The GEF Tiam1 acts as a novel molecular link to the VE-cadherin–p67phox–Par3 polarity complex, leading to localized activation of Rac1 and NADPH oxidase in response to fluid flow.

SUMMARY PDZ (PSD-95/Dlg/ZO-1) domains are protein-protein interaction modules often regulated by ligand phosphorylation. Here, we investigated the specificity, structure, and dynamics of Tiam1 PDZ domain/ligand interactions. We show that the PDZ domain specifically binds syndecan1 (SDC1), phosphorylated SDC1 (pSDC1), and SDC3 but not other syndecan isoforms. The crystal structure of the PDZ/SDC1 complex indicates that syndecan affinity is derived from amino acids beyond the four C-terminal residues. Remarkably, the crystal structure of the PDZ/pSDC1 complex reveals a binding pocket that accommodates the phosphoryl group. Methyl relaxation experiments of PDZ/SCD1 and PDZ/pSDC1 complexes reveal that PDZ-phosphoryl interactions dampen dynamic motions in a distal region of the PDZ domain by decoupling them from the ligand-binding site. Our data are consistent with a selection model by which specificity and phosphorylation regulate PDZ/syndecan interactions and signaling events. Importantly, our relaxation data demonstrate that PDZ/phospho-ligand interactions regulate protein dynamics and their coupling to distal sites.

Guanine nucleotide exchange factor proteins of the Tiam family are activators of the Rho GTPase Rac1 and critical for cell morphology, adhesion, migration, and polarity. These proteins are modular and contain a variety of interaction domains, including a single post-synaptic density-95/ discs large/zonula occludens-1 (PDZ) domain. Previous studies suggest that the specificities of the Tiam1 and Tiam2 PDZ domains are distinct. Here, we sought to conclusively define these specificities and determine their molecular origin. Using a combinatorial peptide library, we identified a consensus binding sequence for each PDZ domain. Analysis of these consensus sequences and binding assays with peptides derived from native proteins indicated that these two PDZ domains have overlapping, but distinct specificities. We also identified residues in two regions (S 0 and S -2 pockets) of the Tiam1 PDZ domain that are important determinants of ligand specificity. Site-directed mutagenesis of four non-conserved residues in these two regions along with peptide binding analyses confirmed that these residues are crucial for ligand affinity and specificity. Furthermore, double-mutant cycle analysis of each region revealed energetic couplings that were dependent on the ligand being investigated. Remarkably, a Tiam1 PDZ domain quadruple mutant had the same specificity as the Tiam2 PDZ domain. Finally, analysis of Tiamfamily PDZ domain sequences indicated that the PDZ domains segregate into four distinct families based on the residues studied here. Collectively, our data suggest that Tiam-family proteins have highly evolved PDZ-ligand interfaces with distinct specificities, and that they have disparate PDZ-dependent biological functions.The T-cell lymphoma invasion and metastasis 1 (Tiam1) protein and its homolog Tiam2, also known as STEF (SIF-and Tiam1-like Exchange Factor), are guanine exchange factor proteins that specifically activate the Rho-family GTPase Rac1 (1,2). Tiam1 is important for the integrity of adherens junctions (3,4), tight junctions (5,6), and cell-matrix interactions † This work was supported by funds from National Science Foundation (MCB-0624451 to EJF), the American Heart Association (0835261N to EJF), and the National Institutes of Health (GM062820 to DP). TRS was supported in part by an NIH predoctoral fellowship in Pharmacological Sciences (GM067795) and by a University of Iowa Graduate Student Fellowship sponsored by the Center for Biocatalysis and Bioprocessing. RLH was supported by a predoctoral fellowship from the NIH Chemistry-Biology Interface Training Program (GM08512). * To whom correspondence should be addressed. Telephone: (319) . ernesto-fuentes@uiowa.edu. ± These two authors contributed equally to this work. Tables S1 and S2 show putative Tiam1 PDZ and Tiam2 PDZ binding proteins, respectively. Figure S1 shows binding curves for various peptides identified in the combinatorial peptide screen. This material is available free of charge via the Internet at http://pubs.acs.org. Supporting Informat...

