title: Structure-based functional characterization of repressor of toxin (Rot), a central regulator of Staphylococcus aureus virulence.
abstract: Staphylococcus aureus is responsible for a large number of diverse infections worldwide. In order to support its pathogenic lifestyle, S. aureus has to regulate the expression of virulence factors in a coordinated fashion. One of the central regulators of the S. aureus virulence regulatory networks is the transcription factor repressor of toxin (Rot). Rot plays a key role in regulating S. aureus virulence through activation or repression of promoters that control expression of a large number of critical virulence factors. However, the mechanism by which Rot mediates gene regulation has remained elusive. Here, we have determined the crystal structure of Rot and used this information to probe the contribution made by specific residues to Rot function. Rot was found to form a dimer, with each monomer harboring a winged helix-turn-helix (WHTH) DNA-binding motif. Despite an overall acidic pI, the asymmetric electrostatic charge profile suggests that Rot can orient the WHTH domain to bind DNA. Structure-based site-directed mutagenesis studies demonstrated that R(91), at the tip of the wing, plays an important role in DNA binding, likely through interaction with the minor groove. We also found that Y(66), predicted to bind within the major groove, contributes to Rot interaction with target promoters. Evaluation of Rot binding to different activated and repressed promoters revealed that certain mutations on Rot exhibit promoter-specific effects, suggesting for the first time that Rot differentially interacts with target promoters. This work provides insight into a precise mechanism by which Rot controls virulence factor regulation in S. aureus.
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, NYU School of Medicine, New York, New York, USA.
authors.forename: April
authors.initials: A
authors.lastname: Killikelly
authors.affiliation: Department of Microbiology, NYU School of Medicine, New York, New York, USA.
authors.forename: Meredith A
authors.initials: MA
authors.lastname: Benson
authors.affiliation: Department of Microbiology, NYU School of Medicine, New York, New York, USA.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Ohneck
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, NYU School of Medicine, New York, New York, USA.
authors.forename: Jared M
authors.initials: JM
authors.lastname: Sampson
authors.affiliation: Photon Sciences Directorate, Brookhaven National Laboratory, Upton, New York, USA.
authors.forename: Jean
authors.initials: J
authors.lastname: Jakoncic
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, NYU School of Medicine, New York, New York, USA.
authors.forename: Brett
authors.initials: B
authors.lastname: Spurrier
authors.affiliation: Department of Microbiology, NYU School of Medicine, New York, New York, USA Victor.Torres@nyumc.org Xiangpeng.Kong@med.nyu.edu.
authors.forename: Victor J
authors.initials: VJ
authors.lastname: Torres
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, NYU School of Medicine, New York, New York, USA Victor.Torres@nyumc.org Xiangpeng.Kong@med.nyu.edu.
authors.forename: Xiang-Peng
authors.initials: XP
authors.lastname: Kong
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D008565:Membrane Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D014774:Virulence
chemical_list: C438144:ASI1 protein, S cerevisiae; D001426:Bacterial Proteins; D008565:Membrane Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C408643:rot protein, Staphylococcus aureus