Biomedical subjects

Klaus Fütterer

Publications and source records attributed to Klaus Fütterer.

10 recordsLinked to original sources

PubMed2006

Designed high affinity Cu2+-binding alpha-helical foldamer.

The design, synthesis, and characterization of a folded high-affinity metal-binding peptide is described. Based on the previously described folded peptide NTH-18, in which an alpha-helix was constrained through two disulfide bonds to a C-terminal extension of noncanonical secondary structure, a peptide (1) was designed to contain two histidine residues in positions 3 and 7. Air oxidation of 1 led to the formation of peptide 2, which contained two intramolecular disulfide bonds. The presence of the two histidines significantly destabilized the alpha-helical structure of 2 when compared to NTH-18. However, CD spectroscopy revealed that the addition of certain transition metal ions allowed the reformation of a stable alpha-helix. CD, NMR, and EPR spectroscopy as well as MALDI-TOF mass spectrometry indicated that 2 bound to Cu2+ to form a 1:1 complex via the imidazoles of the two histidine side chains. A glycine displacement assay revealed a dissociation constant for this complex of 5 nM at pH 8, which is the lowest reported value for a designed Cu2+-binding peptide. This peptide displayed more than 100-fold selectivity for Cu2+ over Zn2+, Ni2+, and Co2+. The 1.05 A crystal structure of the Cu(II)-complex of 2 revealed a square-pyramidal coordination geometry and confirmed that 2 bound to copper in an alpha-helical conformation via its two histidine side chains. The high affinity metal binding of peptide 2 demonstrates that metals can be used for the selective nucleation of alpha-helices.

Copper↗

PubMed2006

Molecular structure of EmbR, a response element of Ser/Thr kinase signaling in Mycobacterium tuberculosis.

Ser/Thr phosphorylation has emerged as a critical regulatory mechanism in a number of bacteria, including Mycobacterium tuberculosis. This problematic pathogen encodes 11 eukaryotic-like Ser/Thr kinases, yet few substrates or signaling targets have been characterized. Here, we report the structure of EmbR (2.0 A), a putative transcriptional regulator of key arabinosyltransferases (EmbC, -A, and -B), and an endogenous substrate of the Ser/Thr-kinase PknH. EmbR presents a unique domain architecture: the N-terminal winged-helix DNA-binding domain forms an extensive interface with the all-helical central bacterial transcriptional activation domain and is positioned adjacent to the regulatory C-terminal forkhead-associated (FHA) domain, which mediates binding to a Thr-phosphorylated site in PknH. The structure in complex with a phospho-peptide (1.9 A) reveals a conserved mode of phospho-threonine recognition by the FHA domain and evidence for specific recognition of the cognate kinase. The present structures suggest hypotheses as to how EmbR might propagate the phospho-relay signal from its cognate kinase, while serving as a template for the structurally uncharacterized Streptomyces antibiotic regulatory protein family of transcription factors.

Bacterial Proteins↗

PubMed2005

Structural basis of filopodia formation induced by the IRSp53/MIM homology domain of human IRSp53.

The scaffolding protein insulin receptor tyrosine kinase substrate p53 (IRSp53), a ubiquitous regulator of the actin cytoskeleton, mediates filopodia formation under the control of Rho-family GTPases. IRSp53 comprises a central SH3 domain, which binds to proline-rich regions of a wide range of actin regulators, and a conserved N-terminal IRSp53/MIM homology domain (IMD) that harbours F-actin-bundling activity. Here, we present the crystal structure of this novel actin-bundling domain revealing a coiled-coil domain that self-associates into a 180 A-long zeppelin-shaped dimer. Sedimentation velocity experiments confirm the presence of a single molecular species of twice the molecular weight of the monomer in solution. Mutagenesis of conserved basic residues at the extreme ends of the dimer abrogated actin bundling in vitro and filopodia formation in vivo, demonstrating that IMD-mediated actin bundling is required for IRSp53-induced filopodia formation. This study promotes an expanded view of IRSp53 as an actin regulator that integrates scaffolding and effector functions.

Amino Acid Sequence↗

PubMed2004

Crystal structure of the TetR/CamR family repressor Mycobacterium tuberculosis EthR implicated in ethionamide resistance.