The synthesis and characterization are reported of vanadium complexes of pyridine-2-thiolate (pyt-) with the metal in oxidation states II, III, and IV. The reaction of VOClaCTHFh with 2 equiv of Na(pyt) in THF leads to formation of [VaOalpyfL] (1). Complex l^THF-VsCeHn crystallizes in hexagonal space group 53 with the following cell dimensions at -171 °C: a = b = 30.888(21) Á, c = 8.960(6) Á, Z = 9, and V = 7402.8 Á3. A total of 1310 unique reflections with F > 2.33a(F) were employed for structure solution and refinement to R (5W) values of 0.0613 (0.0563). The structure consists of two VO(pyt)a units joined together by two monoatomic bridges provided by S atoms of two pytgroups. The V* *V distance is 3.989(2) A. The reaction of VCI3-(THF)3 with four equivalents of Na(pyt) in THF gives [VNa(pyt)4(THF)2] (2). Complex 2 crystallizes in triclinic space group PI with the following unit cell dimensions at -171 °C: a = 10.442(2) k,b= 16.476(4) Á, c -9.465(2)Á , a = 100.73(4)°, ß = 109.20(4)°, = 87.86(3)°, Z = 2, and V = 1510.3 Á3. A total of 3391 unique reflections with F > 2.33a{F) were employed for structure solution and refinement to R (5W) values of 0.0412 (0.0431). The structure is best described as a heterodinuclear complex with the metals monoatomically bridged by S atoms from three pytgroups. The V is seven-coordinate, and the Na is six-coordinate. The V• 'Na distance is 3.516(1) Á. The reaction of VCLOmedah (tmeda = tetramethylethylenediamine) with 2 equiv of Na(pyt) in THF or CH2CI2 yields [V(pyt)2(tmeda)j (3). Complex 3 crystallizes in orthorhombic space group 52212 with the following cell dimensions at -170 °C: a = 14.338(5) k, b = 17.755(7) Á, c = 7.255(2) Á, Z = 4, and V = 1846.9Á3. A total of 1192 unique reflections with F > 2.33o(F) were employed for structure solution and refinement to R (5W) values of 0.0220 (0.0261). The complex contains chelating pyt-and tmeda groups with severely distorted octahedral geometry. Complexes 1, 2, and 3 have been characterized by EPR and/or IR spectroscopy. In addition, mass spectral studies are described for 1 and 2, directed toward identifying fragmentation pathways involving C-S bond cleavage within the pytgroup; the latter cleavage is detected in the mass spectra of both compounds.to contain S, N, and/or O-donor ligands.4 5The presence of these vanadyl impurities leads to the formation of vanadium sulfides (primarily V2S3 and V3S4) under the reducing and sulfur-rich hydrotreating conditions.5-9 These sulfides have deleterious

Biopsy from the edge of a septal perforation is performed to diagnose potentially significant aetiological conditions, including malignant pathology and Wegener's granulomatosis. However, it is common for the histological result to be undiagnostic and unhelpful in planning an individual patient's management. To clarify the role of septal perforation biopsy, we reviewed all biopsies performed on our patients over a 13-year period. Of the 65 biopsies performed, in 63 patients, none had a histological result which changed the clinical diagnosis made before biopsy. Forty-three patients had clinically benign perforations biopsied, and no histology other than chronic inflammation was obtained. In 16 patients with a clinical vasculitis, none had this diagnosis confirmed histologically. One positive biopsy was obtained from two patients with clinically malignant perforations yet both were treated for T-cell lymphoma. The subsequent management of all patients was based on their clinical diagnosis rather than their biopsy results. We suggest only clinically malignant perforations are worthwhile biopsying.