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F Jacob-Dubuisson

Publications and source records attributed to F Jacob-Dubuisson.

27 records · Page 2Linked to original sources
PubMed1994

Stable fiber-forming and nonfiber-forming chaperone-subunit complexes in pilus biogenesis.

The P pilus is a composite fiber consisting of a thin adhesive tip fibrillum joined to the pilus rod that mediates specific adherence of uropathogenic Escherichia coli to human uroepithelial cells via the PapG tip adhesin. P pilus assembly depends upon the periplasmic chaperone PapD. The interaction of PapD with different pilus subunits was investigated to gain further insight into pilus assembly. PapA, the major subunit of the pilus rod, formed two periplasmic complexes (DA2 and DA) with PapD. PapK, an adaptor protein that joins the tip fibrillum to the pilus rod, formed only one complex with PapD (DK). Only "fiber forming" or homopolymeric subunits, PapA in the rod and PapE in the tip fibrillum, were able to form subunit-subunit interactions in the periplasm. Subunits that are present in single or low copy in the pilus (PapK and PapG) did not form periplasmic intersubunit interactions. A pulse-chase analysis revealed that a chaperone-PapA complex is a true periplasmic intermediate in pilus assembly.

Bacterial Proteins↗
PubMed1993

Outer-membrane PapC molecular usher discriminately recognizes periplasmic chaperone-pilus subunit complexes.

P pili are highly ordered composite structures consisting of thin fibrillar tips joined end-to-end to rigid helical rods. The production of these virulence-associated structures requires a periplasmic chaperone (PapD) and an outer membrane protein (PapC) that is the prototype member of a newly recognized class of proteins that we have named "molecular ushers." Two in vitro assays showed that the preassembly complexes that PapD forms with the three most distal tip fibrillar proteins (PapG, PapF, and PapE) bound to PapC. The relative affinity of each complex for PapC was found to correlate with the final position of the subunit type in the tip fibrillum. In contrast, the complexes PapD forms with the major component of the pilus rod, PapA, or the pilus rod initiating protein, PapK, did not recognize PapC. The in vitro data argue that differential targeting of chaperone-subunit complexes to PapC may be part of a mechanism to ensure the correctly ordered assembly of adhesive composite pili.

Adhesins, Escherichia coli↗
PubMed1993

Initiation of assembly and association of the structural elements of a bacterial pilus depend on two specialized tip proteins.

Uropathogenic Escherichia coli produce heteropolymeric surface fibers called P pili, which present an adhesin at their tip that specifically recognizes globoside receptors on the host uroepithelium. The initial attachment step is thought to be essential for pathogenesis. P pili are composite fibers consisting of a thin tip fibrillum joined end to end to a rigid helical rod. Here we show that the ordered assembly of these structures requires the activity of two proteins that are minor components of the tip fibrillum, PapF and PapK. PapF is required for the correct presentation of the adhesin at the distal end of the tip fibrillum. PapK regulates the length of the tip fibrillum and joins it to the pilus rod. We propose that these subunits function as adaptors, by providing complementary surfaces to different substructures of the pilus and promoting their proper associations. In addition, the conversion of chaperone-subunit complexes into pili depends on PapF and PapK since a papF- papK- double mutation abolishes piliation. We suggest that in addition to the adaptor functions of PapF and PapK, they are also required to initiate the formation of tip fibrillae and pilus rods.

Adhesins, Escherichia coli↗
PubMed1993

A novel secretion apparatus for the assembly of adhesive bacterial pili.

The biogenesis of most types of bacterial pili requires two specialized proteins: a chaperone that caps the pilus subunits in the periplasm, and an outer membrane usher that receives the subunits and serves as an assembly platform. This secretion and assembly machinery is proposed to be a novel export apparatus found widely in Gram-negative pathogens.

Bacterial Adhesion↗
PubMed1993

PapD and superfamily of periplasmic immunoglobulin-like pilus chaperones.

The formation of a P pilus requires a molecular chaperone in the periplasm and a molecular usher in the outer membrane. Each pilus is composed of six different types of proteins that are assembled into a composite fiber in a defined order. The correct folding of subunits into domains that can serve as assembly modules requires an association with the periplasmic chaperone. PapD is the prototype member of the family of bacterial pilus chaperones that have a three-dimensional structure consistent with an immunoglobulin fold. In general, proteins with an immunoglobulin fold structure have molecular recognition functions in eukaryotic cells that are often integrated with effector functions. PapD has also a recognition function, binding nascently translocated pilus subunits and maintaining them in assembly-competent conformations. The association of the chaperone with the subunit triggers the targeting of the latter to an outer membrane usher. The usher serves as a molecular gatekeeper, allowing the ordered incorporation of the pilus subunits into the pilus structure from the periplasmic chaperone complexes. The two immunoglobulin-like domains of PapD are oriented to form a cleft that contains the subunit binding site. This is a different binding paradigm from that used by either antibodies or the growth hormone receptor. The blend of genetics, biochemistry, X-ray crystallography, and carbohydrate chemistry in the study of pili biogenesis will continue to give insight into some of the most basic intellectual challenges in molecular biology concerning how proteins fold into domains that serve as modules for the formation of larger assemblies, and relating these processes to microbial pathogenesis.

Adhesins, Escherichia coli↗
PubMed1992

Streptomyces albus G serine beta-lactamase. Probing of the catalytic mechanism via molecular modelling of mutant enzymes.

In previous studies, several amino acids of the active site of class A beta-lactamases have been modified by site-directed mutagenesis. On the basis of the catalytic mechanism proposed for the Streptomyces albus G beta-lactamase [Lamotte-Brasseur, Dive, Dideberg, Charlier, Frère & Ghuysen (1991) Biochem. J. 279, 213-221], the influence that these mutations exert on the hydrogen-bonding network of the active site has been analysed by molecular mechanics. The results satisfactorily explain the effects of the mutations on the kinetic parameters of the enzyme's activity towards a set of substrates. The present study also shows that, upon binding a properly structured beta-lactam compound, the impaired cavity of a mutant enzyme can readopt a functional hydrogen-bonding-network configuration.

Amino Acid Sequence↗
PubMed1991

Arginine 220 is a critical residue for the catalytic mechanism of the Streptomyces albus G beta-lactamase.

Residue Arg220 was found to be important for the acylation of the Streptomyces albus G beta-lactamase by classical penicillins and cephalosporins bearing a carboxylate on C3 or C4. The R220L mutant exhibited strongly decreased kcat/Km values for those compounds. Conversely the acylation rates by benzylpenicillin methylester and deacetylcephalosporin C lactone were little affected, indicating a direct or indirect role of that positively charged residue in the interaction of the enzyme cavity with the negative charge of the substrate. Surprisingly that residue is not conserved in all class A beta-lactamases but when it is not present it can be seen in the known tertiary structures that the guanidinium group of another arginine side chain (Arg244) is similarly positioned. The mutation affected the behaviour of the enzyme towards cephaloridine much less than towards cephalothin. This might represent an example of substrate-assisted catalysis where the disappearance of a positive charge on the enzyme is partly compensated by the presence of a similarly charged group on one of the substrate side chains. All the experimental results are nicely explained by computer-modelling of the enzyme-substrate interactions.

Acylation↗