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Robert Huber

Publications and source records attributed to Robert Huber.

At least 55 records · Page 3Linked to original sources
PubMed2004

Structure of the streptococcal endopeptidase IdeS, a cysteine proteinase with strict specificity for IgG.

Pathogenic bacteria have developed complex and diverse virulence mechanisms that weaken or disable the host immune defense system. IdeS (IgG-degrading enzyme of Streptococcus pyogenes) is a secreted cysteine endopeptidase from the human pathogen S. pyogenes with an extraordinarily high degree of substrate specificity, catalyzing a single proteolytic cleavage at the lower hinge of human IgG. This proteolytic degradation promotes inhibition of opsonophagocytosis and interferes with the killing of group A Streptococcus. We have determined the crystal structure of the catalytically inactive mutant IdeS-C94S by x-ray crystallography at 1.9-A resolution. Despite negligible sequence homology to known proteinases, the core of the structure resembles the canonical papain fold although with major insertions and a distinct substrate-binding site. Therefore IdeS belongs to a unique family within the CA clan of cysteine proteinases. Based on analogy with inhibitor complexes of papain-like proteinases, we propose a model for substrate binding by IdeS.

Amino Acid Sequence↗
PubMed2004

Inhibitors of the eukaryotic 20S proteasome core particle: a structural approach.

The ubiquitin-proteasome pathway is particularly important for the regulated degradation of various proteins which control a vast array of biological processes. Therefore, proteasome inhibitors are promising candidates for anti-tumoral or anti-inflammatory drugs. N-Acetyl-Leu-Leu-Norleucinal (Ac-LLN-al, also termed calpain inhibitor I) was one of the first proteasome inhibitors discovered and has been widely used to study the 20S proteasome core particle (CP) function in vivo, despite its lack of specificity. Vinyl sulfones, like Ac-PRLN-vs, show covalent binding of the beta-carbon atom of the vinyl sulfone group to the Thr1Ogamma only of subunit beta2. However, vinyl sulfones have similar limitations as peptide aldehydes as they have been reported also to bind and block intracellular cysteine proteases. A more specific proteasome inhibitor is the natural product lactacystin, which can be isolated from Streptomyces. It was found that this compound forms an ester bond only to the Thr1Ogamma of the chymotrypsin-like active subunit beta5 due to specific P1 interactions. In contrast to most other proteasome inhibitors, the natural alpha',beta'-epoxyketone peptide epoxomicin binds specifically to the small class of N-terminal nucleophilic (Ntn) hydrolases (CPs belong to this protease family) with the formation of a morpholino adduct. All previously described proteasome inhibitors bind covalently to the proteolytic active sites. However, as the proteasome is involved in a variety of biological important functions, it is of particular interest to block the CP only for limited time in order to reduce cytotoxic effects. Recently, the binding mode of the natural specific proteasome inhibitor TMC-95 obtained from Apiospora montagnei was investigated. The crystal structure revealed that the TMC-95 blocks the active sites of the CP noncovalently in the low nanomolar range. This review summarizes the current structural knowledge of inhibitory compounds bound to the CP, showing the proteasome as a potential target for drug development in medical research.

Amidohydrolases↗
PubMed2004

Evolution of vitamin B2 biosynthesis. A novel class of riboflavin synthase in Archaea.

