Premium
The mutation G145S in PrfA, a key virulence regulator of Listeria monocytogenes , increases DNA‐binding affinity by stabilizing the HTH motif
Author(s) -
Eiting Marina,
Hagelüken Gregor,
Schubert WolfDieter,
Heinz Dirk W.
Publication year - 2005
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2005.04561.x
Subject(s) - biology , listeria monocytogenes , mutant , virulence , dna , effector , wild type , dna binding protein , transcription factor , virulence factor , binding site , gene , genetics , biochemistry , bacteria
Summary 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 α‐helices deviates from Cap) and by surface plasmon resonance analyses ( K D = 0.9 µM). 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 β‐barrel and the DNA‐binding domain.