Heinrich Sticht

The interleukin-10 family of cytokines

A family of interleukin-10 (IL-10)-related cytokines has emerged, comprising a series of herpesviral and poxviral members and several cellular sequence paralogs, including IL-19, IL-20, IL-22 [IL-10-related T-cell-derived inducible factor (IL-TIF)], IL-24 [melanoma differentiation-associated antigen 7 (MDA-7)] and IL-26 (AK155). Although the predicted helical structure of these homodimeric molecules is conserved, certain receptor-binding residues are variable and define the interaction with specific heterodimers of different type-2 cytokine receptors. This leads, through the activation of signal transducer and activator of transcription (STAT) factors, to diverse biological effects. For example, whereas IL-10 is a well-studied pleiotropic immunosuppressive and immunostimulatory cytokine, IL-22/IL-TIF mediates acute-phase response signals in hepatocytes and IL-20 induces the hyperproliferation of keratinocytes, which has been proposed as a pathogenic mechanism of psoriasis.

Structure Determination of Human and Murine Beta-Defensins Reveals Structural Conservation in the Absence of Significant Sequence Similarity

Defensins are cationic and cysteine‐rich peptides that play a crucial role in the host defense against microorganisms of many organisms by their capability to permeabilize bacterial membranes. The low sequence similarity among the members of the large mammalian β‐defensin family suggests that their antimicrobial activity is largely independent of their primary structure. To investigate to what extent these defensins share a similar fold, the structures of the two human β‐defensins, hBD‐1 and hBD‐2, as well as those of two novel murine defensins, termed mBD‐7 and mBD‐8, were determined by nuclear magnetic resonance spectroscopy. All four defensins investigated share a striking similarity on the level of secondary and tertiary structure including the lack of a distinct hydrophobic core, suggesting that the fold is mainly stabilized by the presence of three disulfide bonds. In addition to the overall shape of the molecules, the ratio of solvent‐exposed polar and hydrophobic side chains is also very similar among the four defensins investigated. It is significant that β‐defensins do not exhibit a common pattern of charged and hydrophobic residues on the protein surface and that the β‐defensin‐specific fold appears to accommodate a wide range of different amino acids at most sequence positions. In addition to the implications for the mode of biological defensin actions, these findings are of particular interest because β‐defensins have been suggested as lead compounds for the development of novel peptide antibiotics for the therapy of infectious diseases.

The structure of iron–sulfur proteins

Ferredoxins are a group of iron-sulfur proteins for which a wealth of structural and mutational data have recently become available. Previously unknown structures of ferredoxins which are adapted to halophilic, acidophilic or hyperthermophilic environments and new cysteine patterns for cluster ligation and non-cysteine cluster ligation have been described. Site-directed mutagenesis experiments have given insight into factors that influence the geometry, stability, redox potential, electronic properties and electron-transfer reactivity of iron-sulfur clusters.

Structural rearrangements of HIV-1 Tat-responsive RNA upon binding of neomycin B.

Binding of human immunodeficiency virus type 1 (HIV-1) transactivator (Tat) protein to Tat-responsive RNA (TAR) is essential for viral replication and is considered a promising starting point for the design of anti-HIV drugs. NMR spectroscopy indicated that the aminoglycosides neomycin B and ribostamycin bind to TAR and that neomycin is able to inhibit Tat binding to TAR. The solution structure of the neomycin-bound TAR has been determined by NMR spectroscopy. Chemical shift mapping and intermolecular nuclear Overhauser effects define the binding region of the aminoglycosides on TAR and give strong evidence for minor groove binding. Based on 15 nuclear Overhauser effect-derived intermolecular distance restraints, a model structure of the TAR-neomycin complex was calculated. Neomycin is bound in a binding pocket formed by the minor groove of the lower stem and the uridine-rich bulge of TAR, which adopts a conformation different from those known. The neamine core of the aminoglycoside (rings I and II) is covered with the bulge, explaining the inhibition of Tat by an allosteric mechanism. Neomycin reduces the volume of the major groove in which Tat is bound and thus impedes essential protein-RNA contacts.

CD and NMR Studies of Prion Protein (PrP) Helix 1 NOVEL IMPLICATIONS FOR ITS ROLE IN THE PrPC→ PrPSc CONVERSION PROCESS

The conversion of prion helix 1 from an α-helical into an extended conformation is generally assumed to be an essential step in the conversion of the cellular isoform PrPC of the prion protein to the pathogenic isoform PrPSc. Peptides encompassing helix 1 and flanking sequences were analyzed by nuclear magnetic resonance and circular dichroism. Our results indicate a remarkably high instrinsic helix propensity of the helix 1 region. In particular, these peptides retain significant helicity under a wide range of conditions, such as high salt, pH variation, and presence of organic co-solvents. As evidenced by a data base search, the pattern of charged residues present in helix 1 generally favors helical structures over alternative conformations. Because of its high stability against environmental changes, helix 1 is unlikely to be involved in the initial steps of the pathogenic conformational change. Our results implicate that interconversion of helix 1 is rather representing a barrier than a nucleus for the PrPC→ PrPSc conversion.

