Biomedical subjects

Matthias Wilmanns

Publications and source records attributed to Matthias Wilmanns.

At least 19 recordsLinked to original sources

PubMed2006

Recognition of a functional peroxisome type 1 target by the dynamic import receptor pex5p.

Peroxisomes require the translocation of folded and functional target proteins of various sizes across the peroxisomal membrane. We have investigated the structure and function of the principal import receptor Pex5p, which recognizes targets bearing a C-terminal peroxisomal targeting signal type 1. Crystal structures of the receptor in the presence and absence of a peroxisomal target, sterol carrier protein 2, reveal major structural changes from an open, snail-like conformation into a closed, circular conformation. These changes are caused by a long loop C terminal to the 7-fold tetratricopeptide repeat segments. Mutations in residues of this loop lead to defects in peroxisomal import in human fibroblasts. The structure of the receptor/cargo complex demonstrates that the primary receptor-binding site of the cargo is structurally and topologically autonomous, enabling the cargo to retain its native structure and function.

Amino Acid Sequence↗

PubMed2006

Structural diversity in the six-fold redundant set of acyl-CoA carboxyltransferases in Mycobacterium tuberculosis.

Mycobacterium tuberculosis contains multiple versions of the accA and accD genes that encode the alpha- and beta-subunits of at least three distinct multi-functional acyl-CoA carboxylase complexes. Because of its proposed involvement in pathogenic M. tuberculosis survival, the high-resolution crystal structure of the beta-subunit gene accD5 product has been determined and reveals a hexameric 356 kDa complex. Analysis of the active site properties of AccD5 and homology models of the other five M. tuberculosis AccD homologues reveals unexpected differences in their surface composition, providing a molecular rational key for a sorting mechanism governing correct acyl-CoA carboxylase holo complex assembly in M. tuberculosis.

Acyl Coenzyme A↗

PubMed2006

Dynamic architecture of the peroxisomal import receptor Pex5p.

The majority of peroxisomal matrix proteins are recognized by the import receptor Pex5p. The receptor is dynamic in terms of its overall architecture and association with the peroxisomal membrane. It participates in different protein complexes during the translocation of cargos from the cytosol to the peroxisomal matrix. Its sequence comprises two structurally and functionally autonomous parts. The N-terminal segment interacts with several peroxins that assemble into distinct protein complexes during cargo translocation. Despite evidence for alpha-helical binding motifs for some of these components (Pex13p, Pex14p) its overall appearance is that of a molten globule and folding/unfolding transitions may play a critical role in its function. In contrast, most of the C-terminal part of the receptor folds into a ring-like alpha-helical structure and binds folded and functionally intact peroxisomal targets that bear a C-terminal peroxisomal targeting signal type-1. Some of these targets also bind to secondary binding sites of the receptor.

Humans↗

PubMed2006

The impact of protein characterization in structural proteomics.

Protein characterization plays a role in two key aspects of structural proteomics. The first is the quality assessment of the produced protein preparations. Obtaining well diffracting crystals is one of the major bottlenecks in the structure-determination pipeline. Often, this is caused by the poor quality of the protein preparation used for crystallization trials. Hence, it is essential to perform an extensive quality assessment of the protein preparations prior to crystallization and to use the results in the evaluation of the process. Here, a protein-production and crystallization strategy is proposed with threshold values for protein purity (95%) and monodispersity (85%) below which a further optimization of the protein-production process is strongly recommended. The second aspect is the determination of protein characteristics such as domains, oligomeric state, post-translational modifications and protein-protein and protein-ligand interactions. In this paper, applications and new developments of protein-characterization methods using MS, fluorescence spectroscopy, static light scattering, analytical ultracentrifugation and small-angle X-ray scattering within the EC Structural Proteomics in Europe contract are described. Examples of the application of the various methods are given.

Crystallization↗

PubMed2006

The Mycobacterium tuberculosis LipB enzyme functions as a cysteine/lysine dyad acyltransferase.

Lipoic acid is essential for the activation of a number of protein complexes involved in key metabolic processes. Growth of Mycobacterium tuberculosis relies on a pathway in which the lipoate attachment group is synthesized from an endogenously produced octanoic acid moiety. In patients with multiple-drug-resistant M. tuberculosis, expression of one gene from this pathway, lipB, encoding for octanoyl-[acyl carrier protein]-protein acyltransferase is considerably up-regulated, thus making it a potential target in the search for novel antiinfectives against tuberculosis. Here we present the crystal structure of the M. tuberculosis LipB protein at atomic resolution, showing an unexpected thioether-linked active-site complex with decanoic acid. We provide evidence that the transferase functions as a cysteine/lysine dyad acyltransferase, in which two invariant residues (Lys-142 and Cys-176) are likely to function as acid/base catalysts. Analysis by MS reveals that the LipB catalytic reaction proceeds by means of an internal thioesteracyl intermediate. Structural comparison of LipB with lipoate protein ligase A indicates that, despite conserved structural and sequence active-site features in the two enzymes, 4'-phosphopantetheine-bound octanoic acid recognition is a specific property of LipB.

