title: Dynamics and flexibility of human aromatase probed by FTIR and time resolved fluorescence spectroscopy.
abstract: Human aromatase (CYP19A1) is a steroidogenic cytochrome P450 converting androgens into estrogens. No ligand-free crystal structure of the enzyme is available to date. The crystal structure in complex with the substrate androstenedione and the steroidal inhibitor exemestane shows a very compact conformation of the enzyme, leaving unanswered questions on the conformational changes that must occur to allow access of the ligand to the active site. As H/D exchange kinetics followed by FTIR spectroscopy can provide information on the conformational changes in proteins where solvent accessibility is affected, here the amide I region was used to measure the exchange rates of the different elements of the secondary structure for aromatase in the ligand-free form and in the presence of the substrate androstenedione and the inhibitor anastrozole. Biphasic exponential functions were found to fit the H/D exchange data collected as a function of time. Two exchange rates were assigned to two populations of protons present in different flexible regions of the protein. The addition of the substrate androstenedione and the inhibitor anastrozole lowers the H/D exchange rates of the α-helices of the enzyme when compared to the ligand-free form. Furthermore, the presence of the inhibitor anastrozole lowers exchange rate constant (k1) for β-sheets from 0.22±0.06 min(-1) for the inhibitor-bound enzyme to 0.12±0.02 min(-1) for the free protein. Dynamics effects localised in helix F were studied by time resolved fluorescence. The data demonstrate that the fluorescence lifetime component associated to Trp224 emission undergoes a shift toward longer lifetimes (from ≈5.0 to ≈5.5 ns) when the substrate or the inhibitor are present, suggesting slower dynamics in the presence of ligands. Together the results are consistent with different degrees of flexibility of the access channel and therefore different conformations adopted by the enzyme in the free, substrate- and inhibitor-bound forms.
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Di Nardo
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy.
authors.forename: Maximilian
authors.initials: M
authors.lastname: Breitner
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy.
authors.forename: Sheila J
authors.initials: SJ
authors.lastname: Sadeghi
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Castrignanò
authors.affiliation: Department of Experimental Medicine and Surgery, University of Rome 'Tor Vergata', Italy.
authors.forename: Giampiero
authors.initials: G
authors.lastname: Mei
authors.affiliation: Department of Experimental Medicine and Surgery, University of Rome 'Tor Vergata', Italy.
authors.forename: Almerinda
authors.initials: A
authors.lastname: Di Venere
authors.affiliation: Department of Experimental Medicine and Surgery, University of Rome 'Tor Vergata', Italy.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Nicolai
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Allegra
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy.
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Gilardi
mesh_terms: D001141:Aromatase / Q000737:chemistry / Q000378:metabolism*; D041961:Deuterium Exchange Measurement; D017666:Deuterium Oxide / Q000378:metabolism; D004795:Enzyme Stability; D006801:Humans; D008024:Ligands; D050505:Mutant Proteins / Q000737:chemistry; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D008024:Ligands; D050505:Mutant Proteins; D011994:Recombinant Proteins; D001141:Aromatase; D017666:Deuterium Oxide