title: Nanoelectrospray ionization mass spectrometric study of Mycobacterium tuberculosis CYP121-ligand interactions.
abstract: Nondenaturing nanoelectrospray ionization mass spectrometry (nanoESI MS) of intact protein complexes was used to study CYP121, one of the 20 cytochrome P450s in Mycobacterium tuberculosis (Mtb) and an enzyme that is essential for bacterial viability. The results shed new light on both ligand-free and ligand-bound states of CYP121. Isolated unbound CYP121 is a predominantly dimeric protein, with a minor monomeric form present. High affinity azoles cause the dissociation of dimeric CYP121 into monomer, whereas weaker azole binders induce partial dimer dissociation or do not significantly destabilize the dimer. Complexes of CYP121 with azoles were poorly detected by nanoESI MS, indicating kinetically labile complexes that are easily prone to gas-phase dissociation. Unlike with the azoles, CYP121 forms a stable complex with its natural substrate cYY that does not undergo gas-phase dissociation. In addition, a series of potential ligands from fragment-based studies were used as a test for nanoESI MS work against CYP121. Most of these ligands formed stable complexes with CYP121, and their binding did not promote dimer dissociation. On the basis of binding to the monomer and/or CYP121 dimer it was possible to determine the relative order of their CYP121 binding affinities. The top nanoESI MS screening hit was confirmed by heme absorbance shift assay to have a Kd of 40 μM.
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom.
authors.forename: Katie M
authors.initials: KM
authors.lastname: Duffell
authors.forename: Sean A
authors.initials: SA
authors.lastname: Hudson
authors.forename: Kirsty J
authors.initials: KJ
authors.lastname: McLean
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Munro
authors.forename: Chris
authors.initials: C
authors.lastname: Abell
authors.forename: Dijana
authors.initials: D
authors.lastname: Matak-Vinković
mesh_terms: D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000378:metabolism*; D008024:Ligands; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000378:metabolism*; D036103:Nanotechnology / Q000379:methods*; D011485:Protein Binding / Q000502:physiology; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D008024:Ligands; C464825:cytochrome P-450 CYP121; D003577:Cytochrome P-450 Enzyme System