Abstract
The precise control of signaling pathways is essential for biological homeostasis. Protein tyrosine phosphatases (PTP) coordinate with protein tyrosine kinases (PTK) to tune various signal responses such as cell growth, metabolism, and immune function. PTPs remove the phosphoryl group from phosphorylated tyrosine (pTyr) residues in substrate proteins, reversing the signaling effect of PTKs in the network. Most of the attention has long been placed on PTKs as primary drug targets in treating proliferative diseases, but their phosphatase counterparts are equally important in modulating a multitude of signal cascades and the potential to develop better therapeutic targets. The human protein tyrosine phosphatase, SHP2, is a multidomain enzyme where the PTP domain is preceded by two SH2 regulatory domains that bind pTyr-containing peptides. The enzyme interconverts between a closed, inactive state (90%) and an open, active state (10%). The autoinhibited closed state has its N-terminal SH2 docked into the PTP catalytic pocket. This equilibrium is often perturbed by oncogenic mutations, such as E76K and E76D, destabilizing the interaction of the N-SH2 domain with the PTP, and leading to a constitutively open and active phosphatase. Normally, wild-type SHP2 is allosterically activated by the binding of bis-phosphorylated peptides to the distal faces of the N- and C-SH2 domains in a controlled ligand-dependent manner to overcome the autoinhibition. The gain-of-function cancer mutants eliminate the regulation of phosphatase activity and result in rogue dephosphorylation of nearby substrates. PTPs are especially difficult to target with orthosteric inhibitors because of the highly conserved and charged active site. The allosteric inhibitor, SHP099, binds to and stabilizes a pocket formed between the N-SH2, C-SH2, and PTP domains in the closed conformation with a tight nanomolar affinity via a conformational selection mechanism. We used nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, enzyme kinetics, isothermal titration calorimetry (ITC), and stopped-flow kinetics to show the dynamics of SHP2 conformational states and how the potent activating E76K mutation drives the equilibrium to the open conformation; drastically increasing the amount of SHP099 required to return SHP2 to its basal activity. We show that an allosteric inhibitor, such as SHP099, may have the selectivity of binding to SHP2 over other homologous phosphatases, but its binding mechanism makes it a poor choice to treat the active cancer mutants. Most mechanistic studies on SHP2 have used these activating mutants to access its open conformation, but by doing so, they bypass the intriguing allosteric activation process to specifically unlock the phosphatase.
Naturally, the open/closed SHP2 equilibrium is transiently shifted towards the open by the allosteric pTyr peptide activators. In this work, we investigate the mechanism of SHP2 activation by four biological activators: the programmed-cell death receptor (PD-1), the B and T lymphocyte attenuator (BTLA), the insulin substrate receptor 1 (IRS-1), and Grb2-associated binder 1 (Gab1). The activators are essential for maintaining immune responses (PD-1 and BTLA) or cellular growth and metabolism pathways (IRS-1 and GAB1). SHP2 activators are found in the unstructured tails of receptors and soluble proteins and feature two phosphotyrosine motifs separated by a linker. We are the first to successfully produced stable, bis-phosphorylated peptides to a high yield for probing the dynamics of the natural SHP2 activation process. By using NMR, steady-state enzyme kinetics, ITC, and stopped-flow kinetics, we solved the complex mechanism of SHP2 allosteric regulation.
The diversity of activators and the biological selectivity between SHP2 and its close paralog SHP1 motivated us to study the evolution of protein tyrosine phosphatase regulation. It is well known that both phosphatases can co-exist, but rarely have overlap in their activators and the signaling cascades in which they function. SHP2 and SHP1 share approximately 50% sequence identity, an extremely similar domain architecture, and a conserved active site. This alludes to non-trivial but impactful evolutionary steps that may have occurred to develop and distinguish between the pair of PTPs. We gained insights into the origins of allosteric regulation by comparing phosphatase activity from extant organisms in several lineages. We performed ancestral sequence reconstruction (ASR) and resurrected extinct SHPs to study the ancestors’ abilities to self-regulate and be allosterically activated by modern bis-tyrosyl phosphorylated peptides. We experimentally characterized eleven extant and extinct SHPs to determine the evolutionary trajectory of the phosphatases and that all ancestors were well regulated by the autoinhibition of the N-SH2 -PTP domain interaction. This finding suggests that an affinity between the N-SH2 and PTP existed before the gene fusion event and the origin of SHPs. Regulation of PTP activity was clearly essential for the health of primordial organisms as it developed quickly during the evolutionary process. The allosteric activation via bis-phosphorylated peptides was a metazoan innovation, and activators likely co-evolved with SHP2. As organisms became more complex, the need to differentiate between signaling responses became apparent. This led to developing the newer SHP1 and its highly specific interaction with BTLA. We show that the discrete preference for BTLA is possibly liked to the C-SH2 domain orientation. While the C-SH2 is not directly responsible for the autoinhibition of SHP2, it plays an important role in activator recognition and specificity.
Allostery is at the core of enzyme regulation and has remained largely unexplored. In this work, we characterized the regulatory mechanisms that acted as the molecular switch for SHP2 and SHP1 activity and illustrated how this sophisticated system evolved.