Substrate-induced conformational changes of the mitochondrial oxoglutarate carrier: a spectroscopic and molecular modelling study.
The structural and dynamic properties of the oxoglutarate carrier were investigated by introducing a single tryptophan in the Trp-devoid carrier in position 184, 190 or 199 and by monitoring the fluorescence spectra in the presence and absence of the substrate oxoglutarate. In the absence of substrate, the emission maxima of Arg190Trp, Cys184Trp and Leu199Trp are centered at 342, 345 and 348 nm, respectively, indicating that these residues have an increasing degree of solvent exposure. The emission intensity of the Arg190Trp and Cys184Trp mutants is higher than that of Leu199Trp. Addition of substrate increases the emission intensity of Leu199Trp, but not that of Cys184Trp and Arg190Trp. A 3D model of the oxoglutarate carrier was built using the structure of the ADP/ATP carrier as a template and was validated with the experimental results available in the literature. The model identifies Lys122 as the most likely candidate for the quenching of Trp199. Consistently, the double mutant Lys122Ala-Leu199Trp exhibits a higher emission intensity than Leu199Trp and does not display further fluorescence enhancement in response to substrate addition. Substitution of Lys122 with Cys and evaluation of its reactivity with a sulphydryl reagent in the presence and absence of substrate confirms that residue 122 is masked by the substrate, likely through a substrate-induced conformational change.
Morozzo Della Rocca
Blasco
B
INFM and Department of Biology, University of Rome "Tor Vergata", via della Ricerca Scientifica, Rome.
Miniero
Daniela V
DV
Tasco
Gianluca
G
Dolce
Vincenza
V
Falconi
Mattia
M
Ludovico
Anna
A
Cappello
Anna R
AR
Sanchez
Paloma
P
Stipani
Italo
I
Casadio
Rita
R
Desideri
Alessandro
A
Palmieri
Ferdinando
F
D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D006801:Humans; D007656:Ketoglutaric Acids / Q000737:chemistry; D008239:Lysine / Q000737:chemistry / Q000235:genetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics
D007656:Ketoglutaric Acids; D026901:Membrane Transport Proteins; D024101:Mitochondrial Proteins; C021723:oxoglutarate translocator; D014443:Tyrosine; D014364:Tryptophan; D008239:Lysine; D003545:Cysteine