Acyltransferases↗

PubMed2006

Evidence for a dimeric assembly of two titin/telethonin complexes induced by the telethonin C-terminus.

The Z-disk region defines the lateral boundary of the sarcomere and requires a high level of mechanical strength to provide a stable framework for large filamentous muscle proteins. The level of complexity at this area is reflected by a large number of protein-protein interactions. Recently, we unraveled how the N-terminus of the longest filament component, the giant muscle protein titin, is assembled into an antiparallel (2:1) sandwich complex by the N-terminal titin-binding segment of the Z-disk ligand telethonin/T-cap [Zou, P., Pinotsis, N., Lange, S., Song, Y.H., Popov, A., Mavridis, I., Mayans, O.M., Gautel, M., Wilmanns, M., 2006. Palindromic assembly of the giant muscle protein titin in the sarcomeric Z-disk. Nature 439, 229-233]. In this contribution, we present structural data of a related complex of the titin N-terminus with full-length telethonin. The C-terminus of telethonin remains invisible, suggesting that it does not fold into a defined structure even in the presence of titin. In contrast to the structure with truncated telethonin, a dimer of two titin/telethonin complexes is formed within the crystal environment, potentially indicating the formation of higher oligomers. We further investigated the structure and dynamics of this assembly by small-angle X-ray scattering, circular dichroism, and in vivo complementation data. The data consistently indicate the involvement of the C-terminal part of telethonin into the assembly of two titin/telethonin complexes.

Animals↗

PubMed2006

The structure of the 2[4Fe-4S] ferredoxin from Pseudomonas aeruginosa at 1.32-A resolution: comparison with other high-resolution structures of ferredoxins and contributing structural features to reduction potential values.

The structure of the 2[4Fe-4S] ferredoxin (PaFd) from Pseudomonas aeruginosa, which belongs to the Allochromatium vinosum (Alvin) subfamily, has been determined by X-ray crystallography at 1.32-A resolution, which is the highest up to now for a member of this subfamily of Fds. The main structural features of PaFd are similar to those of AlvinFd. However, the significantly higher resolution of the PaFd structure makes possible a reliable comparison with available high-resolution structures of [4Fe-4S]-containing Fds, in an effort to rationalize the unusual electrochemical properties of Alvin-like Fds. Three major factors contributing to the reduction potential values of [4Fe-4S]2+/+ clusters of Fds, namely, the surface accessibility of the clusters, the N-H...S hydrogen-bonding network, and the volume of the cavities hosting the clusters, are extensively discussed. The volume of the cavities is introduced in the present work for the first time, and can in part explain the very negative potential of cluster I of Alvin-like Fds.

Amino Acid Sequence↗

PubMed2006

Palindromic assembly of the giant muscle protein titin in the sarcomeric Z-disk.

The Z-disk of striated and cardiac muscle sarcomeres is one of the most densely packed cellular structures in eukaryotic cells. It provides the architectural framework for assembling and anchoring the largest known muscle filament systems by an extensive network of protein-protein interactions, requiring an extraordinary level of mechanical stability. Here we show, using X-ray crystallography, how the amino terminus of the longest filament component, the giant muscle protein titin, is assembled into an antiparallel (2:1) sandwich complex by the Z-disk ligand telethonin. The pseudosymmetric structure of telethonin mediates a unique palindromic arrangement of two titin filaments, a type of molecular assembly previously found only in protein-DNA complexes. We have confirmed its unique architecture in vivo by protein complementation assays, and in vitro by experiments using fluorescence resonance energy transfer. The model proposed may provide a molecular paradigm of how major sarcomeric filaments are crosslinked, anchored and aligned within complex cytoskeletal networks.

Amino Acid Sequence↗

PubMed2006

Mechanical strength of the titin Z1Z2-telethonin complex.

Using molecular dynamics simulations, we have explored the mechanical strength of the titin Z1Z2-telethonin complex, namely, its ability to bear strong forces such as those encountered during passive muscle stretch. Our results show that not only does this complex resist considerable mechanical force through beta strand crosslinking, suggesting that telethonin is an important component of the N-terminal titin anchor, but also that telethonin distributes these forces between its two joined titin Z2 domains to protect the proximal Z1 domains from bearing too much stress. Our simulations also reveal that without telethonin, apo-titin Z1Z2 exhibits significantly decreased resistance to mechanical stress, and that the N-terminal segment of telethonin (residues 1-89) does not exhibit a stable fold conformation when it is unbound from titin Z1Z2. Consequently, our study sheds light on a key but little studied architectural feature of biological cells-the existence of strong mechanical links that glue separate proteins together.

