In vitro and in silico : two complementary approaches to elucidate the molecular mechanisms of Medium-Chain Acyl-CoA dehydrogenase deficiency (MCADD)

Tese de mestrado, Ciências Biofarmaêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2015The medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most common genetic disorder affecting the mitochondrial fatty acid β-oxidation (mFAO) pathway, in particular the first dehydrogenation reaction of C4-C14 fatty acyl substrates. The mature MCAD enzyme is a homotetramer with Flavin Adenine Dinucleotide (FAD) as its natural cofactor. The most common mutation found in MCADD patients (c.985A>G in ACADM gene) is translated in the substitution of lysine by a glutamic acid residue at position 304 (p.K304E) of the mature protein. Accordingly, this substitution has been widely related with protein misfolding and tetramer instability. To better understand the molecular basis of the pathogenicity of the p.K304E variant in MCADD, in vitro and in silico approaches were undertaken. Experimental in vitro studies comprised the heterologous expression of human MCAD wild-type (hMCADwt) and p.K304E variant proteins, followed by the recombinant proteins’ purification and functional and structural characterization. The results obtained show that, when compared to hMCADwt, the p.K304E variant is structurally unstable, presenting an alteration in its oligomeric profile, thermal instability and a higher susceptibility to trypsin limited proteolysis, the latter pointing to a higher conformational flexibility of the variant protein. The addition of FAD in the initial step of the purification of the recombinant proteins seems to increase the tetrameric fraction of the variant protein and recover its deficient enzymatic activity. Moreover, FAD also incremented the thermal stability of both hMCADwt and p.K304E proteins, as expected being FAD the MCAD’s cofactor. Promising results were also obtained with glycerol and TMAO tested, among other compounds, as chemical chaperones for the p.K304E variant. To complement the in vitro data, molecular dynamics (MD) simulations of the MCAD enzyme were also performed and both porcine and human structures were studied. The initial coordinates were obtained, respectively, from the crystallographic structure of the porcine MCAD protein (PDB: 1UDY; pMCADwt), and by reversing the crystallographic structure of the E376G/T255E mutant of the human MCAD protein (PDB: 1EGC; hMCADwt). In a following step, both pMCADwt and hMCADwt proteins were used to generate the corresponding p.K304E variants. The results obtained showed that both wild-type proteins were stable during the simulation time. The FAD seems to have a structural role in the catalytic pockets and in the tetramer stability. The catalytic pockets are highly flexible and dynamic to better accommodate the substrate. The p.K304E variant seems to induce a different behavior over MD simulations regarding both pMCADwt and hMCADwt. The p.K304E mutation seems also to induce structural modifications in the catalytic pockets, ultimately affecting their volume. Finally, the mutation seems to asymmetrically affect the FAD and substrate binding affinities in both pMCAD and hMCAD variants, which may explain the decrease of the enzymatic activity and catalytic efficiency observed in the p.K304E/hMCAD variant (~50% residual activity in respect to hMCADwt). Altogether, the in vitro and in silico data obtained contribute to better clarify the structural and functional abnormalities of the p.K304E variant of hMCAD thereby opening perspectives for the search for small molecules as protein stabilizers towards the development of pharmacological strategies for the treatment of MCADD