Ethionamide has been used for more than 30 years as a second-line chemotherapeutic to treat tuberculosis patients who have developed resistance to first-line drugs, such as isoniazid (INH) and rifampicin. Activation of the pro-drug ethionamide is regulated by the Baeyer-Villiger monooxygenase EthA and the TetR/CamR family repressor EthR, whose open reading frames are separated by 75 bp on the Mycobacterium tuberculosis genome. EthR has been shown to repress transcription of the activator gene ethA by binding to this intergenic region, thus contributing to ethionamide resistance. We have determined the crystal structure of EthR, to 1.7A resolution, revealing a dimeric two-domain molecule with an overall architecture typical for TetR/CamR repressor proteins. A 20A long hydrophobic tunnel-like cavity in the "drug-binding" domain of EthR is occupied by two 1,4-dioxane molecules, a component of the crystallisation buffer. Comparing the present structure to those of the homologues Staphylococcus aureus QacR and Escherichia coli TetR leads to the hypothesis that the hydrophobic cavity constitutes a binding site for an as yet unknown ligand that might regulate DNA-binding of EthR.

Amino Acid Sequence↗

PubMed2004

The C-terminal domain of full-length E. coli SSB is disordered even when bound to DNA.

The crystal structure of full-length homotetrameric single-stranded DNA (ssDNA)-binding protein from Escherichia coli (SSB) has been determined to 3.3 A resolution and reveals that the entire C-terminal domain is disordered even in the presence of ssDNA. To our knowledge, this is the first experimental evidence that the C-terminal domain of SSB may be inherently disordered. The N-terminal DNA-binding domain of the protein is well ordered and is virtually indistinguishable from the previously determined structure of the chymotryptic fragment of SSB (SSBc) in complex with ssDNA. The absence of observable interactions with the core protein and the crystal packing of SSB together suggest that the disordered C-terminal domains likely extend laterally away from the DNA- binding domains, which may facilitate interactions with components of the replication machinery in vivo. The structure also reveals the conservation of molecular contacts between successive tetramers mediated by the L(45) loops as seen in two other crystal forms of SSBc, suggesting a possible functional relevance of this interaction.

Crystallography, X-Ray↗

PubMed2004

The structure of Mycobacterium tuberculosis MPT51 (FbpC1) defines a new family of non-catalytic alpha/beta hydrolases.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is known to secrete a number of highly immunogenic proteins that are thought to confer pathogenicity, in part, by mediating binding to host tissues. Among these secreted proteins are the trimeric antigen 85 (Ag85) complex and the related MPT51 protein, also known as FbpC1. While the physiological function of Ag85, a mycolyltransferase required for the biosynthesis of the cell wall component alpha,alpha'-trehalose dimycolate (or cord factor), has been identified recently, the function of the closely related MPT51 (approximately 40% identity with the Ag85 components) remains to be established. The crystal structure of M.tuberculosis MPT51, determined to 1.7 A resolution, shows that MPT51, like the Ag85 components Ag85B and Ag85C2, folds as an alpha/beta hydrolase, but it does not contain any of the catalytic elements required for mycolyltransferase activity. Moreover, the absence of a recognizable alpha,alpha'-trehalose monomycolate-binding site and the failure to detect an active site suggest that the function of MPT51 is of a non-enzymatic nature and that MPT51 may in fact represent a new family of non-catalytic alpha/beta hydrolases. Previous experimental evidence and the structural similarity to some integrins and carbohydrate-binding proteins led to the hypothesis that MPT51 might have a role in host tissue attachment, whereby ligands may include the serum protein fibronectin and small sugars.

Acyltransferases↗

PubMed2003

Crystallization and preliminary X-ray diffraction data of Mycobacterium tuberculosis FbpC1 (Rv3803c).

