Development of a technique to determine bicyclomycin-rho binding and stoichiometry by isothermal titration calorimetry and mass spectrometry.
Bicyclomycin (1) is the only natural product inhibitor of the transcription termination factor rho. Rho is a hexameric helicase that terminates nascent RNA transcripts utilizing ATP hydrolysis and is an essential protein for many bacteria. The paucity of information concerning the 1-rho interaction stems from the weak binding affinity of 1. We report a novel technique using imine formation with rho to enhance the affinity of a bicyclomycin analogue and determine the binding stoichiometry by isothermal titration calorimetry (ITC) and mass spectrometry (MS). Our designed bicyclomycin ligand, 5a-(3-formyl-phenylsulfanyl)-dihydrobicyclomycin (2) (apparent I(50) = 4 muM), inhibits rho an order of magnitude more efficiently than 1 (I(50) = 60 muM). MS shows that 2 selectively forms an imine with K181 in rho. We found that despite the heterogeneity of ATP binding (three tight and three weak) imposed on the rho hexamer, the nearby bicyclomycin binding pocket is not affected, and both 1 and 2 bind with equal affinity to all six subunits.
Brogan
Andrew P
AP
Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599-7360, USA.
Widger
William R
WR
Bensadek
Dalila
D
Riba-Garcia
Isabel
I
Gaskell
Simon J
SJ
Kohn
Harold
H
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D000447:Aldehydes / Q000737:chemistry; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000737:chemistry* / Q000378:metabolism; D002151:Calorimetry; D007700:Kinetics; D011485:Protein Binding; D012234:Rho Factor / Q000737:chemistry* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D017186:Titrimetry
D000447:Aldehydes; D000900:Anti-Bacterial Agents; D019086:Bridged Bicyclo Compounds, Heterocyclic; D012234:Rho Factor; D000251:Adenosine Triphosphatases; C024504:bicozamycin