The pentapeptide-repeat protein, MfpA, interacts with mycobacterial DNA gyrase as a DNA T-segment mimic

Area of Science:

• Molecular biology
• Biochemistry
• Structural biology

Background:

• DNA gyrase, a type II topoisomerase, is essential for DNA supercoiling and a target for fluoroquinolone (FQ) antibiotics.
• Mycobacterial pentapeptide-repeat protein MfpA is known to confer resistance to FQs, but its mechanism is unclear.

Purpose of the Study:

• To elucidate the molecular mechanism by which MfpA protects DNA gyrase from FQ inhibition.
• To investigate the interaction between MfpA and DNA gyrase.

Main Methods:

• Biochemical assays to measure gyrase supercoiling and ATPase activity.
• X-ray crystallography to determine the structure of the MfpA-gyrase complex.
• Site-directed mutagenesis to probe protein interactions.

Main Results:

• MfpA inhibits negative supercoiling by M. smegmatis gyrase (Msgyrase) independently of FQs.
• MfpA reduces FQ-induced DNA cleavage by Msgyrase in the presence of FQs.
• MfpA directly interacts with the ATPase domain of Msgyrase (MsGyrB47), stimulating its ATPase activity.
• Structural and mutational analyses indicate MfpA mimics a DNA segment.

Conclusions:

• MfpA protects mycobacterial DNA gyrase from FQs by directly binding to the enzyme's ATPase domain.
• This interaction modulates gyrase activity and confers FQ resistance.
• The findings provide a molecular basis for MfpA's protective role and suggest a general mechanism for pentapeptide-repeat proteins.