Binding Sites↗

PubMed2006

The Ig doublet Z1Z2: a model system for the hybrid analysis of conformational dynamics in Ig tandems from titin.

Titin is a gigantic elastic filament that determines sarcomere ultrastructure and stretch response in vertebrate muscle. It folds into numerous Ig and FnIII domains connected in tandem. Data on interdomain arrangements and dynamics are essential for understanding the function of this filament. Here, we report a mechanistic analysis of the conformational dynamics of two Ig domains from the N terminus of titin, Z1Z2, by using X-ray crystallography, SAXS, NMR relaxation data, and residual dipolar couplings in combination. Z1Z2 preferentially adopts semiextended conformations in solution, with close-hinge arrangements representing low-probability states. Although interdomain contacts are not observed, the linker appears to acquire moderate rigidity via small, local hydrophobic interactions. Thus, Z1Z2 constitutes an adaptable modular system with restricted dynamics. We speculate that its preexistent conformation contributes to the selective recruitment of the binding partner telethonin onto the repetitive surface of the filament. The structural interconversion of four Z1Z2 conformers is analyzed.

Connectin↗

PubMed2006

Cellular target genes of Epstein-Barr virus nuclear antigen 2.

Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) is a key determinant in the EBV-driven B-cell growth transformation process. By activating an array of viral and cellular target genes, EBNA-2 initiates a cascade of events which ultimately cause cell cycle entry and the proliferation of the infected B cell. In order to identify cellular target genes that respond to EBNA-2 in the absence of other viral factors, we have performed a comprehensive search for EBNA-2 target genes in two EBV-negative B-cell lines. This screen identified 311 EBNA-2-induced and 239 EBNA-2-repressed genes that were significantly regulated in either one or both cell lines. The activation of most of these genes had not previously been attributed to EBNA-2 function and will be relevant for the identification of EBNA-2-specific contributions to EBV-associated malignancies. The diverse spectrum of EBNA-2 target genes described in this study reflects the broad spectrum of EBNA-2 functions involved in virus-host interactions, including cell signaling molecules, adapters, genes involved in cell cycle regulation, and chemokines.

B-Lymphocytes↗

PubMed2005

Crystal structure of the PB1 domain of NBR1.

The scaffold protein NBR1 is involved in signal transmission downstream of the serine/protein kinase from the giant muscle protein titin. Its N-terminal Phox and Bem1p (PB1) domain plays a critical role in mediating protein-protein interactions with both titin kinase and with another scaffold protein, p62. We have determined the crystal structure of the PB1 domain of NBR1 at 1.55A resolution. It reveals a type-A PB1 domain with two negatively charged residue clusters. We provide a structural perspective on the involvement of NBR1 in the titin kinase signalling pathway.

Connectin↗

PubMed2005

Human alpha-defensins neutralize anthrax lethal toxin and protect against its fatal consequences.

Anthrax caused by Bacillus anthracis represents a major bioterroristic threat. B. anthracis produces lethal toxin (LeTx), a combination of lethal factor (LF) and protective antigen that plays a major role in anthrax pathogenesis. We demonstrate that human neutrophil alpha-defensins are potent inhibitors of LF. The inhibition of LF by human neutrophil protein (HNP-1) was noncompetitive. HNP-1 inhibited cleavage of a mitogen-activated protein kinase kinase and restored impaired mitogen-activated protein kinase signaling in LeTx-treated macrophages. HNP-1 rescued murine macrophages from B. anthracis-induced cytotoxicity, and in vivo treatment with HNP-1-3 protected mice against the fatal consequences of LeTx.

Animals↗

PubMed2005

Two-fold repeated (betaalpha)4 half-barrels may provide a molecular tool for dual substrate specificity.

Some bacterial genomes contain an incomplete set of genes encoding phosphoribosyl isomerases, raising the question of whether there exists broadened substrate specificity for the missing gene products. To investigate the underlying molecular principles of this hypothesis, we have determined the crystal structure of the bifunctional enzyme PriA from Streptomyces coelicolor at 1.8 A resolution. It consists of a (betaalpha)(8)-barrel fold that is assembled by two symmetric (betaalpha)(4) half-barrels. The structure shows how its active site may catalyse the isomerization reactions of two different substrates, and we provide a plausible model of how the smaller of the two substrates could be bound in two different orientations. Our findings expand the half-barrel ancestor concept by demonstrating that symmetry-related half-barrels could provide a smart solution to cope with dual substrate specificity. The data may help to unravel molecular rationales regarding how organisms with miniature genomes can keep central biological pathways functional.

