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The nucleotide sequence of a 2510 base pair chromosomal fragment containing the aerolysin gene aerA, and its regulatory region aerC, from a clinical isolate of Aeromonas sobria was determined. The aerolysin gene coded for a 54.5 kD polypeptide and had a G + C content of 59%, indicating that it is endogenous to the genus Aeromonas. In contrast, the aerC region was characterized by its high A + T content (61%) and the presence of a core motif, aATAAAa, repeated eight times within 300 base pairs. A 12 base pair repeat, 5′ AATAAAACCGGG3′ present within this region occurred as a direct repeat 544 base pairs away, within the coding region of aerolysin. RNA polymerase binding studies and S1 mapping allowed the detection of two divergent non-overlapping promoters within aerC. Despite having identical transcriptional start sites in both A. sobria and Escherichia coli, the amount of aerolysin transcript produced in E. coli is 30–40 times less than that found in A. sobria. The signal peptide of preproaerolysin was shown by deletion to be essential for export of the toxin to the external medium. The mature toxin is a hydrophilic protein with no hydrophobic stretches long enough to cross a membrane. A search for similarities to the primary sequence of aerolysin revealed that the toxin may share a functional similarity to haemolysin (hlyA) of E. coli. [ABSTRACT FROM AUTHOR]

The vasodilator-stimulated phosphoprotein (VASP) is a key regulator of actin dynamics. We have determined the 1.3-A resolution crystal structure of the 45-residue-long tetramerization domain (TD) from human VASP. This domain forms a right-handed alpha-helical coiled-coil structure with a similar degree of supercoiling as found in the widespread left-handed coiled coils with heptad repeats. The basis for the right-handed geometry of VASP TD is a 15-residue repeat in its amino acid sequence, which reveals a characteristic pattern of hydrophobic residues. Hydrophobic interactions and a network of salt bridges render VASP TD highly thermostable with a melting point of 120 degrees C.

Vasodilator-stimulated phosphoprotein (VASP) associates with cytoskeletal structures and promotes F-actin formation. RhoA, a member of the Ras superfamily of proteins, activates serum response element (SRE)-dependent transcription through changes in actin dynamics. We now show that the F-actin binding region of VASP is required for VASP stimulation of SRE-dependent transcription, and that VASP is downstream of RhoA in stimulating SRE-dependent transcription. The isolated carboxyl-terminal coiled-coil region of VASP mediates protein tetramerization and has been used as a dominant negative form of VASP; we found that it forms complexes with endogenous VASP in vivo and inhibits in a dose-dependent fashion serum-, RhoA-, and VASP-stimulated SRE-dependent transcription. Cyclic GMP-dependent protein kinase (G-kinase) inhibits RhoA activation of SRE-dependent transcription (Gudi, T., Chen, J. C., Casteel, D. E., Seasholtz, T. M., Boss, G. R., and Pilz, R. B. (2002) J. Biol. Chem. 277, 37382-37393). We now show that the G-kinase inhibition that occurs downstream of RhoA can be explained, at least in part, by G-kinase phosphorylation of VASP on Ser(239) at the carboxyl-terminal end of the G-actin binding site, with some contribution by phosphorylation of Ser(157), which is proximal to the profilin binding site. A phosphorylation-deficient VASP mutant can partly prevent cGMP/G-kinase inhibition of serum- and RhoA-induced SRE-dependent transcription. These studies show that VASP, an important component of the cellular microfilament system, plays a major role in regulating SRE-dependent transcription, and that G-kinase regulates VASP activity.

Vasodilator-stimulated phosphoprotein (VASP) and mammalian enabled (Mena) are actin cytoskeleton and signaling modulators. Ena-VASP proteins share an identical domain organization with an NH2-terminal Ena VASP homology (EVH1) domain, which mediates the binding of these proteins to FPPPP-motif containing partners such as zyxin and vinculin. VASP and Mena are abundantly expressed in the heart. However, previous studies showed that disruption by gene targeting of VASP or Mena genes in mice did not reveal any cardiac phenotype, whereas mice lacking both VASP and Mena died during embryonic development. To determine the in vivo function of Ena-VASP proteins in the heart, we used a dominant negative strategy with cardiac-specific expression of the VASP-EVH1 domain. Transgenic mice with cardiac myocyte-restricted, alpha-myosin heavy chain promoter-directed expression of the VASP-EVH1 domain were generated. Overexpression of the EVH1 domain resulted in specific displacement of both VASP and Mena from cardiac intercalated disks. VASP-EVH1 transgenic mice developed dilated cardiomyopathy with myocyte hypertrophy and bradycardia, which resulted in early postnatal lethality in mice with high levels of transgene expression. The results demonstrate that Ena-VASP proteins may play an important role in intercalated disk function at the interface between cardiac myocytes.

