Claus Urbanke

Structure of Internalin, a Major Invasion Protein of Listeria monocytogenes, in Complex with Its Human Receptor E-Cadherin

Listeria monocytogenes, a food-borne bacterial pathogen, enters mammalian cells by inducing its own phagocytosis. The listerial protein internalin (InlA) mediates bacterial adhesion and invasion of epithelial cells in the human intestine through specific interaction with its host cell receptor E-cadherin. We present the crystal structures of the functional domain of InlA alone and in a complex with the extracellular, N-terminal domain of human E-cadherin (hEC1). The leucine rich repeat (LRR) domain of InlA surrounds and specifically recognizes hEC1. Individual interactions were probed by mutagenesis and analytical ultracentrifugation. These include Pro16 of hEC1, a major determinant for human susceptibility to L. monocytogenes infection that is essential for intermolecular recognition. Our studies reveal the structural basis for host tro-pism of this bacterium and the molecular deception L. monocytogenes employs to exploit the E-cadherin system.

Clustering of VASP actively drives processive, WH2 domain-mediated actin filament elongation

Vasodilator‐stimulated phosphoprotein (VASP) is a key regulator of dynamic actin structures like filopodia and lamellipodia, but its precise function in their formation is controversial. Using in vitro TIRF microscopy, we show for the first time that both human and Dictyostelium VASP are directly involved in accelerating filament elongation by delivering monomeric actin to the growing barbed end. In solution, DdVASP markedly accelerated actin filament elongation in a concentration‐dependent manner but was inhibited by low concentrations of capping protein (CP). In striking contrast, VASP clustered on functionalized beads switched to processive filament elongation that became insensitive even to very high concentrations of CP. Supplemented with the in vivo analysis of VASP mutants and an EM structure of the protein, we propose a mechanism by which membrane‐associated VASP oligomers use their WH2 domains to effect both the tethering of actin filaments and their processive elongation in sites of active actin assembly.

Isolation and characterization of heparin- and phosphorylcholine-binding proteins of boar and stallion seminal plasma. Primary structure of porcine pB1

In the bovine, seminal plasma heparin‐binding proteins bind to sperm lipids containing the phosphorylcholine group and mediate the capacitating effects of heparin‐like glycosaminoglycans during sperm residence in the female genital tract. We report the characterization of heparin‐ and phosphorylcholine‐binding proteins of stallion and boar seminal plasma. orse eminal lasma proteins HSP‐1 and HSP‐2, and boar protein pB1, belong to the same family as the bull heparin‐ and phosphorylcholine‐binding proteins BSP‐A1/2, BSP‐A3, and BSP‐30K. We have determined the amino acid sequence and posttranslational modifications of boar glycoprotein pB1. It contains 105 amino acids arranged into a mosaic structure consisting of a N‐terminal 18‐residue O‐glycosylated polypeptide followed by two tandemly organized 40–45‐residue fibronectin type II domains. pB1 displays 60–65% amino acid sequence similarity with its equine and bovine homologues. However, in their respective seminal plasmas, the BSP and the HSP proteins associate into 90–150‐kDa oligomeric complexes, whereas pB1 forms a 35–40‐kDa complex with spermadhesin AQN‐1. In addition, pB1 appears to be identical to the recently described leukocyte adhesion regulator of porcine seminal fluid pAIF‐1. Our results tie in with the hypothesis that homologous proteins from different mammalian species may display distinct biological activities, which may be related to species‐specific aspects of sperm physiology.

Formation of nucleoprotein filaments by mammalian DNA methyltransferase Dnmt3a in complex with regulator Dnmt3L

The C-terminal domains of Dnmt3a and Dnmt3L form elongated heterotetramers (3L-3a-3a-3L). Analytical ultracentrifugation confirmed the Dnmt3a-C/3L-C complex exists as a 2:2 heterotetramer in solution. The 3a–3a interface is the DNA-binding site, while both interfaces are essential for AdoMet binding and catalytic activity. Hairpin bisulfite analysis shows correlated methylation of two CG sites in a distance of ∼8-10 bp in the opposite DNA strands, which corresponds to the geometry of the two active sites in one Dnmt3a-C/3L-C tetramer. Correlated methylation was also observed for two CG sites at similar distances in the same DNA strand, which can be attributed to the binding of two tetramers next to each other. DNA-binding experiments show that Dnmt3a-C/3L-C complexes multimerize on the DNA. Scanning force microscopy demonstrates filament formation rather than binding of single tetramers and shows that protein–DNA filament formation leads to a 1.5-fold shortening of the DNA length.

A common core for binding single-stranded DNA: structural comparison of the single-stranded DNA-binding proteins (SSB) from E. coli and human mitochondria.

The crystal structure of the DNA‐binding domain of E. coli SSB (EcoSSB) has been determined to a resolution of 2.5 Å. This is the first reported structure of a prokaryotic SSB. The structure of the DNA‐binding domain of the E. coli protein is compared to that of the human mitochondrial SSB (HsmtSSB). In spite of the relatively low sequence identity between them, the two proteins display a high degree of structural similarity. EcoSSB crystallises with two dimers in the asymmetric unit, unlike HsmtSSB which contains only a dimer. This is probably a consequence of the different polypeptide chain lengths in the EcoSSB heterotetramer. Crucial differences in the dimer‐dimer interface of EcoSSB may account for the inability of EcoSSB and HsmtSSB to form cross‐species heterotetramers, in contrast to many bacterial SSBs.

