleated sheet structure. When the pD and concentration of NaCl are in a physiological range, the secondary structure of H2B and H3 contains about 15% of alpha-helix, 4% of parallel pleated sheet structure, 14% of antipatallel pleated sheet structure in H2B and 18% in H3. For mixtures in all cases, except H2A-H4, there is an interaction between molecules of different histones followed by a reduction of the antiparallel pleated sheet structure content. The data on the secondary structure of histones in different states (under self-association, in mixtures, in nucleosomes, and in chromatin) have been discussed and it is suggested that: 1) the secondary structure of histones in chromatin is essentially similar to that in the state of self-association; 2) in the core nucleosome particle the quantity of DNA (in nucleotide pairs), and the quantities of alpha-helix and antiparallel pleated sheet structure (in peptide groups) satisfy the relation 1 : 1 : 1.     

Secondary structure of human interleukin 2 from 3D heteronuclear NMR experiments.

Recombinant 15N-labeled human interleukin 2 (IL-2) has been studied by 2D and 3D NMR using uniformly 15N-labeled protein. Assignment of the backbone resonances has enabled the secondary structure of the protein to be defined. The secondary structure was found to consist of four alpha-helical regions and a short section of antiparallel beta-sheet. This structure is more similar to recent published structures of interleukin 4 and granulocyte-macrophage colony-stimulating factor than to a structure of IL-2 previously obtained from low-resolution X-ray diffraction data.     

Secondary structure and membrane topology of dengue virus NS4B N-terminal 125 amino acids

The transmembrane NS4B protein of dengue virus (DENV) is a validated antiviral target that plays important roles in viral replication and invasion of innate immune response. The first 125 amino acids of DENV NS4B are sufficient for inhibition of alpha/beta interferon signaling. Resistance mutations to NS4B inhibitors are all mapped to the first 125 amino acids. In this study, we expressed and purified a protein representing the first 125 amino acids of NS4B (NS4B_1–125). This recombinant NS4B_1–125 protein was reconstituted into detergent micelles. Solution NMR spectroscopy demonstrated that there are five helices (α1 to α5) present in NS4B_1–125. Dynamic studies, together with a paramagnetic relaxation enhancement experiment demonstrated that four helices, α2, α3, α4, and α5 are embedded in the detergent micelles. Comparison of wild type and V63I mutant (a mutation that confers resistance to NS4B inhibitor) NS4B_1–125 proteins demonstrated that V63I mutation did not cause significant conformational changes, however, V63 may have a molecular interaction with residues in the α5 transmembrane domain under certain conditions. The structural and dynamic information obtained in study is helpful to understand the structure and function of NS4B.     

Human interleukin 4. The solution structure of a four-helix bundle protein.

Heteronuclear 13C and 15N three-dimensional nuclear magnetic resonance (n.m.r.) techniques have been used to determine the solution structure of human interleukin 4, a four-helix bundle protein. A dynamical simulated annealing protocol was used to calculate an ensemble of structures from an n.m.r. data set of 1735 distance restraints, 101 phi angle restraints and 27 pairs of hydrogen bond restraints. The protein structure has a left-handed up-up-down-down topology for the four helices with the two long overhand loops in the structure being connected by a short section of irregular antiparallel beta-sheet. Analysis of the side-chains in the protein shows a clustering of hydrophobic residues, particularly leucines, in the core of the bundle with the side-chains of charged residues being located on the protein surface. The solution structure has been compared with a recent structure prediction for human interleukin 4 and with crystal structures of other helix bundle proteins.     

