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H Sticht

Publications and source records attributed to H Sticht.

At least 19 recordsLinked to original sources
PubMed2004

Functional analysis of chemically synthesized derivatives of the human CC chemokine CCL15/HCC-2, a high affinity CCR1 ligand.

The CCL15 is a human CC chemokine that activates the receptors, CCR1 and CCR3. Unlike other chemokines, it contains an unusually long N-terminal domain of 31 amino acids preceding the first cysteine residue and a third disulfide bond. To elucidate the functional role of distinct structural determinants, a series of sequential amino-terminal truncated and point-mutated CCL15 derivatives as well as mutants lacking the third disulfide bond and the carboxy-terminal alpha-helix were synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. We demonstrate that a truncation of 24 amino acid residues (delta24-CCL15) converts the slightly active 92-residue delta0-CCL15 into a potent agonist of CC chemokine receptor 1 (CCR1) and a weak agonist of CCR3 in cell-based assays. The biological activity decreases from delta24-CCL15 to delta29-CCL15, and re-increases from delta29-CCL15 to delta30-CCL15. Thus, an exocyclic N-terminal region of only one amino acid residue is sufficient for efficient CCR1 activation. As none of the peptides investigated except for delta24-CCL15 activates CCR3, we suggest that CCR1 is the major receptor for CCL15 in vivo. Further we demonstrate that the third disulfide bond of CCL15 and an exchange of tyrosine in position 70 by a leucine residue, which is conserved in CXC chemokines, do not alter the interaction with CCR1. In contrast, a CCL15 derivative lacking the carboxy-terminal alpha-helix exhibits a complete loss of tertiary structure and hence loss of CCR1 agonistic and binding activity. This study demonstrates that specific protein residues in chemokines, which contribute to receptor-ligand interaction, vary significantly between chemokines and cannot be extrapolated using data from functionally related chemokines.

Amino Acid Sequence↗
PubMed2001

The structure of the coliphage HK022 Nun protein-lambda-phage boxB RNA complex. Implications for the mechanism of transcription termination.

Nun protein from coliphage HK022 binds to phage boxB RNA and functions, in contrast to phage lambda N protein, as a transcriptional terminator. The basic Nun-(10-44) peptide contains the boxB RNA binding arginine rich motif, ARM. The peptide binds boxB RNA and competes with the phage lambda ARM peptide N-(1-36) as indicated by nuclear magnetic resonance (NMR) spectroscopy titrations. In two-dimensional nuclear Overhauser enhancement spectroscopy experiments boxB RNA in complex with Nun-(20-44) exhibits the same pattern of resonances as it does in complex with N peptides containing the ARM, and we could show that Nun-(20-44) forms a bent alpha-helix upon binding to the boxB RNA. The structure of the boxB RNA-bound Nun-(20-44) was determined on the basis of 191 intra- and 30 intermolecular distance restraints. Ser-24 is anchored to the lower RNA stem, and stacking of Tyr-39 and A7 is clearly experimentally indicated. Arg-28 shows numerous contacts to the RNA stem. Leu-22, Ile-30, Trp-33, Ile-37, and Leu-41 form a hydrophobic surface, which could be a recognition site for additional host factors such as NusG. Such a hydrophobic surface area is not present in N-(1-36) bound to boxB RNA.

Amino Acid Sequence↗
PubMed2001

Allergic cross-reactivity made visible: solution structure of the major cherry allergen Pru av 1.

