In order to design an atomistic model that may explain the activation mechanism of SHP2, mediated by the ligand-induced conformational changes, it is essential to clarify what are the conformational states that the N-SH2 domain may adopt. For that reason, molecular dynamics simulations were initially performed on the isolated N-SH2 domain, bound to a set of phosphopeptides, differing in length and sequence, and chosen according their high experimental binding affinities. The analysis of such simulations clearly showed that the N-SH2 domain adopts two distinct conformations, that we called α and β. These conformational states essentially differ in the structure of two sites, responsible respectively for the binding of the phosphotyrosine (pY site) and of the flanking residues, downstream respect to that (+5 site). These sites adopt two arrangements, respectively open and closed. Simulations clearly assessed an allosteric mechanism between these two sites. It was observed that the N-SH2 domain binds a phosphopeptide both in α and in β conformations, though the binding is stronger in the α state, when the pY site is closed. However, the fact that N-SH2 domain may prevalently populate one between the α and the β state depends on the phosphopeptide sequence and on the particular arrangement of the ligand that gives rise to a distinct binding mode. It was observed that in absence of a ligand, the N-SH2 is in the β state, both in its isolated form and in the autoinhibited structure of SHP2. For that reason, it was assumed that the β state is consistent with the autoinhibited structure of SHP2 whereas the α state is the activating conformation. In addition, it was postulated that in the context of SHP2 activation, the phosphopeptide sequence is not only essential for modulating the binding affinity but also for regulating the peptide potency of selecting between the activating and not activating conformation of the N-SH2 domain.
In order to assess if the α state is effectively the activating conformation, we calculated the free energy landscapes of the opening of SHP2, while the N-SH2 was forced to populate either the α or the β state. The β state resulted to be the conformation requiring the higher free energy for the activation of SHP2. On the other hand, the α state resulted to be effectively activating, as after the overcoming of a free energy barrier, SHP2 rearranged into a stable open state.
In order to assess the importance of the amino acid sequence in modulating the peptide potency of selecting between the activating α state and the inhibiting β state, free energy calculations were performed with alchemical methods. It was demonstrated that a phosphopeptide, or one of its binding modes, initially selecting for one of the conformational states, turned its preference by rationally replacing a single amino acid. It was also observed that for the selection potency the residue in position +5 respect to the phosphotyrosine plays a crucial role.
A further validation of the model was obtained explaining the effect of pathogenetic T42A mutation in terms of altered equilibrium between the α and the β state.
Finally, enhanced sampling MD simulations were performed on SHP2 complexed with a phosphopeptide, in order to observe the opening of the phosphatase under stimulated conditions.
These results were disseminated by oral presentation in an international workshop. In addition, a paper is in preparation, and it is foreseen to be published in a high impact journal.
The knowledge of the activation mechanism of SHP2, and the possibility to induce its opening in silico, allows us to address further questions, first of all, an effective comparison with the SAXS spectra in solution, that are currently available in literature. In this manner, it may be possible validating the activation pathway proposed or identifying the three-dimensional structure of the open state of SHP2. In addition, the MD simulations in stimulated conditions may be replicated on a series of peptides, in order to measure their activation potency. Then, it is possible translating the same approach so far employed on single phosphorylated sequences to the case of BTAMs, shedding light on the role of the BTAM linker in favoring the rearrangement of SHP2 during the activation. Finally, the structures of SHP2 observed at the early stages of the activation may be used for the design of molecules that can be inserted between N-SH2 and PTP, stabilizing the interdomain interface and avoiding a further opening and containing the activation.
