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Computational and experimental approaches for assessing the interactions between the model calycin β‐lactoglobulin and two antibacterial fluoroquinolones
Author(s) -
Eberini Ivano,
Fantucci Piercarlo,
Rocco Alessandro Guerini,
Gianazza Elisabetta,
Galluccio Lara,
Maggioni Daniela,
Ben Ilaria Dal,
Galliano Monica,
Mazzitello Rosalba,
Gaiji Noura,
Beringhelli Tiziana
Publication year - 2006
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.21109
Subject(s) - chemistry , docking (animal) , norfloxacin , stereochemistry , molecule , molecular dynamics , crystallography , computational chemistry , organic chemistry , ciprofloxacin , biochemistry , medicine , nursing , antibiotics
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 β‐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. Proteins 2006. © 2006 Wiley‐Liss, Inc.