The open reading frame MJ1184 of Methanococcus jannaschii with similarity to riboflavin synthase of Methanothermobacter thermoautotrophicus was cloned into an expression vector but was poorly expressed in an Escherichia coli host strain. However, a synthetic open reading frame that was optimized for expression in E.coli directed the synthesis of abundant amounts of a protein with an apparent subunit mass of 17.5 kDa. The protein was purified to apparent homogeneity. Hydrodynamic studies indicated a relative mass of 88 kDa suggesting a homopentamer structure. The enzyme was shown to catalyze the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a rate of 24 nmol mg(-1) min(-1) at 40 degrees C. Divalent metal ions, preferably manganese or magnesium, are required for maximum activity. In contrast to pentameric archaeal type riboflavin synthases, orthologs from plants, fungi and eubacteria are trimeric proteins characterized by an internal sequence repeat with similar folding patterns. In these organisms the reaction is achieved by binding the two substrate molecules in an antiparallel orientation. With the enzyme of M.jannaschii, 13C NMR spectroscopy with 13C-labeled 6,7-dimethyl-8-ribityllumazine samples as substrates showed that the regiochemistry of the dismutation reaction is the same as observed in eubacteria and eukaryotes, however, in a non-pseudo-c2 symmetric environment. Whereas the riboflavin synthases of M.jannaschii and M.thermoautotrophicus are devoid of similarity with those of eubacteria and eukaryotes, they have significant sequence similarity with 6,7-dimethyl-8-ribityllumazine synthases catalyzing the penultimate step of riboflavin biosynthesis. 6,7-Dimethyl-8-ribityllumazine synthase and the archaeal riboflavin synthase appear to have diverged early in the evolution of Archaea from a common ancestor. Some Archaea have eubacterial type riboflavin synthases which may have been acquired by lateral gene transfer.

Amino Acid Sequence↗
PubMed2004

Molecular structure of the rod domain of dictyostelium filamin.

Dictyostelium discoideum filamin (ddFLN) is a two-chain F-actin crosslinking protein with an N-terminal actin-binding domain and a rod domain constructed from six tandem repeats of a 100 residue motif that has an immunoglobulin (Ig) fold. We report the 2.8 A resolution crystal structure of a homodimer of rod repeats 4, 5 and 6. The two chains are arranged in an antiparallel fashion and form an elongated element, which is shortened, however, compared to a fully extended, linear configuration because the long axis of each Ig domain is arranged at an angle to the long axis of the rod. Same arrangement of repeats should also be present in the rod domain of human FLNa, much longer than Dictyostelium FLN, which forms an extended structure able to crosslink F-actin chains over distances of more than 1000 A.

Actins↗
PubMed2004

Structural basis for the interaction of Escherichia coli NusA with protein N of phage lambda.

The C terminus of transcription factor NusA from Escherichia coli comprises two repeat units, which bind during antitermination to protein N from phage lambda. To delineate the structural basis of the NusA-lambdaN interaction, we attempted to crystallize the NusA C-terminal repeats in complex with a lambdaN peptide (residues 34-47). The two NusA domains became proteolytically separated during crystallization, and crystals contained two copies of the first repeat unit in contact with a single lambdaN fragment. The NusA modules employ identical regions to contact the peptide but approach the ligand from opposite sides. In contrast to the alpha-helical conformation of the lambdaN N terminus in complex with boxB RNA, residues 34-40 of lambdaN remain extended upon interaction with NusA. Mutational analyses indicated that only one of the observed NusA-lambdaN interaction modes is biologically significant, supporting an equimolar ratio of NusA and lambdaN in antitermination complexes. Solution studies indicated that additional interactions are fostered by the second NusA repeat unit, consistent with known compensatory mutations in NusA and lambdaN. Contrary to the RNA polymerase alpha subunit, lambdaN binding does not stimulate RNA interaction of NusA. The results demonstrate that lambdaN serves as a scaffold to closely oppose NusA and the mRNA in antitermination complexes.

Amino Acid Sequence↗
PubMed2004

Binding mode of TMC-95A analogues to eukaryotic 20S proteasome.

The complex thermodynamics that govern noncovalent protein-ligand interactions are still not fully understood, despite the exponential increase in experimental structural data available from X-ray crystallography and NMR spectroscopy. The eukaryotic 20S proteasome offers an ideal system for such studies as it contains in duplicate three proteolytically active sites with different substrate specificities. The natural product TMC-95A inhibits these proteolytic centers noncovalently with distinct affinities. X-ray crystallographic analysis of the complexes of the yeast proteasome core particle with this natural inhibitor and two synthetic analogues clearly revealed highly homologous hydrogen-bonding networks involving mainly the peptide backbone despite the strongly differentiated binding affinities to the three active sites of the 20S proteasome. The natural product and the two analogues are constrained in a rigid beta-type extended conformation by the endocyclic biaryl clamp, which preorganizes the peptide backbone for optimal adaptation of the ligands to the active site clefts and thus favors the binding processes entropically. However, the biaryl clamp also dictates the orientation of the P1 and P3 residues and their mode of interaction with the protein binding subsites. This limitation is optimally solved in TMC-95A with the conformationally restricted (Z)-prop-1-enyl group acting as P1 residue, at least for the chymotrypsin-like active site; however, it critically affects the inhibitory potencies of the analogues, thus suggesting the use of less-rigid endocyclic clamps in the design of proteasome inhibitors that allow for a better presentation of residues interacting with the active site clefts of the enzyme.

