An allosteric interaction controls the activation mechanism of SHP2 tyrosine phosphatase

Abstract

SHP2 is a protein tyrosine phosphatase with a key role in multiple signaling pathways, including the RAS-MAPK cascade. Germline mutations in PTPN11, the gene encoding SHP2, occur in 50% of individuals affected by Noonan syndrome, whereas somatic mutations in this gene cause more than 30% of cases of juvenile myelomonocytic leukemia (JMML), and are variably found in other pediatric hematologic malignancies and tumors. Inhibition of the wild-type protein has been recently demonstrated as a novel effective therapeutic strategy for many forms of cancer. Under basal conditions, wild-type SHP2 is inactive because its N-terminal Src homology 2 (N-SH2) domain blocks the active site of the protein tyrosine phosphatase (PTP) domain. Previous work established that binding of a phosphopeptide ligand to the N-SH2 domain causes SHP2 activation by favoring dissociation of the N-SH2 and PTP domains, however the conformational transitions of N-SH2 controlling ligand affinity and PTP dissociation are not well understood. Based on molecular simulations and free-energy calculations, we revealed a 20Å-spanning allosteric network restraining the N-SH2 domain to two distinct states, a SHP2-activating and a stabilizing state. We find that ligand binding is necessary but not sufficient for SHP2 activation, as only ligands selecting for the activating conformation of N-SH2, depending on ligand sequence and binding mode, are predicted to be effective activators. The proposed model of SHP2 activation is further validated by rationalizing modified basal activity and responsiveness to ligand stimulation of N-SH2 variations at residue Tyr 42 . This study provides mechanistic and energetic insight into SHP2 activation and may open novel routes for SHP2 regulation.

The loops of the N-SH2 binding cleft do not serve as allosteric switch in SHP2 activation

“…Notably, the "allosteric switch" model has been so far unable to convincingly explain the pathogenicity of the Thr42Ala substitution, in terms of either enhancement of the binding cleft opening or formation of additional favorable contacts with the phosphopeptide [15]. Instead, owing to the reduced propensity of alanine for β-sheet as compared to threonine [57], the α-β model attributes to the Thr42Ala substitution the effect of destabilizing the central β-sheet, thereby rendering N-SH2 more prone to adopt the activating α-state [43].…”

“…activation. In fact, previous molecular dynamics simulations suggested that the complete displacement of N-SH2 away from the PTP active site requires an energy in the order of 70 kJ/mol [41,43]. Hence, it is unlikely that rearrangement of the binding cleft are sufficient to drive SHP2 activation.…”

“…Recently, an allosteric interaction in N-SH2 has been proposed as an alternative mechanism of SHP2 activation [43]. Molecular dynamics (MD) simulations have shown that the N-SH2 domain may adopt two distinct conformations, denoted as α-and β-state, which differ primarily in the conformation of the central β-sheet.…”

“…(B) Correlation between the N-SH2 binding cleft opening, d cleft (Gly 67 C α -Lys 89 C α distance) and the N-SH2 blocking loop distance from the catalytic PTP loop, d block (Asp 61 C α -Ala 460 C α distance), as taken from crystal structures of autoinhibited SHP2, comprising either wild type or functional mutants[16,32,49,50].on the other face of N-SH2.In particular, Lys 91 side chain in BG loop forms salt bridges with charged residues of another replica, whereas the motion of the Tyr 66 side chain in EF loop is sterically hindered by the presence of other bulky side chains. Even the pY loop, which in absence of a phosphate group is typically modelled in a partially closed conformation, corresponding to an intermediate conformation between the α-and β-state[43], interacts with acidic side chains of Glu 313 and Glu 348 belonging to another protein chain. Therefore, the partially closed loops in the N-SH2Considering that the activation of SHP2 presumably involves a free energy difference of several tens of kJ/mol[41,43], it is mandatory to evaluate the energy required to open the binding cleft in different conditions.In order to quantify the intrinsic flexibility of the binding cleft, we used umbrella sampling to compute the potential of mean force (PMF, also referred to as 'free energy profile') of the binding cleft opening for the isolated N-SH2 domain(Figure 5A/B).…”

“…Even the pY loop, which in absence of a phosphate group is typically modelled in a partially closed conformation, corresponding to an intermediate conformation between the α-and β-state[43], interacts with acidic side chains of Glu 313 and Glu 348 belonging to another protein chain. Therefore, the partially closed loops in the N-SH2Considering that the activation of SHP2 presumably involves a free energy difference of several tens of kJ/mol[41,43], it is mandatory to evaluate the energy required to open the binding cleft in different conditions.In order to quantify the intrinsic flexibility of the binding cleft, we used umbrella sampling to compute the potential of mean force (PMF, also referred to as 'free energy profile') of the binding cleft opening for the isolated N-SH2 domain(Figure 5A/B). As reaction coordinate, we used the distance d cleft defined as the C α -C α distance between Gly 67 (belonging to the EG loop) and Lys 89 (belonging to the BG loop).…”