title: Computational and experimental approaches for assessing the interactions between the model calycin beta-lactoglobulin and two antibacterial fluoroquinolones.
abstract: Norfloxacin and levofloxacin, two fluoroquinolones of different bulk, rigidity and hydrophobicity taken as model ligands, were docked to one apo and two holo crystallographic structures of bovine beta-lactoglobulin (BLG) using different computational approaches. BLG is a member of the lipocalin superfamily. Lipocalins show a typical b-barrel structure encompassing an internal cavity where small hydrophobic molecules are usually bound. Our studies allowed the identification of two putative binding sites in addition to the calyx. The rigid docking approximation resulted in strong repulsive forces when the ligands were docked into the calyx of the apo form. On the contrary, hindrance was not experienced in flexible docking protocols whether on the apo or on the holo BLG forms, due to allowance for side chain rearrangement. K(i) between 10(-7) and 10(-6) M were estimated for norfloxacin at pH 7.4, smaller than 10(-5) M for levofloxacin. Spectroscopic and electrophoretic techniques experimentally validated the occurrence of an interaction between norfloxacin and BLG. Changes in chemical shift and dynamic parameters were observed between the (19)F NMR spectra of the complex and of the ligand. A K(i) (ca 10(-7) M) comparable with the docking results was estimated through a NMR relaxation titration. Stabilization against unfolding was demonstrated by denaturant gradient gel electrophoresis on the complex versus apo BLG. NMR experimental evidence points to a very loose interaction for ofloxacin, the racemic mixture containing levofloxacin. Furthermore, we were able to calculate in silico K(i)'s comparable to the published experimental values for the complexes of palmitic and retinoic acid with BLG.
authors.affiliation: Gruppo di Studio per la Proteomica e la Struttura delle Proteine, Dipartimento di Scienze Farmacologiche, Università degli Studi di Milano, Milano, Italia.
authors.forename: Ivano
authors.initials: I
authors.lastname: Eberini
authors.forename: Piercarlo
authors.initials: P
authors.lastname: Fantucci
authors.forename: Alessandro Guerini
authors.initials: AG
authors.lastname: Rocco
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Gianazza
authors.forename: Lara
authors.initials: L
authors.lastname: Galluccio
authors.forename: Daniela
authors.initials: D
authors.lastname: Maggioni
authors.forename: Ilaria Dal
authors.initials: ID
authors.lastname: Ben
authors.forename: Monica
authors.initials: M
authors.lastname: Galliano
authors.forename: Rosalba
authors.initials: R
authors.lastname: Mazzitello
authors.forename: Noura
authors.initials: N
authors.lastname: Gaiji
authors.forename: Tiziana
authors.initials: T
authors.lastname: Beringhelli
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002417:Cattle; D019295:Computational Biology; D018360:Crystallography, X-Ray; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007782:Lactoglobulins / Q000737:chemistry*; D064704:Levofloxacin; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009643:Norfloxacin / Q000737:chemistry* / Q000378:metabolism; D015242:Ofloxacin / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D000900:Anti-Bacterial Agents; D007782:Lactoglobulins; D008024:Ligands; D064704:Levofloxacin; D015242:Ofloxacin; D009643:Norfloxacin