Fig. 4

Structure of GTP-γS-bound Gsα protein fragment with indicated amino acid subsitution (A, B). The structure was generated with PyMol and derived from Sunahara et al. (1997) [28] (PDB: 1AZT). NGS analysis highlighted the presence of a missense mutation (c.677G > A; p.G226D) in the GNAS gene causing an amino acid change from nonpolar glycine (G226) to negatively charged aspartic acid (D) that may affect Gsα protein conformation and function. Moreover, G226 is in the Switch II region, one of the two loops undergoing structural changes upon GTP binding essential for binding and activation of adenylyl cyclase [8]. The G3 box (DXXG), that overlaps this region, is involved in binding a Mg²⁺ through Asp223 and, more importantly, in a hydrogen bonding with GTP through Gly226 [22]. Thus, Gsα G226D mutant could be likely present in an inactive (GDP-bound) state and, due to the disruption of a hydrogen bond network, unable to bind GTP. Prediction of clinical significance of G226D mutation (C). The prediction obtained using the Functional Analysis through Hidden Markov Models v2.3 tool [26], indicated the G226D mutation as a potentially cancer-associated alteration, showing a high probability of the prediction with a score − 3.29 (cutoff: −0.75)