Ena/VASP proteins are implicated in cytoskeletal reorganization during actin-dependent motility processes. Recruitment to subcellular sites of actin polymerization is mediated by the highly conserved N-terminal EVH1 domain, which interacts with target proteins containing proline-rich motifs. The VASP EVH1 domain specifically binds peptides with the consensus motif FPPPP present in all its binding partners, including the Listerial ActA protein. Previous studies have shown that the Phe and first and final Pro residues are highly conserved and cannot be substituted with any other natural amino acid without significant loss of binding affinity. We have incorporated peptoid building blocks (sarcosine derived, non-natural amino acids) into the peptide SFEFPPPPTEDEL from the Listerial ActA protein and were able to substitute the most highly conserved residues of this motif while maintaining binding to the VASP EVH1 domain with affinities in the range of 45-180 microm. We then used NMR chemical shift perturbations to locate specific domain residues involved in particular interactions. These studies may open up the way for designing selective modulators of VASP function for biological studies and for the development of novel therapeutics for diseases involving pathologically altered cell adhesion or cell motility.

An investigation of the structural and dynamic properties of the C-terminal fragment of the human protein VASP (VASP 336-380) has been performed. Full length VASP has been shown to be tetrameric in solution, and the C-terminal 45 residues of the protein have been suggested to be responsible for the oligomerization. We have expressed and purified a C-terminal fragment of the human VASP protein from residue 336-380. It was found to form a stable domain in its own right. The fragment was shown by CD spectroscopy to form a helical structure, stable under a wide range of temperature and pH conditions. A (15)N-HSQC-experiment exhibits only one set of peaks, suggesting a high degree of symmetry for a putative oligomer. Measurements of the rotational correlation time tau(C) of the molecule and analytical ultracentrifugation data show VASP (336-380) to be entirely tetrameric in solution. The secondary structure was confirmed from a (15)N-NOESY-HSQC experiment and is completely alpha-helical. We conclude that VASP (336-380) forms a tetramer in solution via a coiled coil arrangement and is solely responsible for tetramerization of full-length VASP.

Drosophila enabled/vasodilator-stimulated phosphoprotein homology 1 (EVH1) domains are 115 residue protein-protein interaction modules which provide essential links for their host proteins to various signal transduction pathways. Many EVH1-containing proteins are associated closely with actin-based structures and are involved in re-organization of the actin cytoskeleton. EVH1 domains are also present in proteins enriched in neuronal tissue, thus implicating them as potential mediators of synaptic plasticity, linking them to memory formation and learning. Like Src homology 3, WW and GYF domains and profilin, EVH1 domains recognize and bind specific proline-rich sequences (PRSs). The binding is of low affinity, but tightly regulated by the high specificity encoded into residues in the protein:peptide interface. In general, a small (3-6 residue) 'core' PRS in the target protein binds a 'recognition pocket' on the domain surface. Further affinity- and specificity-increasing interactions are then formed between additional domain epitopes and peptide 'core-flanking' residues. The three-dimensional structures of EVH1:peptide complexes now reveal, in great detail, some of the most important features of these interactions and allow us to better understand the origins of specificity, ligand orientation and sequence degeneracy of target peptides, in low affinity signalling complexes.

We propose a normalization of symbols and terms used to describe, accurately and succinctly, the detailed interactions between amino acid residues of pairs of interacting proteins at protein:protein (or protein:peptide) interfaces. Our aim is to unify several diverse descriptions currently in use in order to facilitate communication in the rapidly progressing field of signaling by protein domains. In order for the nomenclature to be convenient and widely used, we also suggest a parallel set of symbols restricted to the ASCII format allowing accurate parsing of the nomenclature to a computer-readable form. This proposal will be reviewed in the future and will therefore be open for the inclusion of new rules, modifications and changes.