Secondary structure and membrane topology of cytochrome P450s

The secondary structure prediction of 19 microsomal cytochrome P450s from two different families was made on the basis of their amino acid sequences. It was shown that there is structural similarity between the heme-binding sites in these enzymes and those in the bacterial P450cam. An average predicted secondary structure of cytochrome P450 proteins with 70% accuracy contains about 46% alpha-helices, 12% beta-sheets, 9% beta-turns, and 33% random coils. In the region of residues 35-120 in microsomal P450s two adjacent beta alpha beta-units (the Rossmann domain), were recognized and may be available to interact with the NADPH-cytochrome P450 reductase. Using the procedure for identification of hydrophobic and membrane-associated alpha-helical segments, only one N-terminal transmembrane anchor was predicted. Also the heme-binding site may include the surface-bound helix. A model for vertebrate microsomal P450s having an amphipathic membrane protein located on the cytoplasmic side of the endoplasmic reticulum membrane, with their active center lying outside or on the bilayer border, is proposed.     

Secondary structure of human apolipoprotein A-I(1-186) in lipid-mimetic solution

The solution structure of an apoA-I deletion mutant, apoA-I(1-186) was determined by the chemical shift index (CSI) method and the torsion angle likelihood obtained from shift and sequence similarity (TALOS) method, using heteronuclear multidimensional NMR spectra of [u-(13)C, u-(15)N, u-50% (2)H]apoA-I(1-186) in the presence of sodium dodecyl sulfate (SDS). The backbone resonances were assigned from a combination of triple-resonance data (HNCO, HNCA, HN(CO)CA, HN(CA)CO and HN(COCA)HA), and intraresidue and sequential NOEs (three-dimensional (3D) and four-dimensional (4D) 13C- and 15N-edited NOESY). Analysis of the NOEs, H(alpha), C(alpha) and C' chemical shifts shows that apoA-I(1-186) in lipid-mimetic solution is composed of alpha-helices (which include the residues 8-32, 45-64, 67-77, 83-87, 90-97, 100-140, 146-162, and 166-181), interrupted by short irregular segments. There is one relatively long, irregular and mostly flexible region (residues 33-44), that separates the N-terminal domain (residues 1-32) from the main body of protein. In addition, we report, for the first time, the structure of the N-terminal domain of apoA-I in a lipid-mimetic environment. Its structure (alpha-helix 8-32 and flexible linker 33-44) would suggest that this domain is structurally, and possibly functionally, separated from the other part of the molecule.     

Resonance assignments, secondary structure and topology of leukaemia inhibitory factor in solution

The chemical shift assignments and secondary structure of a murine–human chimera,MH35, of leukaemia inhibitory factor (LIF), a 180-residue protein of molecular mass 20 kDa,have been determined from multidimensional heteronuclear NMR spectra acquired on auniformly 13C,15N-labelled sample. Secondary structure elements were defined on the basisof chemical shifts, NH-CH coupling constants, medium-range NOEs and the location ofslowly exchanging amide protons. The protein contains four -helices, the relativeorientations of which were determined on the basis of long-range, interhelical NOEs. The fourhelices are arranged in an up-up-down-down orientation, as found in other four-helical bundlecytokines. The overall topology of MH35-LIF is similar to that of the X-ray crystallographicstructure for murine LIF [Robinson et al. (1994) Cell, 77, 1101–1116]. Differencesbetween the X-ray structure and the solution structure are evident in the N-terminal tail, wherethe solution structure has a trans-Pro17 compared with the cis-Pro17 found in the crystalstructure and the small antiparallel -sheet encompassing residues in the N-terminus andCD loop in the crystal structure is less stable.     

Induction of in vitro human lymphocyte migration by interleukin 3, interleukin 4, and interleukin 6.