Binding Sites↗
PubMed2004

Cloning, expression, crystallization and initial crystallographic analysis of the C-terminal domain of the amyloid precursor protein APP.

Alzheimer's disease is associated with typical brain deposits (senile plaques) consisting mainly of neurotoxic amyloid beta-peptides. These are proteolytically derived from the large type I transmembrane protein amyloid precursor protein (APP), which is possibly involved in signal transduction. The large C-terminal domain CAPPD of the human APP ectodomain has been cloned, expressed in large amounts in Escherichia coli and purified to homogeneity. Well diffracting tetragonal crystals have been obtained and native data have been collected to 2.1 A. Initial experimental phases from a three-wavelength MAD experiment using (NH(4))(2)OsCl(6)-derivatized crystals are of good quality and show mostly alpha-helical conformations.

Amyloid beta-Protein Precursor↗
PubMed2004

Potential anti-infective targets in pathogenic yeasts: structure and properties of 3,4-dihydroxy-2-butanone 4-phosphate synthase of Candida albicans.

A synthetic gene specifying a putative 3,4-dihydroxy-2-butanone 4-phosphate synthase of Candida albicans directed the synthesis of a 22.5 kDa peptide in a recombinant Escherichia coli strain. The recombinant protein was purified to apparent homogeneity by two chromatographic steps and was shown to catalyze the formation of L-3,4-dihydroxy-2-butanone 4-phosphate from ribulose 5-phosphate at a rate of 332 nmol mg(-1) min(-1). Hydrodynamic studies indicated a native molecular mass of 41 kDa in line with a homodimer structure. The protein was crystallized in its apoform. Soaking yielded crystals in complex with the substrate ribulose 5-phosphate. The structures were solved at resolutions of 1.6 and 1.7 angstroms, respectively, using 3,4-dihydroxy-2-butanone 4-phosphate synthase of E. coli for molecular replacement. Structural comparison with the orthologs of Magnaporthe grisea and Methanococcus jannaschii revealed a hitherto unknown conformation of the essential acidic active-site loop.

Amino Acid Sequence↗
PubMed2004

Ethological analyses of crayfish behavior: a new invertebrate system for measuring the rewarding properties of psychostimulants.

Recent investigations in invertebrate neurobiology have opened up a new line of research into the basic behavioral, neurochemical and genomic alterations that accompany psychostimulant drug exposure. However, the extent to which such findings relate to changes in motivational and learning processes, such as those that typify drug addictions, remains unclear. The present study addressed this issue in the crayfish, Orconectes rusticus. The first set of experiments demonstrated that intramuscular injections of cocaine and amphetamine have robust and distinguishable effects on crayfish behavior. In the second part of the study, the reinforcing properties of psychostimulants were tested in a series of conditioned place preference experiments. Amphetamine and, to a lesser extent, cocaine were both found to serve as rewards when their intra-circulatory infusion was coupled to a distinct visual environment. The monoaminergic regulation of behavior has been extensively studied in decapod crustaceans and the present experiments demonstrated that (mammalian) drugs of abuse, capable of interfering with monoamine chemistry, are similarly rewarding to crayfish. Behavioral studies in crayfish can provide a complementary approach to using other invertebrate species in addiction research.

Amphetamine↗
PubMed2004

Gender-selective patterns of aggressive behavior in Drosophila melanogaster.

