title: Functional dissection of intersubunit interactions in the EspR virulence regulator of Mycobacterium tuberculosis.
abstract: The nucleoid-associated protein EspR, a chromosome organizer, has pleiotropic effects on expression of genes associated with cell wall function and pathogenesis in Mycobacterium tuberculosis. In particular, EspR binds to several sites upstream of the espACD locus to promote its expression, thereby ensuring full function of the ESX-1 secretion system, a major virulence determinant. The N terminus of EspR contains the helix-turn-helix DNA-binding domain, whereas the C-terminal dimerization domain harbors residues involved in intersubunit interactions. While direct binding to DNA appears to be mediated by an EspR dimer-of-dimers, where two helix-turn-helix motifs remain free for long-range interactions, the mechanism of EspR higher-order organization and its impact on chromosome structure and gene expression are not understood. To investigate these processes, we identified seven amino acid residues using molecular dynamics and replaced them with Ala in order to probe interactions at either the dimer or the dimer-of-dimers interfaces. Arg70, Lys72, and Arg101 were important for protein stability and optimal DNA-binding activity. Moreover, the Arg70 mutant showed decreased dimerization in a mycobacterial two-hybrid system. To correlate these defects with higher-order organization and transcriptional activity, we used atomic force microscopy to observe different EspR mutant proteins in complex with the espACD promoter region. In addition, complementation of an M. tuberculosis espR knockout mutant was performed to measure their impact on EspA expression. Our results pinpoint key residues required for EspR function at the dimer (Arg70) and the dimer-of-dimers (Lys72) interface and demonstrate that EspR dimerization and higher-order oligomerization modulate espACD transcriptional activity and hence pathogenesis.
authors.affiliation: Global Health Institute, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Blasco
authors.forename: Aleksandre
authors.initials: A
authors.lastname: Japaridze
authors.forename: Marco
authors.initials: M
authors.lastname: Stenta
authors.forename: Basile I M
authors.initials: BI
authors.lastname: Wicky
authors.forename: Giovanni
authors.initials: G
authors.lastname: Dietler
authors.forename: Matteo
authors.initials: M
authors.lastname: Dal Peraro
authors.forename: Florence
authors.initials: F
authors.lastname: Pojer
authors.forename: Stewart T
authors.initials: ST
authors.lastname: Cole
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004269:DNA, Bacterial / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits; D014158:Transcription, Genetic; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D021122:Protein Subunits