PMC:4570552 / 9574-12413 JSONTXT

{"project":"2_test","denotations":[{"id":"25865493-18042454-2044017","span":{"begin":583,"end":585},"obj":"18042454"},{"id":"25865493-17682063-2044017","span":{"begin":583,"end":585},"obj":"17682063"},{"id":"25865493-24442204-2044018","span":{"begin":752,"end":754},"obj":"24442204"},{"id":"25865493-14529625-2044019","span":{"begin":1559,"end":1561},"obj":"14529625"},{"id":"25865493-25017102-2044020","span":{"begin":1758,"end":1760},"obj":"25017102"},{"id":"25865493-15264259-2044021","span":{"begin":1857,"end":1859},"obj":"15264259"},{"id":"25865493-9020976-2044021","span":{"begin":1857,"end":1859},"obj":"9020976"}],"text":"Similar to other “large” MAF subfamily members (i.e., MAFA, MAFB, and NRL), MAF’s structure is characterized by a C-terminal extended homology region and bZIP domain mediating DNA binding, and a N-terminal transactivation domain required for transcriptional activity and regulatory function (Figure 1B). The latter contains four GSK3 phosphorylation motifs, highly conserved among large MAF proteins (Figure 1C). In MAFA, the sequential phosphorylation of these serine/threonine residues promotes ubiquitination and rapid degradation, but also increases transactivation potential.24,25 Remarkably, all identified MAF mutations clustered within these motifs. Three affected residues, Ser54, Thr58, and Ser62, are known GSK3 phosphorylation target sites.26 The remainder did not involve phosphorylatable residues, but were predicted to affect GSK3-mediated phosphorylation by altering proline residues adjacent to either a phosphorylation site (Thr58) or the C-terminal priming site (Ser70), whose phosphorylation is absolutely required for GSK3 function. To explore the impact of the p.Pro59His, p.Pro59Leu, and p.Pro69Arg changes, we performed molecular dynamics (MD) simulations on complexes formed by full-length GSK3 and ten residue-long peptides of MAF corresponding to the segment that interacts directly with the GSK3 binding cleft, encompassing both the GSK3 target and pSer/pThr primed residues (Table S6). The starting coordinates for the ATP-bound GSK3 were taken from the crystallographic structure of GSK3B complexed with AMP-PNP (PDB entry 1pyx).27 Each decapeptide was set in an extended conformation along the catalytic cleft of GSK3 as specified in Table S6. The MD simulations were carried out according to the protocol previously described.10 The Gromos 53a6 force field was used, with the exception of the partial charges of pSer/pThr,28,29 and the parameters for ATP, obtained from quantum mechanical calculation of the molecular system reported in Figure S4. For p.Pro59His and p.Pro59Leu decamers, the conformation of the trimer comprised between the substrate and primed residues was rearranged considerably during the simulations (Figure 2A and Figure S5), with the correct orientation of the substrate residue in the GSK3 active site being destabilized (Figure 2A). p.Pro69Arg, introducing a cationic residue in the proximity of the positively charged GSK3 priming pocket formed by residues Arg96, Arg180, and Lys205, caused a general rearrangement of the adjacent pSer70, pulling it away from the binding pocket (Figure 2B). Overall, our simulations indicated consistently that all disease-causing MAF mutations inhibit GSK3-mediated phosphorylation through impaired association and/or catalysis, by perturbing the interaction with the priming site (p.Pro69Arg) or the active site (substitutions affecting Pro59)."}