Birch pollinosis is often accompanied by hypersensitivity to fruit as a consequence of the cross-reaction of pollen allergen-specific IgE antibodies with homologous food proteins. To provide a basis for examining the cross-reactivity on a structural level, we used heteronuclear multidimensional NMR spectroscopy to determine the high-resolution three-dimensional structure of the major cherry allergen, Pru av 1, in solution. Based on a detailed comparison of the virtually identical structures of Pru av 1 and Bet v 1, the major birch pollen allergen, we propose an explanation for a significant aspect of the observed cross-reactivity pattern among the family of allergens under consideration. The large hydrophobic cavity expected to be important for the still unknown physiological function of Bet v 1 is conserved in Pru av 1. Structural homology to a domain of human MLN64 associated with cholesterol transport suggests phytosteroids as putative ligands for Pru av 1. NMR spectroscopy provides experimental evidence that Pru av 1 interacts with phytosteroids, and molecular modeling shows that the hydrophobic cavity is large enough to accommodate two such molecules.

Allergens↗
PubMed2001

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 beta-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 beta-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 beta-defensins do not exhibit a common pattern of charged and hydrophobic residues on the protein surface and that the beta-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 beta-defensins have been suggested as lead compounds for the development of novel peptide antibiotics for the therapy of infectious diseases.

Amino Acid Sequence↗
PubMed2000

Eukaryotically encoded and chloroplast-located rubredoxin is associated with photosystem II.

We analyzed a eukaryotically encoded rubredoxin from the cryptomonad Guillardia theta and identified additional domains at the N- and C-termini in comparison to known prokaryotic paralogous molecules. The cryptophytic N-terminal extension was shown to be a transit peptide for intracellular targeting of the protein to the plastid, whereas a C-terminal domain represents a membrane anchor. Rubredoxin was identified in all tested phototrophic eukaryotes. Presumably facilitated by its C-terminal extension, nucleomorph-encoded rubredoxin (nmRub) is associated with the thylakoid membrane. Association with photosystem II (PSII) was demonstrated by co-localization of nmRub and PSII membrane particles and PSII core complexes and confirmed by comparative electron paramagnetic resonance measurements. The midpoint potential of nmRub was determined as +125 mV, which is the highest redox potential of all known rubredoxins. Therefore, nmRub provides a striking example of the ability of the protein environment to tune the redox potentials of metal sites, allowing for evolutionary adaption in specific electron transport systems, as for example that coupled to the PSII pathway.

Biological Transport↗
PubMed2000

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.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2000

Identification and characterization of a eukaryotically encoded rubredoxin in a cryptomonad alga.

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 NH(2)-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.

Amino Acid Sequence↗
PubMed2000

Antitermination in bacteriophage lambda. The structure of the N36 peptide-boxB RNA complex.

The solution structure of a 15-mer nutRboxB RNA hairpin complexed with the 36-mer N-terminal peptide of the N protein (N36) from bacteriophage lambda was determined by 2D and 3D homonuclear and heteronuclear magnetic resonance spectroscopy. These 36 amino acids include the arginine-rich motif of the N protein involved in transcriptional antitermination of phage lambda. Upon complex formation with boxB RNA, the synthetic N36 peptide binds tightly to the major groove of the boxB hairpin through hydrophobic and electrostatic interactions forming a bent alpha helix. Four nucleotides of the GAAAA pentaloop of the boxB RNA adopt a GNRA-like tetraloop fold in the complex. The formation of a GAAA tetraloop involves a loop-closing sheared base pair (G6-A10), base stacking of three adenines (A7, A8, and A10), and extrusion of one nucleotide (A9) from the loop, as observed previously for the complex of N(1-22) peptide and the nutLboxB RNA [Legault, P., Li, J., Mogridge, J., Kay, L.E. & Greenblatt, J. (1998) Cell 93, 289-299]. Stacking of the bases is extended by the indole-ring of Trp18 which also forms hydrophobic contacts to the side-chains of Leu24, Leu25, and Val26. Based on the structure of the complex, three mutant peptides were synthesized and investigated by CD and NMR spectroscopy in order to determine the role of particular residues for complex formation. These studies revealed very distinct amino-acid requirements at positions 3, 4, and 8, while replacement of Trp18 with tyrosine did not result in any gross structural changes.

