Biomedical subjects

Han Remaut

Publications and source records attributed to Han Remaut.

9 recordsLinked to original sources

PubMed2006

Rationally designed small compounds inhibit pilus biogenesis in uropathogenic bacteria.

A chemical synthesis platform with broad applications and flexibility was rationally designed to inhibit biogenesis of adhesive pili assembled by the chaperone-usher pathway in Gram-negative pathogens. The activity of a family of bicyclic 2-pyridones, termed pilicides, was evaluated in two different pilus biogenesis systems in uropathogenic Escherichia coli. Hemagglutination mediated by either type 1 or P pili, adherence to bladder cells, and biofilm formation mediated by type 1 pili were all reduced by approximately 90% in laboratory and clinical E. coli strains. The structure of the pilicide bound to the P pilus chaperone PapD revealed that the pilicide bound to the surface of the chaperone known to interact with the usher, the outer-membrane assembly platform where pili are assembled. Point mutations in the pilicide-binding site dramatically reduced pilus formation but did not block the ability of PapD to bind subunits and mediate their folding. Surface plasmon resonance experiments confirmed that the pilicide interfered with the binding of chaperone-subunit complexes to the usher. These pilicides thus target key virulence factors in pathogenic bacteria and represent a promising proof of concept for developing drugs that function by targeting virulence factors.

Anti-Bacterial Agents↗

PubMed2006

Molecular mechanism of P pilus termination in uropathogenic Escherichia coli.

P pili are important adhesive fibres that are assembled by the conserved chaperone-usher pathway. During pilus assembly, the subunits are incorporated into the growing fibre by the donor-strand exchange mechanism, whereby the beta-strand of the chaperone, which complements the incomplete immunoglobulin fold of each subunit, is displaced by the amino-terminal extension of an incoming subunit in a zip-in-zip-out exchange process that is initiated at the P5 pocket, an exposed hydrophobic pocket in the groove of the subunit. In vivo, termination of P pilus growth requires a specialized subunit, PapH. Here, we show that PapH is incorporated at the base of the growing pilus, where it is unable to undergo donor-strand exchange. This inability is not due to a stronger PapD-PapH interaction, but to a lack of a P5 initiator pocket in the PapH structure, suggesting that PapH terminates pilus growth because it is lacking the initiation point by which donor-strand exchange proceeds.

Bacterial Adhesion↗

PubMed2006

Protein-protein interaction through beta-strand addition.

Protein-protein interactions have essential roles at almost every level of organization and communication in living cells. During complex formation, proteins can interact via covalent, surface-surface or peptide-surface contacts. Many protein complexes are now known to involve the binding of linear motifs in one of the binding partners. An emerging mechanism of such non-covalent peptide-surface interaction involves the donation or addition of a beta strand in the ligand to a beta sheet or a beta strand in the receptor. Such 'beta-strand addition' contacts can dictate or modulate binding specificity and affinity, or can be used in more promiscuous protein-protein contacts. Three main classes of beta-strand addition can be distinguished: beta-sheet augmentation; beta-strand insertion and fold complementation; and beta-strand zippering. A survey of protein-protein complexes in the protein data bank identifies beta-strand additions in many important metabolic pathways. Targeting these interactions might, thus, provide novel routes for rational drug design.

Animals↗

PubMed2006

Donor-strand exchange in chaperone-assisted pilus assembly proceeds through a concerted beta strand displacement mechanism.

Gram-negative pathogens commonly use the chaperone-usher pathway to assemble adhesive multisubunit fibers on their surface. In the periplasm, subunits are stabilized by a chaperone that donates a beta strand to complement the subunits' truncated immunoglobulin-like fold. Pilus assembly proceeds through a "donor-strand exchange" (DSE) mechanism whereby this complementary beta strand is replaced by the N-terminal extension (Nte) of an incoming pilus subunit. Using X-ray crystallography and real-time electrospray ionization mass spectrometry (ESI-MS), we demonstrate that DSE requires the formation of a transient ternary complex between the chaperone-subunit complex and the Nte of the next subunit to be assembled. The process is crucially dependent on an initiation site (the P5 pocket) needed to recruit the incoming Nte. The data also suggest a capping reaction displacing DSE toward product formation. These results support a zip-in-zip-out mechanism for DSE and a catalytic role for the usher, the molecular platform at which pili are assembled.

Bacterial Proteins↗

PubMed2004

Fiber assembly by the chaperone-usher pathway.

Bacterial pathogens utilize the chaperone-usher pathway to assemble extracellular multi-subunit fibers essential for virulence. The periplasmic chaperone facilitates the initial folding of fiber subunits but then traps them in activated folding transition states. Chaperone dissociation releases the folding energy that drives subunit incorporation into the fiber, which grows through a pore formed by the outer-membrane usher.

Bacterial Proteins↗

PubMed2004

Crystal structure of T state aspartate carbamoyltransferase of the hyperthermophilic archaeon Sulfolobus acidocaldarius.

