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The pneumococcal σ^X activator, ComW, is a DNA-binding protein critical for natural transformation.

ABSTRACT: Natural genetic transformation via horizontal gene transfer enables rapid adaptation to dynamic environments and contributes to both antibiotic resistance and vaccine evasion among bacterial populations. In Streptococcus pneumoniae (pneumococcus), transformation occurs when cells enter competence, a transient state in which cells express the competence master regulator, SigX (σ^Χ), an alternative σ factor (σ), and a competence co-regulator, ComW. Together, ComW and σ^X facilitate expression of the genes required for DNA uptake and genetic recombination. SigX activity depends on ComW, as ΔcomW cells transcribe late genes and transform at levels 10- and 10,000-fold below that of WT cells, respectively. Previous findings suggest that ComW functions during assembly of the RNA polymerase-σ^X holoenzyme to help promote transcription from σ^X-targeted promoters. However, it remains unknown how ComW facilitates holoenzyme assembly. As ComW seems to be unique to Gram-positive cocci and has no sequence similarity with known transcriptional activators, here we used Rosetta to generate an ab initio model of pneumococcal ComW's 3D-structure. Using this model as a basis for further biochemical, biophysical, and genetic investigations into the molecular features important for its function, we report that ComW is a predicted globular protein and that it interacts with DNA, independently of DNA sequence. We also identified conserved motifs in ComW and show that key residues in these motifs contribute to DNA binding. Lastly, we provide evidence that ComW's DNA-binding activity is important for transformation in pneumococcus. Our findings begin to fill the gaps in understanding how ComW regulates σ^Χ activity during bacterial natural transformation.

SUBMITTER: Inniss NL

PROVIDER: S-EPMC6643036 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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The pneumococcal σX activator, ComW, is a DNA-binding protein critical for natural transformation.

Inniss Nicole L NL Prehna Gerd G Morrison Donald A DA

The Journal of biological chemistry 20190603 29

Natural genetic transformation via horizontal gene transfer enables rapid adaptation to dynamic environments and contributes to both antibiotic resistance and vaccine evasion among bacterial populations. In Streptococcus pneumoniae (pneumococcus), transformation occurs when cells enter competence, a transient state in which cells express the competence master regulator, SigX (σΧ), an alternative σ factor (σ), and a competence co-regulator, ComW. Together, ComW and σX ...[more]

PMID: 31160340

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Project description:Binding free energies are calculated for the phosphorylated and unphosphorylated complexes between the kinase inducible domain (KID) of the DNA transcriptional activator cAMP response element binding (CREB) protein and the KIX domain of its coactivator, CREB-binding protein (CBP). To our knowledge, this is the first application of a method based on a potential of mean force (PMF) with restraining potentials to compute the binding free energy of protein-protein complexes. The KID:KIX complexes are chosen here because of their biological relevance to the DNA transcription process and their relatively small size (81 residues for the KIX domain of CBP, and 28 residues for KID). The results for pKID:KIX and KID:KIX are -9.55 and -4.96 kcal/mol, respectively, in good agreement with experimental estimates (-8.8 and -5.8 kcal/mol, respectively). A comparison between specific contributions to protein-protein binding for the phosphorylated and unphosphorylated complexes reveals a dual role for the phosphorylation of KID at Ser-133 in effecting a more favorable free energy of the bound system: 1), stabilization of the unbound conformation of phosphorylated KID due to favorable intramolecular interactions of the phosphate group of Ser-133 with the charged groups of an arginine-rich region spanning both α-helices, which lowers the configurational entropy; and 2), more favorable intermolecular electrostatic interactions between pSer-133 and Arg-131 of KID, and Lys-662, Tyr-658, and Glu-666 of KIX. Charge reduction through ligand phosphorylation emerges as a possible mechanism for controlling the unbound state conformation of KID and, ultimately, gene expression. This work also demonstrates that the PMF-based method with restraining potentials provides an added benefit in that important elements of the binding pathway are evidenced. Furthermore, the practicality of the PMF-based method for larger systems is validated by agreement with experiment. In addition, we provide a somewhat differently structured exposition of the PMF-based method with restraining potentials and outline its generalization to systems in which both protein and ligand may adopt unbound conformations that are different from those of the bound state.

Dataset Information

The pneumococcal σ^X activator, ComW, is a DNA-binding protein critical for natural transformation.

Publications

The pneumococcal σ<sup>X</sup> activator, ComW, is a DNA-binding protein critical for natural transformation.

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