Josée Plamondon

Mutagenesis analysis of the membrane-proximal ligand binding site of the TGF-beta receptor type III extracellular domain.

There are two TGF‐β binding subdomains in the extracellular domain of receptor type III (proximal and distal in relation to the transmembrane domain). Here we present an extension of our analysis of the proximal binding site of receptor type III. Due to the original deletion mutagenesis strategy, our proximal binding site contained 19 amino acids from the N‐terminal part of the receptor. By deleting these, we demonstrated that they did not contribute to the binding ability of the proximal binding site. We also produced a soluble, secreted form of the proximal binding site and demonstrated that it was able to bind TGF‐β. Finally, we analyzed the role of the three asparagine residues (580, 591, 595) that are located in the region of the receptor that is necessary for expression of a functional proximal binding site, and found that mutation of these residues individually to alanine did not affect ligand binding.

Three-dimensional structure and ligand interactions of the low molecular weight protein tyrosine phosphatase from Campylobacter jejuni

A putative low molecular weight protein tyrosine phosphatase (LMW‐PTP) was identified in the genome sequence of the bacterial pathogen, Campylobacter jejuni. This novel gene, cj1258, has sequence homology with a distinctive class of phosphatases widely distributed among prokaryotes and eukaryotes. We report here the solution structure of Cj1258 established by high‐resolution NMR spectroscopy using NOE‐derived distance restraints, hydrogen bond data, and torsion angle restraints. The three‐dimensional structure consists of a central four‐stranded parallel β‐sheet flanked by five α‐helices, revealing an overall structural topology similar to those of the eukaryotic LMW‐PTPs, such as human HCPTP‐A, bovine BPTP, and Saccharomyces cerevisiae LTP1, and to those of the bacterial LMW‐PTPs MPtpA from Mycobacterium tuberculosis and YwlE from Bacillus subtilis. The active site of the enzyme is flexible in solution and readily adapts to the binding of ligands, such as the phosphate ion. An NMR‐based screen was carried out against a number of potential inhibitors and activators, including phosphonomethylphenylalanine, derivatives of the cinnamic acid, 2‐hydroxy‐5‐nitrobenzaldehyde, cinnamaldehyde, adenine, and hypoxanthine. Despite its bacterial origin, both the three‐dimensional structure and ligand‐binding properties of Cj1258 suggest that this novel phosphatase may have functional roles close to those of eukaryotic and mammalian tyrosine phosphatases. The three‐dimensional structure along with mapping of small‐molecule binding will be discussed in the context of developing high‐affinity inhibitors of this novel LMW‐PTP.

Binding of human angiogenin inhibits actin polymerization

Angiogenin is a potent inducer of angiogenesis, a process of blood vessel formation. It interacts with endothelial and other cells and elicits a wide range of cellular responses including migration, proliferation, and tube formation. One important target of angiogenin is endothelial cell-surface actin and their interaction might be one of essential steps in angiogenin-induced neovascularization. Based on earlier indications that angiogenin promotes actin polymerization, we studied the binding interactions between angiogenin and actin in a wide range of conditions. We showed that at subphysiological KCl concentrations, angiogenin does not promote, but instead inhibits polymerization by sequestering G-actin. At low KCl concentrations angiogenin induces formation of unstructured aggregates, which, as shown by NMR, may be caused by angiogenins propensity to form oligomers. Binding of angiogenin to preformed F-actin does not cause depolymerization of actin filaments though it causes their stiffening. Binding of tropomyosin and angiogenin to F-actin is not competitive at concentrations sufficient for saturation of actin filaments. These observations suggest that angiogenin may cause changes in the cell cytoskeleton by inhibiting polymerization of G-actin and changing the physical properties of F-actin.

Baculovirus expression of mammalian G protein α subunits

Complementary DNAs encoding three subtypes of the α subunit (αi−1, αo and αx) of rat guanyl nucleotide regulatory proteins were used to construct recombinant baculoviruses which direct high‐level expression of the corresponding proteins in cultured Sf9 insect cells. The expressed proteins were recognized by polyclonal antisera specific for the different α chains, and co‐migrated with the native proteins from rat brain membranes in immunoblotting analyses. Soluble and particulate forms of all three immunoreactive α chains were observed following ultracentrifugation of cell lysates. Biosynthetic radiolabelling of infected cells with [35S]methionine or [3H]myristate showed that both soluble and particulate forms of αi−1 and αo were myristoylated; in contrast, α, did not incorporate myristate. The soluble fractions from cells expressing α chains showed high levels of GTP‐binding activity over that observed in uninfected cells, or in cells infected with wild‐type virus. The peak expression levels observed at 72 h post‐infection were highest for αo at ca, 400 pmol of GTP‐γ‐35S/mg protein, or roughly 2% of the total soluble protein. The results of this work show that the baculovirus system can be employed for high‐level production of mammalian G protein α chains which retain GTP‐binding activity and are appropriately modified by myristoylation.

A structural genomics pilot project based on gene targets selected from Escherichia coli

A pilot project based on gene targets selected from the genome of E. coli has been initiated with 38 genes for initial cloning. Of these, 18 proteins have been purified to date and some crystals were obtained for twelve of them. Of these, four proteins yielded crystals diffracting to a sufficiently high resolution to warrant structural investigation. We have determined 3-D structures of three of these proteins using Se-Met labeling and MAD methods, while the structure of the fourth one was simultaneously determined by another group. To manage the parallel work on many proteins by several researchers it became necessary to create a searchable database containing the pertinent information about every stage of the work.