Aspartate carbamoyltransferase (ATCase) is a model enzyme for understanding allosteric effects. The dodecameric complex exists in two main states (T and R) that differ substantially in their quaternary structure and their affinity for various ligands. Many hypotheses have resulted from the structure of the Escherichia coli ATCase, but so far other crystal structures to test these have been lacking. Here, we present the tertiary and quaternary structure of the T state ATCase of the hyperthermophilic archaeon Sulfolobus acidocaldarius (SaATC(T)), determined by X-ray crystallography to 2.6A resolution. The quaternary structure differs from the E.coli ATCase, by having altered interfaces between the catalytic (C) and regulatory (R) subunits, and the presence of a novel C1-R2 type interface. Conformational differences in the 240 s loop region of the C chain and the C-terminal region of the R chain affect intersubunit and interdomain interfaces implicated previously in the allosteric behavior of E.coli ATCase. The allosteric-zinc binding domain interface is strengthened at the expense of a weakened R1-C4 type interface. The increased hydrophobicity of the C1-R1 type interface may stabilize the quaternary structure. Catalytic trimers of the S.acidocaldarius ATCase are unstable due to a drastic weakening of the C1-C2 interface. The hyperthermophilic ATCase presents an interesting example of how an allosteric enzyme can adapt to higher temperatures. The structural rearrangement of this thermophilic ATCase may well promote its thermal stability at the expense of changes in the allosteric behavior.

Allosteric Site↗

PubMed2004

Structural biology of bacterial pathogenesis.

Recent years have seen a rapid increase in structural information on proteins implicated in bacterial pathogenesis. The different modes by which bacteria establish contact with their host tissues are exemplified by the structures of bacterial adhesins in complex with their cognate host receptor. A more detailed structural understanding of the various Gram-negative secretion systems has emerged with the determination of the structures of type I and type IV secretion system components, and with the elucidation of the mechanism of fibre formation in the chaperone-usher pathway of pilus biogenesis. Finally, the structures of complexes of secreted virulence factors bound to their host targets have unravelled the mechanisms by which bacterial pathogens exploit cellular processes to their advantage.

Adhesins, Bacterial↗

PubMed2002

Molecular characterization of Bacillus pasteurii UreE, a metal-binding chaperone for the assembly of the urease active site.

The present study describes the cloning, isolation, and thorough biochemical characterization of UreE from Bacillus pasteurii, a novel protein putatively involved in the transport of Ni in the urease assembly process. A DNA fragment of the B. pasteurii urease operon, containing all four accessory genes (ureE, ureF, ureG, and ureD) required for the incorporation of Ni ions into the active site of urease, was cloned, sequenced, and analyzed. B. pasteurii ureE was cloned, and the UreE protein (BpUreE) was over-expressed and purified to homogeneity. The identity of the recombinant protein was determined by N- and C-terminal sequencing and by mass spectrometry. BpUreE has a chain length of 147 amino acids, and features a p I value of 4.7. As isolated, BpUreE contains one Zn(II) ion per dimer, while no Ni(II) is present, as shown by mass spectrometry and atomic absorption spectroscopy. BpUreE behaves as a dimer independently of the presence of Zn(II), as shown by gel filtration and mass spectrometry. Paramagnetic NMR spectroscopy on concentrated (2 mM) UreE solutions reveals a one Ni atom per tetramer stoichiometry, with the Ni(II) ion bound to histidines in an octahedral coordination environment. BpUreE has a high sequence similarity with UreE proteins isolated from different biological sources, while no sequence homology is observed with proteins belonging to different classes. In particular, BpUreE is most similar to UreE from Bacillus halodurans (55% identity). A multiple sequence alignment reveals the presence of four strictly conserved residues (Leu55, Gly97, Asn98, His100; BpUreE numbering), in addition to position 115, conservatively occupied by an Asp or a Glu residue. Several secondary structure elements, including a betaalphabetabetaalphabeta "ferredoxin-like" motif, are highly conserved throughout the UreE sequences.

Amino Acid Sequence↗

PubMed2002

Crystallization and preliminary X-ray crystallographic analysis of glutathione amide reductase from Chromatium gracile.

The Chromatiaceae-specific glutathione amide reductase (GAR) belongs to the well known family of the glutathione reductases, even though differences in both substrate (glutathione amide instead of glutathione) and coenzyme (NADH instead of NADPH) specificities are reported. Crystals of the GAR enzyme from Chromatium gracile have been grown at 294 K by the hanging-drop vapour-diffusion method using lithium sulfate as a precipitant in the presence of nickel ions. The crystals belong to space group P4(1), with unit-cell parameters a = b = 71.93, c = 223.85 A, alpha = beta = gamma = 90 degrees and one dimer per asymmetric unit. A full set of X-ray diffraction data was collected to 2.1 A resolution with a completeness of 95.2%. Structure determination via the method of molecular replacement is under way.

Bacterial Proteins↗