Activation of cyclic GMP-dependent protein kinase (cGK) is an important event in the regulation of blood pressure and platelet function. Upstream signals are the generation of nitric oxide (NO) by NO synthases and the subsequent rise in cyclic GMP levels mediated by NO-dependent guanylyl cyclases (GCs). The identification of new cGK activators by high throughput screening (HTS) may lead to the development of a novel class of therapeutics for the treatment of cardiovascular diseases. Therefore, a homogeneous, nonradioactive assay for cGK activity was developed using a biotinylated peptide derived from vasodilator-stimulated phosphoprotein (VASP), a well-characterized natural cGK substrate. The phosphorylated peptide could be detected by a VASP-specific monoclonal phosphoserine antibody and a fluorescent detection system consisting of a europium-labeled secondary antibody and allophycocyanin (APC)-labeled streptavidin. Fluorescence resonance energy transfer (FRET) from europium to APC was detected in a time-resolved fashion (TR-FRET). Activation and inhibition constants for known substances determined by this new fluorescence-based assay correlated well with published results obtained by conventional radioactive cGK activity assays. The assay proved to be sensitive, robust, highly specific for cGK, and suitable for HTS in 96- and 384-well formats. This assay is applicable to purified enzymes as well as to complex samples such as human platelet extracts.

Proteins of the Ena/VASP (Enabled/vasodilator-stimulated phosphoprotein) family are involved in Abl and/or cyclic nucleotide-dependent protein kinase signaling pathways. These proteins are also crucial factors in regulating actin dynamics and associated processes such as cell-cell adhesion, platelet function and actin-based motility of both cytopathogenic Listeria and their eukaryotic host cells. Although biochemical mechanisms have emerged depicting Ena/VASP proteins as enhancers of actin filament formation, increasing evidence also suggests that these proteins have inhibitory functions in integrin regulation, cell motility and axon guidance.

The Ena-VASP family of proteins act as molecular adaptors linking the cytoskeletal system to signal transduction pathways. Their N-terminal EVH1 domains use groups of exposed aromatic residues to specifically recognize 'FPPPP' motifs found in the mammalian zyxin and vinculin proteins, and ActA protein of the intracellular bacterium Listeria monocytogenes. Here, evidence is provided that the affinities of these EVH1-peptide interactions are strongly dependent on the recognition of residues flanking the core FPPPP motifs. Determination of the VASP EVH1 domain solution structure, together with peptide library screening, measurement of individual K(d)s by fluorescence titration, and NMR chemical shift mapping, revealed a second affinity-determining epitope present in all four ActA EVH1-binding motifs. The epitope was shown to interact with a complementary hydrophobic site on the EVH1 surface and to increase strongly the affinity of ActA for EVH1 domains. We propose that this epitope, which is absent in the sequences of the native EVH1-interaction partners zyxin and vinculin, may provide the pathogen with an advantage when competing for the recruitment of the host VASP and Mena proteins in the infected cell.

Background: Focal adhesion sites are cell-matrix contacts that are regulated by phosphatidylinositol-4,5-bisphosphate (PIP2)-dependent pathways. Vinculin is a major structural component of these sites and is thought to be engaged in multiple ligand interactions at the cytoplasmic face of these contacts. Cytoplasmic vinculin is considered to be inactive due to its closed conformation involving intramolecular head-tail interactions. Recently, the vasodilator-stimulated phosphoprotein (VASP), a substrate of cyclic AMP-dependent or cyclic GMP-dependent kinases and a component of focal adhesion sites, was shown to bind to vinculin., Results: VASP-vinculin complexes could be immunoprecipitated from cell lysates and, using immunofluorescence, both proteins were found to colocalize in nascent focal adhesions. Consistent with the view that vinculin must be activated at these sites, we found that PIP2, levels of which are elevated during the early stages of adhesion, bound to two discrete regions in the vinculin tail, disrupting the intramolecular head-tail interaction and inducing vinculin oligomerization. Vinculin-VASP complex formation was greatly enhanced by PIP2 and both the EVH1 and EVH2 domains of VASP participated in vinculin binding., Conclusions: Focal contact assembly involves interaction between VASP and vinculin, which is enhanced by PIP2-induced vinculin activation and oligomerization. Given that vinculin and VASP both bind to F-actin, vinculin-VASP complexes might bundle the distal ends of actin filaments in focal contacts. We propose that PIP2-dependent signalling modulates microfilament organization at cellular adhesion sites by regulating vinculin-VASP complexes.