MOLECULAR CHARACTERIZATION AND CRYSTALLIZATION OF DIOCLEINAE LECTINS

Molecular characterization of seven Diocleinae lectins was assessed by sequence analysis, determination of molecular masses by mass spectrometry, and analytical ultracentrifugation equilibrium sedimentation. The lectins show distinct pH-dependent dimer-tetramer equilibria, which we hypothesize are due to small primary structure differences at key positions. Lectins from Dioclea guianensis, Dioclea virgata, and Cratylia floribunda seeds have been crystallized and preliminary X-ray diffraction analyses are reported.

Oligomerization and Binding of the Dnmt3a DNA Methyltransferase to Parallel DNA Molecules: HETEROCHROMATIC LOCALIZATION AND ROLE OF Dnmt3L

Structural studies showed that Dnmt3a has two interfaces for protein-protein interaction in the heterotetrameric Dnmt3a/3L C-terminal domain complex: the RD interface (mediating the Dnmt3a-3a contact) and the FF interface (mediating the Dnmt3a-3L contact). Here, we demonstrate that Dnmt3a-C forms dimers via the FF interface as well, which further oligomerize via their RD interfaces. Each RD interface of the Dnmt3a-C oligomer creates an independent DNA binding site, which allows for binding of separate DNA molecules oriented in parallel. Because Dnmt3L does not have an RD interface, it prevents Dnmt3a oligomerization and binding of more than one DNA molecule. Both interfaces of Dnmt3a are necessary for the heterochromatic localization of the enzyme in cells. Overexpression of Dnmt3L in cells leads to the release of Dnmt3a from heterochromatic regions, which may increase its activity for methylation of euchromatic targets like the differentially methylated regions involved in imprinting.

Single-stranded DNA-binding protein of Deinococcus radiodurans: a biophysical characterization

The highly conserved bacterial single-stranded DNA-binding (SSB) proteins play an important role in DNA replication, repair and recombination and are essential for the survival of the cell. They are functional as tetramers, in which four OB(oligonucleotide/oligosaccharide binding)-folds act as DNA-binding domains. The protomer of the SSB protein from the extremely radiation-resistant organism Deinococcus radiodurans (DraSSB) has twice the size of the other bacterial SSB proteins and contains two OB-folds. Using analytical ultracentrifugation, we could show that DraSSB forms globular dimers with some protrusions. These DraSSB dimers can interact with two molecules of E.coli DNA polymerase III χ subunit. In fluorescence titrations with poly(dT) DraSSB bound 47–54 nt depending on the salt concentration, and fluorescence was quenched by more than 75%. A distinct low salt binding mode as for EcoSSB was not observed for DraSSB. Nucleic acid binding affinity, rate constant and association mechanism are quite similar for EcoSSB and DraSSB. In a complementation assay in E.coli, DraSSB took over the in vivo function of EcoSSB. With DraSSB behaving almost identical to EcoSSB the question remains open as to why dimeric SSB proteins have evolved in the Thermus group of bacteria.

Native acridone synthases I and II from Ruta graveolens L. form homodimers

Acridone synthase II cDNA was cloned from irradiated cell suspension cultures of Ruta graveolens L. and expressed in Escherichia coli. The translated polypeptide of M r 42 681 revealed a high degree of similarity to heterologous chalcone and stilbene synthases (70–75%), and the sequence was 94% identical to that of acridone synthase I cloned previously from elicited Ruta cells. Highly active recombinant acridone synthases I and II were purified to apparent homogeneity by a four‐step purification protocol, and the affinities to N‐methylanthraniloyl‐CoA and malonyl‐CoA were determined. The molecular mass of acridone synthase II was estimated from size exclusion chromatography on a Fractogel EMD BioSEC (S) column at about 45 kDa, as compared to a mass of 44±3 kDa found for the acridone synthase I on Superdex 75. Nevertheless, the sedimentation analysis by ultracentrifugation revealed molecular masses of 81±4 kDa for both acridone synthases. It is proposed, therefore, that the acridone synthases of Ruta graveolens are typical homodimeric plant polyketide synthases.

The C-terminal domain is sufficient for endonuclease activity of Neisseria gonorrhoeae MutL.

The mutL gene of Neisseria gonorrhoeae has been cloned and the gene product purified. We have found that the homodimeric N. gonorrhoeae MutL (NgoL) protein displays an endonuclease activity that incises covalently closed circular DNA in the presence of Mn(2+), Mg(2+) or Ca(2+) ions, unlike human MutLalpha which shows endonuclease activity only in the presence of Mn(2+). We report in the present paper that the C-terminal domain of N. gonorrhoeae MutL (NgoL-CTD) consisting of amino acids 460-658 exhibits Mn(2+)-dependent endonuclease activity. Sedimentation velocity, sedimentation equilibrium and dynamic light scattering experiments show NgoL-CTD to be a dimer. The probable endonucleolytic active site is localized to a metal-binding motif, DMHAX2EX4E, and the nicking endonuclease activity is dependent on the integrity of this motif. By in vitro comparison of wild-type and a mutant NgoL-CTD protein, we show that the latter protein exhibits highly reduced endonuclease activity. We therefore suggest that the mode of excision initiation in DNA mismatch repair may be different in organisms that lack MutH protein, but have MutL proteins that harbour the D[M/Q]HAX2EX4E motif.