The effects of interleukin 3 (IL 3), IL 4, IL 6, and interferon-gamma (IFN-gamma) on lymphocyte migration have been investigated and compared with those of transforming growth factor-beta 1 (TGF-beta 1), granulocyte colony stimulating factor (GCSF), and macrophage colony stimulating factor (MCSF). Potent, temperature-dependent stimulation of lymphocyte migration was obtained in response to IL 3 and IL 4 (ED50 less than 10(-11) M and less than 10(-13) M, respectively) and this migration was abolished in the presence of 3 micrograms ml-1 cytochalasin B. IL 6 and IFN-gamma were less active (ED50 greater than or equal to 10(-9) M and greater than or equal to 10(-8) M, respectively), maximal migration in response to IFN-gamma being only 30% above background as compared with approximately 250% for IL 3 and IL 4. TGF-beta 1, GCSF, and MCSF failed to stimulate lymphocyte migration in doses similar to those used for IL 3, IL 4, and IL 6. The presence of antisera to IL 3, IL 4, and IL 6 specifically inhibited lymphocyte migration induced by the corresponding cytokines (IC50 values being 1/10,000, greater than 1/30,000, and greater than 1/30,000 dilution of antibody, respectively). Cross-desensitization experiments using IL 3 and IL 4 demonstrated that neither IL 3 nor IL 4 were able to stimulate dose-related lymphocyte migration in cells preincubated with IL 3. Cells preincubated with IL 4 were only stimulated by a supraoptimal concentration of IL 4 (10(-11) M). The induction of lymphocyte migration by IL 3, IL 4, and IL 6 therefore appears to be a specific and potentially important effect of these cytokines. Cross-desensitization of lymphocytes by IL 3 and IL 4 raises the possibility that the induction of lymphocyte migration by these cytokines may occur through a common postreceptor signal transduction mechanism.     

Disulfide assignments in recombinant mouse and human interleukin 4

The disulfide pairings of mouse and human interleukin 4 (IL-4) proteins have been determined. The purified proteins, synthesized by recombinant DNA technology, are fully active as judged by their ability to stimulate an appropriate biological response in a variety of functional assays. Peptide maps were produced by digesting the proteins with pepsin and separating the resulting fragments by reverse-phase HPLC using linear acetonitrile-TFA gradients. Cystine-containing peptides were identified by determining which reverse-phase peaks showed an altered elution pattern after reduction. These peptides were purified further and defined by composition and sequence analysis. Three sets of disulfide-linked peptides were consistently identified for each protein. For mouse IL-4, the first and fifth, second and fourth, and third and sixth cysteines are joined. The disulfide bonds in human IL-4 are between the first and sixth, second and fourth, and third and fifth cysteines. A large double-loop region within the central three-fifths of each protein is stabilized by these bonds. Sequence analysis of the peptides containing the third and fifth cysteines of human IL-4 also demonstrated that only one of the potential N-glycosylation sites is used by C127 mammary tumor cells. Complete alkylation of mouse IL-4 under mild conditions completely destroyed its biological activity in a hematopoietic precursor cell proliferation assay.     

Secondary structure of human interleukin-3

1. The secondary structure of human interleuken-3 in solution was determined by circular dichroism spectroscopy. 2. The results were then compared with empirical secondary structure predictions based on primary sequence structure of the protein. 3. The two approaches are in extremently close agreement showing the protein to have 40% alpha-helix, 12% total beta-structure and 48% random coil content.     

Heteronuclear NMR studies of human serum apolipoprotein A-I. Part I. Secondary structure in lipid-mimetic solution

Class I aminoacyl-tRNA synthetases have been thought to be single polypeptide enzymes. However, the complete genome sequence of a hyper thermophile Aquifex aeolicus suggests that the gene for leucyl-tRNA synthetases (LeuRS) is probably split into two pieces (leuS and leuS'). In this research, each gene was separately cloned and overexpressed in Escherichia coli and the protein products were examined for LeuRS activity. Leucylation activity was detected only when both gene products coexisted. Gel filtration analysis showed that the active form of A. aeolicus LeuRS has a heterodimeric (alpha/beta type) quaternary structure that is unique among class I aminoacyl-tRNA synthetases.     

Crystallization and preliminary X-ray investigation of recombinant human interleukin 4

Crystals of recombinant human interleukin 4 have been grown from solutions of ammonium sulfate. The crystals are tetragonal, space-group P4(1)2(1)2 or P4(3)2(1)2; the unit cell axes are a = 92.2(1) A and c = 46.4(1) A. The crystals are stable to X-rays for at least three days and diffract beyond 2.8 A resolution. The crystals contain approximately 63% solvent, assuming there is one molecule in the asymmetric unit.     

