title: The mutation G145S in PrfA, a key virulence regulator of Listeria monocytogenes, increases DNA-binding affinity by stabilizing the HTH motif.
abstract: Listeria monocytogenes, a Gram-positive, facultative intracellular human pathogen, causes systemic infections with high mortality rate. The majority of the known pathogenicity factors of L. monocytogenes is regulated by a single transcription factor, PrfA. Hyperhaemolytic laboratory strains of L. monocytogenes express the constitutively active mutant PrfA(G145S) inducing virulence gene overexpression independent of environmental conditions. PrfA belongs to the Crp/Fnr family of transcription factors generally activated by a small effector, such as cAMP or O(2). We present the crystal structures of wild-type PrfA, the first Gram-positive member of the Crp/Fnr family, and of the constitutively active mutant PrfA(G145S). Cap (Crp) has previously been described exclusively in the cAMP-induced (DNA-free and -bound) conformation. By contrast, the PrfA structures present views both of the non-induced state and of the mutationally activated form. The low DNA-binding affinity of wild-type PrfA is supported both structurally (partly disordered helix-turn-helix motif, overall geometry of the HTH alpha-helices deviates from Cap) and by surface plasmon resonance analyses (K(D) = 0.9 microM). In PrfA(G145S) the HTH motifs dramatically rearrange to adopt a conformation comparable to cAMP-induced Cap and hence favourable for DNA binding, supported by a DNA-binding affinity of 50 nM. Finally, the hypothesis that wild-type PrfA, like other Crp/Fnr family members, may require an as yet unidentified cofactor for activation is supported by the presence of a distinct tunnel in PrfA, located at the interface of the beta-barrel and the DNA-binding domain.
authors.affiliation: Division of Structural Biology, German Research Centre for Biotechnology (GBF), Mascheroder Weg 1, D-38124 Braunschweig, Germany.
authors.forename: Marina
authors.initials: M
authors.lastname: Eiting
authors.forename: Gregor
authors.initials: G
authors.lastname: Hagelüken
authors.forename: Wolf-Dieter
authors.initials: WD
authors.lastname: Schubert
authors.forename: Dirk W
authors.initials: DW
authors.lastname: Heinz
mesh_terms: D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D002373:Cyclic AMP Receptor Protein / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005809:Genes, Regulator; D019077:Helix-Turn-Helix Motifs; D008089:Listeria monocytogenes / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D009154:Mutation; D010454:Peptide Termination Factors; D020349:Surface Plasmon Resonance; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D014774:Virulence / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D002373:Cyclic AMP Receptor Protein; D004268:DNA-Binding Proteins; D010454:Peptide Termination Factors; D015534:Trans-Activators; D004247:DNA