Complex behaviors, such as aggression, are comprised of distinct stereospecific behavioral patterns (modules). How such patterns get wired into nervous systems remains unknown. Recently, we reported on a quantitative analysis of fighting behavior in male flies of the common Canton-S strain of Drosophila melanogaster. Here, we report a similar analysis of fighting behavior in females of the same species. Fights were carried out between pairs of virgin and pairs of mated females in competition for a yeast resource. Each fight was videotaped and analyzed by using transition matrices and Markov chain analyses. We observe only small difference in fighting intensity between virgin and mated females. In contrast to what is seen in male fights, however, no clear hierarchical relationship is formed in the female fights. A further comparison of the behavioral patterns making up male and female fights reveals that some modules are shared by both sexes, whereas others are highly selective. Within the shared components, transitions between the modules also show gender-selective differences. By using the powerful genetic methods available for examining behavior in fruit flies, it should be possible to use the gender-selective differences in fighting behavior to address the question of how these behavioral patterns get established in the brains of fruit flies.

Aggression↗
PubMed2004

A fast photoswitch for minimally perturbed peptides: investigation of the trans-->cis photoisomerization of N-methylthioacetamide.

Thio amino acids can be integrated into the backbone of peptides without significantly perturbing their structure. In this contribution we use ultrafast infrared and visible spectroscopy as well as state-of-the-art ab initio computations to investigate the photoisomerization of the trans form of N-methylthioacetamide (NMTAA) as a model conformational photoswitch. Following the S2 excitation of trans-NMTAA in water, the return of the molecule into the trans ground state and the formation of the cis isomer is observed on a dual time scale, with a fast component of 8-9 ps and a slow time constant of approximately 250 ps. On both time scales the probability of isomerization to the cis form is found to be 30-40%, independently of excitation wavelength. Ab initio CASPT2//CASSCF photochemical reaction path calculations indicate that, in vacuo, the trans-->cis isomerization event takes place on the S1 and/or T1 triplet potential energy surfaces and is controlled by very small energy barriers, in agreement with the experimentally observed picosecond time scale. Furthermore, the calculations identify one S2/S1 and four nearly isoenergetic S1/S0 conical intersection decay channels. In line with the observed isomerization probability, only one of the S1/S0 conical intersections yields the cis conformation upon S1-->S0 decay. A substantially equivalent excited-state relaxation results from four T1/S0 intersystem crossing points.

Kinetics↗
PubMed2004

A rapid method for analyzing recombinant protein inclusion bodies by mass spectrometry.

The identification and characterization of a protein overexpressed in insoluble inclusion bodies in Escherichia coli are the first crucial and time-limiting steps in recombinant protein expression. Here, a straightforward approach to the analysis of recombinant proteins in inclusion bodies is presented. Inclusion bodies were dissolved in 8M urea and analyzed by matrix-assisted laser desorption ionization (MALDI)-time of flight mass spectrometry without prior desalting. Mass determination was achieved by direct spotting of the samples onto the MALDI target and serial dilution in the matrix. The masses of four different proteins, expressed in inclusion bodies, were determined with a mass accuracy better than 0.1%. Furthermore, protein modifications, such as N-terminal processing of single amino acids or artificial cyanylation caused by incubation of the inclusion bodies with urea at elevated temperatures, could be detected. Similarly, tryptic digests were directly analyzed in 2M urea to obtain peptide mass fingerprints for identification and more detailed information on the primary protein structure and secondary modifications. Due to the presence of ammonia in the urea-containing buffers, no Na(+) adducts were observed in the peptide mass fingerprint analysis. Taken together, the rapid and robust procedures presented here greatly facilitate the analysis of recombinant proteins.

Antibodies, Monoclonal↗
PubMed2004

The typically disordered N-terminus of PKA can fold as a helix and project the myristoylation site into solution.

Protein kinases comprise the major enzyme family critically involved in signal transduction pathways; posttranslational modifications affect their regulation and determine signaling states. The prototype protein kinase A (PKA) possesses an N-terminal alpha-helix (Helix A) that is atypical for kinases and is thus a major distinguishing feature of PKA. Its physiological function may involve myristoylation at the N-terminus and modulation via phosphorylation at serine 10. Here we describe an unusual structure of an unmyristoylated PKA, unphosphorylated at serine 10, with a completely ordered N-terminus. Using standard conditions (e.g., PKI 5-24, ATP site ligand, MEGA-8), a novel 2-fold phosphorylated PKA variant showed the ordered N-terminus in a new crystal packing arrangement. Thus, the critical factor for structuring the N-terminus is apparently the absence of phosphorylation of Ser10. The flexibility of the N-terminus, its myristoylation, and the conformational dependence on the phosphorylation state are consistent with a functional role for myristoylation.