We previously identified a 42 kDa Ins(1,3,4,5)P4 (InsP4) receptor protein (p42IP4) in brain membranes from several species. Here the cDNA sequence of p42IP4 was obtained by PCR using degenerate primers derived from peptide sequences of proteolytic fragments of the porcine protein and by subsequent screening of a pig brain cDNA library. The derived peptide sequence of 374 amino acids for porcine p42IP4 is 45 amino acids shorter at the C-terminus than centaurin-alpha from rat (84% homology) and has a calculated molecular mass of 43 kDa. From the InsP4 binding activity present in brain tissue homogenate about 25% is found in the cytosolic fraction and 75% associated with microsomes. Both activities are due to p42IP4 since (i) a peptide-specific antiserum recognizing specifically p42IP4 labels the InsP4 receptor protein in membranes and in the cytosol, (ii) the antiserum immunoprecipitates both the membrane protein and the cytosolic protein of 42 kDa, (iii) the InsP4 binding activity released by high salt or by alkaline extraction from membranes is identified immunologically as the 42 kDa protein, and (iv) the affinity for InsP4 and specificity for various inositolphosphates are similar for the membrane-associated and for the soluble p42IP4. The functional importance of p42IP4 is highlighted by the identical affinity for InsP4 and for phosphatidylinositol (3,4,5)P3 (Ki = 1.6 and 0.9 nM, respectively). Thus, the InsP4 receptor, apparently a peripheral membrane protein, which exists also as a cytosolic protein can transfer the signals mediated by InsP4 or by PtdInsP3 between membranes and cytosolic compartment.

Detailed studies of differences in distinct cGMP kinase isoforms are highly dependent on expression of large amounts of these enzyme isoforms that are not easily purified by conventional methods. Here cGMP-dependent protein kinases, the type I beta soluble form from human placenta, and the type II membrane-associated form from rat intestine, were each expressed in a baculovirus/Sf9 cell system and purified in milligram amounts by affinity chromatography. The expressed recombinant proteins displayed characteristics like those of their native counterparts. cGK I beta was expressed as a 76 kDa protein predominantly found in the cytosol fraction, whereas cGK II was expressed as an 86 kDa protein predominantly associated with the membrane fraction. The apparent Ka and Vmax of cGMP for activation of cGK I beta were 0.5 microM and 3.4 mumol/min/mg, and for cGK II were 0.04 microM and 1.8 mumol/min/mg.

Overlapping subsequences in a DNA sequence are not independent even if independence is supposed for the single nucleotides. Therefore the often used geometric distribution for the length of restriction fragments is not exact. The exact distribution of this random variable is derived for non-overlapping restriction sites in a DNA sequence with an infinite (or very large) number of nucleotides. Correction to the finite case is easy. It is shown that the simple geometric distribution is a good approximation as long as the basic probability for the occurrence of the recognition sequence at a given site is small.

The haemolysin protein (HlyA) of Escherichia coli contains 11 tandemly repeated sequences consisting of 9 amino acids each between amino acids 739 and 849 of HlyA. We removed, by oligonucleotide-directed mutagenesis, different single repeats and combinations of several repeats. The resulting mutant proteins were perfectly stable in E. coli and were secreted with the same efficiency as the wild-type HlyA. HlyA proteins which had lost a single repeat only were still haemolytically active (in the presence of HlyC) but required elevated levels of Ca2+ for activity, as compared to the wild-type haemolysin. Removal of three or more repeats led to the complete loss of the haemolytic activity even in the presence of high Ca2+ concentrations. The mutant haemolysins were unable to compete with the wild-type haemolysin for binding to erythrocytes at low Ca2+ concentrations but could still generate ion-permeable channels in artificial lipid bilayer membranes formed of plant asolectin, even in the complete absence of Ca2+. These data indicate that the repeat domain of haemolysin is responsible for Ca2+-dependent binding of haemolysin to the erythrocyte membrane. A model for the possible functional role of Ca2+ in haemolysis is presented.