Adenosine Triphosphatases↗

PubMed2005

Regulation of the hetero-octameric ATP phosphoribosyl transferase complex from Thermotoga maritima by a tRNA synthetase-like subunit.

The molecular structure of the ATP phosphoribosyl transferase from the hyperthermophile Thermotoga maritima is composed of a 220 kDa hetero-octameric complex comprising four catalytic subunits (HisGS) and four regulatory subunits (HisZ). Steady-state kinetics indicate that only the complete octameric complex is active and non-competitively inhibited by the pathway product histidine. The rationale for these findings is provided by the crystal structure revealing a total of eight histidine binding sites that are located within each of the four HisGS-HisZ subunit interfaces formed by the ATP phosphoribosyl transferase complex. While the structure of the catalytic HisGS subunit is related to the catalytic domain of another family of (HisGL)2 ATP phosphoribosyl transferases that is functional in the absence of additional regulatory subunits, the structure of the regulatory HisZ subunit is distantly related to class II aminoacyl-tRNA synthetases. However, neither the mode of the oligomeric subunit arrangement nor the type of histidine binding pockets is found in these structural relatives. Common ancestry of the regulatory HisZ subunit and class II aminoacyl-tRNA synthetase may reflect the balanced need of regulated amounts of a cognate amino acid (histidine) in the translation apparatus, ultimately linking amino acid biosynthesis and protein biosynthesis in terms of function, structure and evolution.

ATP Phosphoribosyltransferase↗

PubMed2004

Structural insights into the catalytic mechanism of phosphate ester hydrolysis by dUTPase.

dUTPase is essential to keep uracil out of DNA. Crystal structures of substrate (dUTP and alpha,beta-imino-dUTP) and product complexes of wild type and mutant dUTPases were determined to reveal how an enzyme responsible for DNA integrity functions. A kinetic analysis of wild type and mutant dUTPases was performed to obtain relevant mechanistic information in solution. Substrate hydrolysis is shown to be initiated via in-line nucleophile attack of a water molecule oriented by an activating conserved aspartate residue. Substrate binding in a catalytically competent conformation is achieved by (i) multiple interactions of the triphosphate moiety with catalysis-assisting Mg2+, (ii) a concerted motion of residues from three conserved enzyme motifs as compared with the apoenzyme, and (iii) an intricate hydrogen-bonding network that includes several water molecules in the active site. Results provide an understanding for the catalytic role of conserved residues in dUTPases.

Amino Acid Motifs↗

PubMed2004

Structural studies of the catalytic reaction pathway of a hyperthermophilic histidinol-phosphate aminotransferase.

In histidine biosynthesis, histidinol-phosphate aminotransferase catalyzes the transfer of the amino group from glutamate to imidazole acetol-phosphate producing 2-oxoglutarate and histidinol phosphate. In some organisms such as the hyperthermophile Thermotoga maritima, specific tyrosine and aromatic amino acid transaminases have not been identified to date, suggesting an additional role for histidinol-phosphate aminotransferase in other transamination reactions generating aromatic amino acids. To gain insight into the specific function of this transaminase, we have determined its crystal structure in the absence of any ligand except phosphate, in the presence of covalently bound pyridoxal 5'-phosphate, of the coenzyme histidinol phosphate adduct, and of pyridoxamine 5'-phosphate. The enzyme accepts histidinol phosphate, tyrosine, tryptophan, and phenylalanine, but not histidine, as substrates. The structures provide a model of how these different substrates could be accommodated by histidinol-phosphate aminotransferase. Some of the structural features of the enzyme are more preserved between the T. maritima enzyme and a related threonine-phosphate decarboxylase from S. typhimurium than with histidinol-phosphate aminotransferases from different organisms.

Amino Acid Sequence↗

PubMed2004

Adaptation of class-13 alpha-amylases to diverse living conditions.

There are currently 35 available nonredundant molecular structures of class-13 alpha-amylases (EC 3.2.1.1), mostly from microbial organisms living under a wide range of environmental conditions. One of the most recent additions has been the first alpha-amylase structure of a hyperthermophilic archaeon [Linden et al., J. Biol. Chem. 2003, 278, 9875-9884]. The structure has been used for comparative analyses with a representative set of three alpha-amylases from thermophilic, mesophilic and psychrophilic sources to identify molecular parameters for environmental adaptation. Our analysis supports generally observed trends such as an increase in structural compactness as well as an increase in salt bridges in order to cope with high-temperature conditions. The two representative thermophilic structures used in this comparative study have independently evolved di-metal centres--not present in the mesophilic and psychrophilic structures--in the vicinity of the active site. These observations may provide impetus for the design of alpha-amylases with improved molecular properties to enhance their utility in biotechnological processes.

Adaptation, Physiological↗