Cyclic AMP-Dependent Protein Kinases↗
PubMed2004

Biosynthesis of tetrahydrofolate in plants: crystal structure of 7,8-dihydroneopterin aldolase from Arabidopsis thaliana reveals a novel adolase class.

Dihydroneopterin aldolase (DHNA) catalyses a retroaldol reaction yielding 6-hydroxymethyl-7,8-dihydropterin, a biosynthetic precursor of the vitamin, tetrahydrofolate. The enzyme is a potential target for antimicrobial and anti-parasite chemotherapy. A gene specifying a dihydroneopterin aldolase from Arabidopsis thaliana was expressed in a recombinant Escherichia coli strain. The recombinant protein was purified to apparent homogeneity and crystallised using polyethylenglycol as the precipitating agent. The crystal structure was solved by X-ray diffraction analysis at 2.2A resolution. The enzyme forms a D(4)-symmetric homooctamer. Each polypeptide chain is folded into a single domain comprising an antiparallel four-stranded beta-sheet and two long alpha-helices. Four monomers are arranged in a tetrameric ring, and two of these rings form a hollow cylinder. Well defined purine derivatives are found at all eight topologically equivalent active sites. The subunit fold of the enzyme is related to substructures of dihydroneopterin triphosphate epimerase, GTP cyclohydrolase I, and pyruvoyltetrahydropterin synthase, which are all involved in the biosynthesis of pteridine type cofactors, and to urate oxidase, although some members of that superfamily have no detectable sequence similarity. Due to structural and mechanistical differences of DHNA in comparison with class I and class II aldolases, a new aldolase class is proposed.

Amino Acid Sequence↗
PubMed2004

Structure of the molybdenum-cofactor biosynthesis protein MoaB of Escherichia coli.

The moaABC operon of Escherichia coli is involved in early steps of the biosynthesis of the molybdenum-binding cofactor molybdopterin, but the precise functions of the cognate proteins are not known. The crystal structure of the MoaB protein from E. coli was determined by multiple anomalous dispersion at 2.1 angstroms A resolution and refined to an R factor of 20.4% (Rfree = 25.0%). The protein is a 32-symmetric hexamer, with the monomers consisting of a central beta-sheet flanked by helices on both sides. The overall fold of the monomer is similar to those of the MogA protein of E. coli, the G-domains of rat and human gephyrin and the G-domains of Cnx1 protein from A. thaliana, all of which are involved in the insertion of an unknown molybdenum species into molybdopterin to form the molybdenum cofactor. Furthermore, the MoaB protein shows significant sequence similarity to the cinnamon protein from Drosophila melanogaster. In addition to other functions, all these proteins are involved in the biosynthesis of the molybdenum cofactor and have been shown to bind molybdopterin. The close structural homology to MogA and the gephyrin and Cnx1 domains suggests that MoaB may bind a hitherto unidentified pterin compound, possibly an intermediate in molybdopterin biosynthesis.

Amino Acid Sequence↗
PubMed2004

The genome sequence of the extreme thermophile Thermus thermophilus.

Thermus thermophilus HB27 is an extremely thermophilic, halotolerant bacterium, which was originally isolated from a natural thermal environment in Japan. This organism has considerable biotechnological potential; many thermostable proteins isolated from members of the genus Thermus are indispensable in research and in industrial applications. We present here the complete genome sequence of T. thermophilus HB27, the first for the genus Thermus. The genome consists of a 1,894,877 base pair chromosome and a 232,605 base pair megaplasmid, designated pTT27. The 2,218 identified putative genes were compared to those of the closest relative sequenced so far, the mesophilic bacterium Deinococcus radiodurans. Both organisms share a similar set of proteins, although their genomes lack extensive synteny. Many new genes of potential interest for biotechnological applications were found in T. thermophilus HB27. Candidates include various proteases and key enzymes of other fundamental biological processes such as DNA replication, DNA repair and RNA maturation.

Genome, Bacterial↗