Amino Acid Sequence↗
PubMed2000

Solution structure of a zinc substituted eukaryotic rubredoxin from the cryptomonad alga Guillardia theta.

The rubredoxin from the cryptomonad Guillardia theta is one of the first examples of a rubredoxin encoded in a eukaryotic organism. The structure of a soluble zinc-substituted 70-residue G. theta rubredoxin lacking the membrane anchor and the thylakoid targeting sequence was determined by multidimensional heteronuclear NMR, representing the first three-dimensional (3D) structure of a eukaryotic rubredoxin. For the structure calculation a strategy was applied in which information about hydrogen bonds was directly inferred from a long-range HNCO experiment, and the dynamics of the protein was deduced from heteronuclear nuclear Overhauser effect data and exchange rates of the amide protons. The structure is well defined, exhibiting average root-mean-square deviations of 0.21 A for the backbone heavy atoms and 0.67 A for all heavy atoms of residues 7-56, and an increased flexibility toward the termini. The structure of this core fold is almost identical to that of prokaryotic rubredoxins. There are, however, significant differences with respect to the charge distribution at the protein surface, suggesting that G. theta rubredoxin exerts a different physiological function compared to the structurally characterized prokaryotic rubredoxins. The amino-terminal residues containing the putative signal peptidase recognition/cleavage site show an increased flexibility compared to the core fold, but still adopt a defined 3D orientation, which is mainly stabilized by nonlocal interactions to residues of the carboxy-terminal region. This orientation might reflect the structural elements and charge pattern necessary for correct signal peptidase recognition of the G. theta rubredoxin precursor.

Amino Acid Sequence↗
PubMed1999

Solution structure of the human CC chemokine 2: A monomeric representative of the CC chemokine subtype.

HCC-2, a 66-amino acid residue human CC chemokine, was reported to induce chemotaxis on monocytes, T-lymphocytes, and eosinophils. The three-dimensional structure of HCC-2 has been determined by 1H nuclear magnetic resonance (NMR) spectroscopy and restrained molecular dynamics calculations on the basis of 871 experimental restraints. The structure is well-defined, exhibiting average root-mean-square deviations of 0.58 and 0.96 A for the backbone heavy atoms and all heavy atoms of residues 5-63, respectively. In contrast to most other chemokines, subtle structural differences impede dimer formation of HCC-2 in a concentration range of 0.1 microM to 2 mM. HCC-2, however, exhibits the same structural elements as the other chemokines, i.e., a triple-stranded antiparallel beta-sheet covered by an alpha-helix, showing that the chemokine fold is not influenced by quaternary interactions. Structural investigations with a HCC-2 mutant prove that a third additional disulfide bond present in wild-type HCC-2 is not necessary for maintaining the relative orientation of the helix and the beta-sheet.

Amino Acid Sequence↗
PubMed1999

Alpha-helix nucleation by a calcium-binding peptide loop.

A 12-residue peptide AcDKDGDGYISAAENH2 analogous to the third calcium-binding loop of calmodulin strongly coordinates lanthanide ions (K = 10(5) M-1). When metal saturated, the peptide adopts a very rigid structure, the same as in the native protein, with three last residues AAE fixed in the alpha-helical conformation. Therefore, the peptide provides an ideal helix nucleation site for peptide segments attached to its C terminus. NMR and CD investigations of peptide AcDKDGDGYISAAEAAAQNH2 presented in this paper show that residues A13-Q16 form an alpha-helix of very high stability when the La3+ ion is bound to the D1-E12 loop. In fact, the lowest estimates of the helix content in this segment give values of at least 80% at 1 degreesC and 70% at 25 degreesC. This finding is not compatible with existing helix-coil transition theories and helix propagation parameters, s, reported in the literature. We conclude, therefore, that the initial steps of helix propagation are characterized by much larger s values, whereas helix nucleation is even more unfavorable than is believed. In light of our findings, thermodynamics of the nascent alpha-helices is discussed. The problem of CD spectra of very short alpha-helices is also addressed.