Solution structure of cytochrome c6 from the thermophilic cyanobacterium Synechococcus elongatus

Cytochrome c6 is a small, soluble electron carrier between the two membrane‐bound complexes cytochrome b6f and photosystem I (PSI) in oxygenic photosynthesis. We determined the solution structure of cytochrome c6 from the thermophilic cyanobacterium Synechococcus elongatus by NMR spectroscopy and molecular dynamics calculations based on 1586 interresidual distance and 28 dihedral angle restraints. The overall fold exhibits four α‐helices and a small antiparallel β‐sheet in the vicinity of Met58, one of the axial heme ligands. The flat hydrophobic area in this cytochrome c6 is conserved in other c6 cytochromes and even in plastocyanin of higher plants. This docking region includes the site of electron transfer to PSI and possibly to the cytochrome b6f complex. The binding of cytochrome c6 to PSI in green algae involves interaction of a negative patch with a positive domain of PSI. This positive domain has not been inserted at the evolutionary level of cyanobacteria, but the negatively charged surface region is already present in S.elongatus cytochrome c6 and may thus have been optimized during evolution to improve the interaction with the positively charged cytochrome f. As the structure of PSI is known in S.elongatus, the reported cytochrome c6 structure can provide a basis for mutagenesis studies to delineate the mechanism of electron transfer between both.

Secondary Structure and Tertiary Fold of the Birch Pollen Allergen Bet v 1 in Solution

Bet v 1 is the major birch pollen allergen and therefore the main cause of type I allergies observed in early spring. It is composed of 159 amino acid residues adding up to a molecular mass of 17 kDa. We determined the secondary structure and tertiary fold of full-length Bet v 1 by NMR spectroscopy. Two- and three-dimensional NMR measurements suggest that Bet v 1 is a globular monomer in solution with a high content of well defined secondary structure. Of the total of 159 residues, 135 could be sequentially assigned, using an improved assignment strategy based mainly on heteronuclear experiments. An improved strategy for structure calculation revealed three helices and two β-sheets as major elements of secondary structure. The globular tertiary structure is mainly stabilized by two antiparallel β-sheets. The two helices at the C terminus are in accordance with the results from the solution structure of the chemically synthesized peptide Bet v 1-(125-154). This peptide is composed of two helices connected by a hinge. The structural features of Bet v 1 are highly similar to those found in the Ambrosia allergen Amb t V.

NMR Spectroscopy Reveals Common Structural Features of the Birch Pollen Allergen Bet v 1 and the cherry allergen Pru a 1

A high percentage of birch pollen allergic patients also experience food hypersensitivity which results from common epitopes on the corresponding allergens. In order to analyze whether this observed cross-reactivity can be attributed to common structural features, the major birch pollen allergen Bet v 1 and the cherry allergen Pru a 1 were investigated by multidimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy. For the 17 kDa Bet v 1 a three-dimensional structure was calculated on the basis of 1344 experimental restraints. The structure is well defined showing average root mean square deviations of 0.67 and 1.15 Å for the backbone heavy atoms and all heavy atoms of residues 1–154, respectively. The major structural features include a seven-stranded antiparallel β-sheet that wraps around a long C-terminal α-helix, thereby forming a large cavity in the interior of the protein. This structure served as template for the generation of an NMR-based model of Pru a 1 by homology modelling in conjunction with 277 experimentally derived distance restraints. Comparison to the structure of Bet v 1 proves both structures to be highly similar concerning the elements of secondary structure as well as the shape and charge distribution of the protein surface. This finding is consistent with the observed cross-reactivity between both proteins and allows the delineation of common cross-reactive B-cell epitopes.

Identification and characterization of a eukaryotically encoded rubredoxin in a cryptomonad alga1

We have identified an open reading frame with homology to prokaryotic rubredoxins (rds) on a nucleomorph chromosome of the cryptomonad alga Guillardia theta. cDNA analysis let us propose that the rd preprotein has an NH2‐terminal extension that functions as a transit peptide for import into the plastid. Compared to rds found in non‐photosynthetic prokaryotes or found in bacteria that exhibit an anoxigenic photosynthesis apparatus, nucleomorph rd has a COOH‐terminal extension, which shows high homology exclusively to the COOH‐termini of cyanobacterial rds as well as to a hypothetical rd in the Arabidopsis genome. This extension can be divided into a putative membrane anchor and a stretch of about 20 amino acids with unknown function linking the common rd fold to this anchor. Overexpression of nucleomorph rd in Escherichia coli using a T7 RNA polymerase/promotor system resulted in a mixture of iron‐containing holorubredoxin and zinc‐substituted protein. Preliminary spectroscopic studies of the iron form of nucleomorph rd suggest the existence of a native rd‐type iron site. One‐dimensional nuclear magnetic resonance spectroscopy of recombinant Zn‐rd suggests the presence of a stable tertiary fold similar to that of other rd structures determined previously.

Structural model of the HIV-1 Tat(46-58)-TAR complex.

The trans-activator protein (Tat) of human immunodeficiency virus type 1 (HIV-1) binds to an uridine-rich bulge of an RNA target (TAR; trans-activation responsive element) predominantly via its basic sequence domain. The structure of the Tat(46-58)-TAR complex has been determined by a novel modeling approach relying on structural information about one crucial arginine residue and crosslink data. The strategy described here solely uses this experimental data without additional modeling assumptions about the structure of the complex in order to avoid human bias. Model building was performed in a fashion similar to structure calculations from nuclear magnetic resonance (NMR)-spectroscopic data using restrained molecular dynamics. The resulting set of structures of Tat(46-58) in its complex with TAR reveals that all models have converged to a common fold, showing a backbone root mean square deviation (RMSD) of 1.36A. Analysis of the calculated structures suggests that HIV-I Tat forms a hairpin loop in its complex with TAR that shares striking similarity to the hairpin formed by the structure of the bovine immunodeficiency virus Tat protein after TAR binding as determined by NMR studies. The outlined approach is not limited to the Tat-TAR complex modeling, but is also applicable to all molecular complexes with sufficient biochemical and biophysical data available.