title: Interactions of the M. tuberculosis UsfX with the cognate sigma factor SigF and the anti-anti sigma factor RsfA.
abstract: M. tuberculosis employs an exquisite cascade consisting of the cognate anti-sigma factor UsfX and anti-anti sigma factors RsfA and RsfB to regulate the functions of the alternate sigma factor SigF. We have purified these proteins to characterize their molecular properties and interactions with UsfX. UsfX forms a stable complex with SigF that could be purified only after co-expressing the proteins in E.coli. Formation of the complex is nucleotide independent and apparently requires unknown in vivo factors. Fluorescence spectroscopy experiments suggest that the nucleotide binding sites of UsfX are distal to the protein-protein interaction interface. RsfA is a novel anti-anti sigma factor whose binding to UsfX is triggered by the reduction of an intrachain disulphide bond between Cys73-Cys109. The reduction is accompanied by an increase in the hydrodynamic radius of the protein. The UsfX-RsfA complex exhibits a novel stoichiometry of 2:1 compared to the 2:2 stoichiometry reported for other anti-anti-sigma factors. The role of the disulphide bond in complex formation was explored using molecular dynamics simulations. These studies support specific conformational changes that occur upon reduction of the Cys73-Cys109 bond of RsfA. This leads to a rearrangement that increases the interactions of a conserved His107 of UsfX with Cys109 of RsfA.
authors.affiliation: Molecular and Structural Biology Division, Central Drug Research Institute, P.O. Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow-226001, India.
authors.forename: Shuja Shafi
authors.initials: SS
authors.lastname: Malik
authors.forename: Amit
authors.initials: A
authors.lastname: Luthra
authors.forename: Ravishankar
authors.initials: R
authors.lastname: Ramachandran
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D002942:Circular Dichroism; D003198:Computer Simulation; D003553:Cystine / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D010084:Oxidation-Reduction; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012808:Sigma Factor / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C064971:FliA protein, Bacteria; D011993:Recombinant Fusion Proteins; C540213:RsfA protein, Mycobacterium tuberculosis; D012808:Sigma Factor; D014157:Transcription Factors; C540214:UsfX protein, Mycobacterium tuberculosis; D003553:Cystine