Cloning and structure of the human interleukin 2 chromosomal gene.

Southern hybridization using 32P-labelled human interleukin 2 (IL2) cDNA probes revealed the existence of a single human IL2 gene. Five clones containing the human IL2 chromosomal gene were isolated from two different human DNA libraries cloned in either lambda Charon 4A or L47 phages. Analysis of the clones showed that they contained different, overlapping portions of human DNA which were derived from the same chromosomal segment. Restriction fragments which hybridized with labelled IL2 cDNA probes were subcloned into plasmid pUR250 and the sequence and organization of the IL2 gene was determined. It contains three introns, 90 bp, +/- 2400 bp and +/- 1900 bp in length, respectively. The organization of the genomic clone resembles that of another lymphokine, interferon-gamma, but no clear homology was found by comparing either the coding sequence or the 5'- and 3'-flanking sequences of the two genes.     

B cell growth-promoting activity of recombinant human interleukin 4

Human interleukin 4 (IL-4), also known as B cell stimulatory factor 1, is a T cell-derived glycoprotein consisting of 129 amino acids for which a cDNA has been recently isolated. IL-4 displays little or no B cell growth factor (BCGF) activity in the standard anti-IgM costimulatory assay using suboptimal concentrations of soluble anti-IgM antibody whereas the low m.w. BCGF is very active. When insolubilized anti-IgM was used as the costimulating agent, both IL-4 and the low m.w. BCGF were found to promote B cell proliferation. Human IL-4 is able to induce the proliferation of B lymphocytes preactivated for either 1 day with insolubilized anti-IgM antibody or for 3 days with Staphylococcus aureus strain Cowan I. However, IL-4 is poorly mitogenic for B cells preactivated for 1 day with the Staphylococcus strain whereas the low m.w. BCGF strongly enhances the proliferation of these B cells. These two findings demonstrate that the preactivation signal necessary to induce human B cells to proliferate in response to IL-4 is critical. The increased tritiated thymidine ([3H]dThd) uptake in preactivated B cell cultures with IL-4 reflects cel proliferation because cell cycle analysis demonstrates that IL-4 induces activated B cells to enter the S and G2/M phases of the cell cycle and the addition of IL-4 to preactivated B cell cultures permits the recovery of three- to fourfold more B cells after 4 days of culture. IL-4 and the low m.w. BCGF act in concert to induce the proliferation of anti-IgM-preactivated B cells as demonstrated by [3H]dThd uptake and cell cycle analysis. In striking contrast to the demonstrated antagonistic effect of interferon-gamma on the IL-4-induced expression of the low affinity receptor for IgE (Fc epsilon RL/CD23), on B cells, it was found that interferon-gamma enhanced the IL-4-induced proliferation of anti-IgM-preactivated B cells. Finally, it was found that IL-4 had to be present continuously during the culture period to exert an optimal growth-promoting effect on B cell blasts. As a conclusion, IL-4 is able to induce the proliferation of an appropriately activated subpopulation of human B cells.     

Gene structure, expression analysis, and membrane topology of RDH4

The murine retinol dehydrogenase RDH4 oxidizes several cis-isomers of retinol into their corresponding aldehydes. We have determined the structure of the murine gene, investigated the temporal and spatial expression of the enzyme, and analyzed the membrane topology of the enzyme. The gene has four translated exons, and several alternatively spliced exons in the 5'-untranslated region were identified. Immunohistochemical analysis showed expression of RDH4 in developing and adult mouse eye, particularly in the retinal pigment epithelium. In nonocular adult tissues, including liver, kidney, lung, and skin, RDH4 expression was widespread. The results suggest that RDH4 may have a dual and tissue-specific role in oxidation of 9-cis- and 11-cis-isomers of retinol into 9-cis-retinal and 11-cis-retinal, respectively. Furthermore, the lumenal orientation of the enzyme domain in the ER suggests that oxidation of both cis-isomers of retinol occurs in the ER.