Structural and function analyses of the global regulatory protein SarA from Staphylococcus aureus

Area of Science:

• Microbiology
• Structural Biology
• Molecular Biology

Background:

• The sarA locus in Staphylococcus aureus regulates numerous virulence genes.
• SarA is a 14.7-kDa protein that binds to target gene promoter regions.

Purpose of the Study:

• To determine the x-ray crystal structure of the dimeric winged helix SarA protein at 2.6 A resolution.
• To investigate the role of specific residues and the metal-binding pocket in SarA's DNA binding and function.

Main Methods:

• X-ray crystallography to determine the 2.6 A-resolution structure of dimeric SarA.
• Site-directed mutagenesis to assess the function of specific residues within the DNA-binding and winged regions, and the metal-binding pocket.

Main Results:

• The determined SarA structure differs significantly from previously published structures.
• Crystal packing reveals SarA dimers forming scaffolds, potentially mediated by divalent cations.
• Mutations in the winged region (R84, R90) critically affect DNA binding.
• Mutations in acidic residues (D88, E89) and cysteine 9 (metal-binding pocket) are essential for SarA function, while the metal-binding pocket itself is only involved in gene function, not DNA binding.

Conclusions:

• The winged region of the winged helix SarA protein is crucial for both DNA binding and activation.
• The putative divalent cation binding pocket is primarily involved in gene function rather than direct DNA binding.