Amino Acid Sequence↗
PubMed1999

Synthesis and characterization of the human CC chemokine HCC-2.

Human CC chemokine 2 (HCC-2) is a novel member of the chemokine peptide family that induces chemotaxis of monocytes, T lymphocytes and eosinophils via activation of the CCR-1 and CCR-3 receptors. Fmoc chemistry was optimized and used to synthesize the biologically active 66-residue peptide HCC-2-(48-113). Introduction of the three disulfide bonds was achieved by oxidative folding in the presence of the redox system cysteine/cystine. Alternatively, a semiselective approach utilizing a mixed Acm/Trt protection scheme for disulfide formation was applied. It was found that, without participation of the two HCC-2-specific cysteine residues in positions 64 and 104, the two typical chemokine disulfides are formed predominantly during oxidative folding. In addition, the mutant [Ala64,104]HCC-2-(48-113) lacking the third disulfide bond that discriminates HCC-2 from most other chemokines was synthesized. For disulfide bond formation, oxidative folding was compared with the use of Acm/Trt protection. HCC-2-(48-113) and the mutant [Ala64,104]HCC-2-(48-113) were further analyzed by CD and one-dimensional 1H NMR-spectroscopy. Both peptides adopt a similar stable secondary and tertiary structure in solution.

Amino Acid Sequence↗
PubMed1998

Equine infectious anemia virus transactivator is a homeodomain-type protein.

Lentiviral transactivator (Tat) proteins are essential for viral replication. Tat proteins of human immunodeficiency virus type 1 and bovine immunodeficiency virus form complexes with their respective RNA targets (Tat responsive element, TAR), and specific binding of the equine anemia virus (EIAV) Tat protein to a target TAR RNA is suggested by mutational analysis of the TAR RNA. Structural data on equine infectious anemia virus Tat protein reveal a helix-loop-helix-turn-helix limit structure very similar to homeobox domains that are known to bind specifically to DNA. Here we report results of gel-shift and footprinting analysis as well as fluorescence and nuclear magnetic resonance spectroscopy experiments that clearly show that EIAV Tat protein binds to DNA specifically at the long terminal repeat Pu.1 (GTTCCTGTTTT) and AP-1 (TGACGCG) sites, and thus suggest a common mechanism for the action of some of the known lentiviral Tat proteins via the AP-1 initiator site. Complex formation with DNA induces specific shifts of the proton NMR resonances originating from amino acids in the core and basic domains of the protein.

Animals↗
PubMed1998

Solution structure of the glycosylated second type 2 module of fibronectin.

Fibronectin is an extracellular matrix glycoprotein that plays a role in a number of physiological processes involving cell adhesion and migration. The modules of the fibronectin monomer are organized into proteolytically resistant domains that in isolation retain their affinity for various ligands. The tertiary structure of the glycosylated second type 2 module (2F2) from the gelatin-binding domain of fibronectin was determined by two-dimensional nuclear magnetic resonance spectroscopy and simulated annealing. The structure is well defined with an overall fold typical of F2 modules, showing two double-stranded antiparallel beta-sheets and a partially solvent-exposed hydrophobic cluster. An N-terminal beta-sheet, that was not present in previously determined F2 module structures, may be important for defining the relative orientation of adjacent F2 modules in fibronectin. This is the first three-dimensional structure of a glycosylated module of fibronectin, and provides insight into the possible role of the glycosylation in protein stability, protease resistance and modulation of collagen binding. Based on the structures of the isolated modules, models for the 1F22F2 pair were generated by randomly changing the orientation of the linker peptide between the modules. The models suggest that the two putative collagen binding sites in the pair form discrete binding sites, rather than combining to form a single binding site.

Amino Acid Sequence↗
PubMed1998

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 alpha-helices and a small antiparallel beta-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.

Amino Acid Sequence↗