title: Structural and Functional Investigation of the Ag(+)/Cu(+) Binding Domains of the Periplasmic Adaptor Protein SilB from Cupriavidus metallidurans CH34.
abstract: Silver ion resistance in bacteria mainly relies on efflux systems, and notably on tripartite efflux complexes involving a transporter from the resistance-nodulation-cell division (RND) superfamily, such as the SilCBA system from Cupriavidus metallidurans CH34. The periplasmic adaptor protein SilB hosts two specific metal coordination sites, located in the N-terminal and C-terminal domains, respectively, that are believed to play a different role in the efflux mechanism and the trafficking of metal ions from the periplasm to the RND transporter. On the basis of the known domain structure of periplasmic adaptor proteins, we designed different protein constructs derived from SilB domains with either one or two metal binding sites per protein chain. ITC data acquired on proteins with single metal sites suggest a slightly higher affinity of Ag(+) for the N-terminal metal site, compared to that for the C-terminal one. Remarkably, via the study of a protein construct featuring both metal sites, nuclear magnetic resonance (NMR) and fluorescence spectroscopies concordantly show that the C-terminal site is saturated prior to the N-terminal one. The C-terminal binding site is supposed to transfer the metal ions to the RND protein, while the transport driven by this latter is activated upon binding of the metal ion to the N-terminal site. Our results suggest that the filling of the C-terminal metal site is a key prerequisite for preventing futile activation of the transport system. Exhaustive NMR studies reveal for the first time the structure and dynamics of the functionally important N-terminal domain connected to the membrane proximal domain as well as of its Ag(+) binding site.
authors.affiliation: Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and Bioinformatics, Université Libre de Bruxelles , B-1050 Bruxelles, Belgium.
authors.forename: Patricia
authors.initials: P
authors.lastname: Urbina
authors.affiliation: Institut de Biologie Structurale (IBS), Univ. Grenoble Alpes, CEA, CNRS , 38044 Grenoble, France.
authors.forename: Beate
authors.initials: B
authors.lastname: Bersch
authors.affiliation: Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and Bioinformatics, Université Libre de Bruxelles , B-1050 Bruxelles, Belgium.
authors.forename: Fabien
authors.initials: F
authors.lastname: De Angelis
authors.affiliation: Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and Bioinformatics, Université Libre de Bruxelles , B-1050 Bruxelles, Belgium.
authors.forename: Kheiro-Mouna
authors.initials: KM
authors.lastname: Derfoufi
authors.affiliation: Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and Bioinformatics, Université Libre de Bruxelles , B-1050 Bruxelles, Belgium.
authors.forename: Martine
authors.initials: M
authors.lastname: Prévost
authors.affiliation: Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and Bioinformatics, Université Libre de Bruxelles , B-1050 Bruxelles, Belgium.
authors.forename: Erik
authors.initials: E
authors.lastname: Goormaghtigh
authors.affiliation: Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and Bioinformatics, Université Libre de Bruxelles , B-1050 Bruxelles, Belgium.
authors.forename: Guy
authors.initials: G
authors.lastname: Vandenbussche
mesh_terms: D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D049921:Cupriavidus / Q000737:chemistry* / Q000378:metabolism; D017136:Ion Transport; D019906:Nuclear Magnetic Resonance, Biomolecular; D019897:Periplasm / Q000737:chemistry* / Q000378:metabolism; D033903:Periplasmic Proteins / Q000737:chemistry* / Q000378:metabolism; D000072417:Protein Domains; D012834:Silver / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D002352:Carrier Proteins; D033903:Periplasmic Proteins; D012834:Silver