title: Structural insight into the mechanism of DNA-binding attenuation of the Neisserial adhesin repressor NadR by the small natural ligand 4-hydroxyphenylacetic acid.
abstract: Neisserial adhesin A (NadA) is a surface exposed trimeric protein present in most hypervirulent meningococcal strains and involved in epithelial cell adhesion and colonization. The expression of nadA is controlled by Neisserial adhesin regulator (NadR), a member of the MarR family, which binds to the nadA promoter and strongly represses the transcription of nadA. It was recently demonstrated that the DNA-binding activity of NadR was attenuated by 4-hydroxyphenylacetic acid (4-HPA), a natural molecule released in human saliva, thus leading to the de-repression of nadA in vivo. To elucidate the mechanism of regulation of NadR by 4-HPA, we used hydrogen-deuterium exchange mass spectrometry in association with in silico docking and site-directed mutagenesis. We show here that 4-HPA binds at the interface between the dimerization and the DNA-binding domains and stabilizes the homodimeric state of NadR without inducing large conformational changes in the DNA-binding lobes. The residues predicted to be in contact with 4-HPA were further selected for mutagenesis to assess their in vitro and in vivo functions in 4-HPA binding. Our results indicate that Arg(40) is critical for DNA-binding and reveal that Tyr(115) plays a key role in the mechanism of regulation of NadR by 4-HPA. Altogether our data suggest that the mechanism of regulation of NadR by 4-HPA mainly involves the stabilization of the dimer in a configuration incompatible with DNA binding.
authors: Research Center, Novartis Vaccines and Diagnostics, via Fiorentina 1, 53100, Siena, Italy.
authors: Sébastien
authors: S
authors: Brier
authors: Luca
authors: L
authors: Fagnocchi
authors: Danilo
authors: D
authors: Donnarumma
authors: Maria
authors: M
authors: Scarselli
authors: Rino
authors: R
authors: Rappuoli
authors: Mikkel
authors: M
authors: Nissum
authors: Isabel
authors: I
authors: Delany
authors: Nathalie
authors: N
authors: Norais
mesh_terms: D018829:Adhesins, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D015964:Gene Expression Regulation, Bacterial; D008024:Ligands; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010648:Phenylacetates / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D008024:Ligands; C501982:NadA protein, Neisseria meningitidis; C117105:NadR protein, bacteria; D010648:Phenylacetates; D012097:Repressor Proteins; C008070:4-hydroxyphenylacetic acid