The heterotrimeric antigen 85 complex (Ag85) is a major component of the cell wall of Mycobacterium tuberculosis and consists of three abundantly secreted proteins (FbpA, FbpB and FbpC2). These play key roles in the pathogenesis of tuberculosis and in maintaining cell-wall integrity. A homologue of the Ag85 subunits ( approximately 40% identity) was recently annotated in the M. tuberculosis genome as FbpC1. Unlike the Ag85-complex components, FbpC1 lacks mycolyltransferase activity and its function remains to be established. In order to aid functional characterization, FbpC1 has been crystallized. At room temperature, tetragonal crystals of FbpC1 were obtained belonging to space group P4(1)2(1)2 (unit-cell parameters a = b = 109.9, c = 61.8 A), yet when frozen the crystals underwent a phase transition to orthorhombic symmetry, space group P2(1)2(1)2(1) (a = 59.9, b = 108.9, c = 109.9 A). Diffraction data complete to 1.7 A resolution were recorded at 100 K at the synchrotron.

Antigens, Bacterial↗

PubMed2003

Structural framework of fructosyl transfer in Bacillus subtilis levansucrase.

Many bacteria and about 40,000 plant species form primary carbohydrate reserves based on fructan; these polymers of beta-D-fructofuranose are thought to confer tolerance to drought and frost in plants. Microbial fructan, the beta(2,6)-linked levan, is synthesized directly from sucrose by levansucrase, which is able to catalyze both sucrose hydrolysis and levan polymerization. The crystal structure of Bacillus subtilis levansucrase, determined to a resolution of 1.5 A, shows a rare five-fold beta-propeller topology with a deep, negatively charged central pocket. Arg360, a residue essential for polymerase activity, lies in a solvent-exposed site adjacent to the central pocket. Mutagenesis data and the sucrose-bound structure of inactive levansucrase E342A, at a resolution of 2.1 A, strongly suggest that three conserved acidic side chains in the central pocket are critical for catalysis, and presumably function as nucleophile (Asp86) and general acid (Glu342), or stabilize the transition state (Asp247).

Amino Acid Sequence↗

PubMed2003

Tetratricopeptide-like repeats in type-III-secretion chaperones and regulators.

Efficient type-III secretion depends on cytosolic molecular chaperones, which bind specifically to the translocators and effectors. In the past there has been a tendency to shoe-horn all type-III-secretion chaperones into a single structural and functional class. However, we have shown that the LcrH/SycD-like chaperones consist of three central tetratricopeptide-like repeats that are predicted to fold into an all-alpha-helical array that is quite distinct from the known structure of the SycE class of chaperones. Furthermore, we predict that this array creates a peptide-binding groove that is utterly different from the helix-binding groove in SycE. We present a homology model of LcrH/SycD that is consistent with existing mutagenesis data. We also report the existence of tetratricopeptide-like repeats in regulators of type-III secretion, such as HilA from Salmonella enterica and HrpB from Ralstonia solanacearum. The discovery of tetratricopeptide-like repeats in type-III-secretion regulators and chaperones provides a new conceptual framework for structural and mutagenesis studies and signals a potential unification of prokaryotic and eukaryotic chaperone biology.

Amino Acid Sequence↗

PubMed2003

Identification and characterisation of a novel human isoform of Arp2/3 complex subunit p16-ARC/ARPC5.

The Arp2/3 complex is an actin filament nucleator that activates regulated actin assembly in response to extracellular signals. The mammalian complex is composed of seven subunits, the smallest of which is known as ARPC5 or p16-Arc. We have identified a human cDNA sequence with homology to ARPC5 and here provide evidence that this encodes a novel ARPC5 isoform. Specific antibodies were generated against the novel protein, which we have termed ARPC5B, as well as the previously characterised ARPC5 isoform, henceforth ARPC5A. The presence of both ARPC5 isoforms was detected in Arp2/3 complex affinity purified from human neutrophil extract. The tissue distribution of ARPC5A and B was analysed using the isoform-specific antibodies and it was found that the two isoforms exhibited significant differences; ARPC5A was found to be highly enriched in spleen and thymus, while ARPC5B exhibits a more regular expression, with levels in the brain being highest. Myc-tagged ARPC5A and B co-localised with the Arp2/3 complex when expressed in C2C12 cells and the cellular distribution of the two isoforms could not be distinguished. Our data show for the first time that mammalian cells contain multiple forms of the Arp2/3 complex.

Actin-Related Protein 2↗