title: Insights into the C-terminal Peptide Binding Specificity of the PDZ Domain of Neuronal Nitric-oxide Synthase: CHARACTERIZATION OF THE INTERACTION WITH THE TIGHT JUNCTION PROTEIN CLAUDIN-3.
abstract: Neuronal nitric-oxide synthase, unlike its endothelial and inducible counterparts, displays a PDZ (PSD-95/Dlg/ZO-1) domain located at its N terminus involved in subcellular targeting. The C termini of various cellular proteins insert within the binding groove of this PDZ domain and determine the subcellular distribution of neuronal NOS (nNOS). The molecular mechanisms underlying these interactions are poorly understood because the PDZ domain of nNOS can apparently exhibit class I, class II, and class III binding specificity. In addition, it has been recently suggested that the PDZ domain of nNOS binds with very low affinity to the C termini of target proteins, and a necessary simultaneous lateral interaction must take place for binding to occur. We describe herein that the PDZ domain of nNOS can behave as a bona fide class III PDZ domain and bind to C-terminal sequences with acidic residues at the P-2 position with low micromolar binding constants. Binding to C-terminal sequences with a hydrophobic residue at the P-2 position plus an acidic residue at the P-3 position (class II) can also occur, although interactions involving residues extending up to the P-7 position mediate this type of binding. This promiscuous behavior also extends to its association to class I sequences, which must display a Glu residue at P-3 and a Thr residue at P-2 By means of site-directed mutagenesis and NMR spectroscopy, we have been able to identify the residues involved in each specific type of binding and rationalize the mechanisms used to recognize binding partners. Finally, we have analyzed the high affinity association of the PDZ domain of nNOS to claudin-3 and claudin-14, two tight junction tetraspan membrane proteins that are essential components of the paracellular barrier.
authors.affiliation: From the Departamentos de Bioquímica y Biología Molecular and.
authors.forename: Javier
authors.initials: J
authors.lastname: Merino-Gracia
authors.affiliation: From the Departamentos de Bioquímica y Biología Molecular and.
authors.forename: Carlos
authors.initials: C
authors.lastname: Costas-Insua
authors.affiliation: Química Orgánica, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Madrid 28040, Spain.
authors.forename: María Ángeles
authors.initials: MÁ
authors.lastname: Canales
authors.affiliation: From the Departamentos de Bioquímica y Biología Molecular and nacho@bbm1.ucm.es.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Rodríguez-Crespo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D062465:Claudin-3 / Q000235:genetics / Q000378:metabolism*; D005454:Fluorescence Polarization; D005455:Fluorescent Antibody Technique; D047468:Immunoprecipitation; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I / Q000235:genetics / Q000378:metabolism*; D054731:PDZ Domains; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D000072417:Protein Domains; D051381:Rats; D017386:Sequence Homology, Amino Acid
chemical_list: D062465:Claudin-3; D010446:Peptide Fragments; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat
keywords: PDZ domain; nitric oxide; nitric oxide synthase; nuclear magnetic resonance (NMR); protein-protein interaction; tight junction