Tyrosine_Phosphatase_SHP2

Non-Receptor Protein Tyrosine Phosphatase SHP2 in Complex with Allosteric Inhibitor SHP099 (PDB ID: 5EHR) from Homo sapiens

Created by Christy Kostka

            Non-receptor Protein Tyrosine Phosphatase SHP2 in Complex with Allosteric Inhibitor SHP099 (PBD ID: 5EHR) from Homo sapiens is a drug complex of Tyrosine Phosphatase SHP-2 (PBD ID: 2SHP) with SHP009, a selective small-molecule inhibitor that stabilize the protein in its auto-inhibited conformation (1). SHP-2 is a ubiquitously expressed cytoplasmic enzyme that removes phosphate groups from receptor tyrosine kinases (RTKs), thus regulates cell proliferation by providing docking sites for signaling proteins in the RAS-ERK signaling pathway, a proliferative cell pathway (2). Activating mutations of SHP-2 have been associated with developmental pathologies such as Noonan syndrome and several cancer types, including leukemia, lung, and breast cancer (1). Therefore, the stabilization of the auto-inhibited state provided by SHP009 in SHP-2 in complex with SHP099 (SHP-2-SHP099) is significant in both treatment of pathologies and further research of proliferative cell pathways (3).

             SHP-2-SHP099 has a molecular weight of 120,490.24 Da and an isoelectric point of 6.69 (3). SHP-2 consists of two identical subunits with three functionally distinct domains each: the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase (PTP) domain (1). The PTP domain (residues 220-525) is primarily responsible for the dephosphorylation of tyrosine-phosphorylated peptides (4). Phosphorylation of Tyr-416 on the PTP domain of SHP-2 unfolds the protein, exposes the positively charged PTP region, and activates it to elicit downstream signaling (5). The N-terminal SH2 domain (residues 3-111) acts as a conformational switch to control the activation of the PTP domain with a surface area contact of 1208 Å (4). The C-terminal SH2 domain (residues 112-216) contains a proline-rich motif, which contributes to the binding energy and substrate specificity to aid in PTP activation (4). The C-terminal SH2 domain does not have a direct role in the inhibition of PTP, its main function is to recruit the SHP-2 to appropriate binding proteins in cells (5).

            The secondary structure of SHP-2 displays a mixed α-helix/β-strand architecture with 25 β-strands and 12 α-helices per subunit. The PTP domain consists of nine α-helices and fourteen β-strands, ten of which form an antiparallel β-sheet. Residues 457-467 contain the PTP signature motif: VHCSAGIGRTG, which contains all residues necessary for phosphate binding. Cys-459 is deprotonated at physiological pH due to the proximal His-458, and its thiolate anion acts as a key nucleophile during substrate dephosphorylation (6). Mutations of Cys-459 to Ser-459 SHP-2 cause it to lose its catalytic function (1). The WPD loop (residues 423-432) of the PTP domain contains Asp-425, which can act as a general acid catalyst (4).

            Arg-465 and several of the backbone amide groups of the PTP signature motif coordinate with the phosphate oxygen atoms of tyrosine-phosphorylated substrates bound to the PTP domain. Arg-465 is highly conserved in all PTPs and greatly influences binding affinity for the phosphoryl substrate (6). In the active conformation, after the WPD loop has closed around the phosphotyrosyl substrate, Cys-459 depohsphorylates the substrate via nucleophilic attack. Then, Asp-425 protonates the phenolate leaving group and promotes the hydrolysis of the cysteinyl phosphate intermediate (7). PTPs are specific for phosphotyrosyl peptides. In SHP-2, the Cys-459 residue is 9 Å below the molecular surface, causing it to exclude both phosphorylated serine and phosphorylated threonine in favor of the longer phosphorylated tyrosine residue for binding (5).

            The SH2 domains consist of a four-stranded β-sheet surrounded on both sides by α-helices and are responsible for the activation of the PTP domain in the presence of phosphopeptides. The phosphopeptide binding sites are located in α-helix A (residues 12-24), β-strands B (28-33) and D (50-57), and the loop connecting β-strands B and C (34-40). Ionic interactions and hydrogen bonds stabilizes the binding of the SH2 domain and the backbone of a phosphotyrosyl peptide at β-strand D. The tyrosine side chains of the phosphotyrosyl peptide forms an ionic interaction with Arg-32 on β-strand B, which is crucial for binding specificity (8).

            The N-terminal SH2 domain is an allosteric regulator of phosphatase activity. In the auto-inhibited structure, the D’E loop (residues 58-62) and the D’ and E β-strands of the N-terminal SH2 domain extend deep into the catalytic cleft and sterically block access of phosphotyrosyl peptides (4). The D’E loop interacts antagonistically with the active site on the PTP domain by hydrogen bonds of Asp-61 and Gly-60 with Cys-459 and hydrophobic interactions between Tyr-62 and Tyr-279. The conformation with the D’E loop antagonistically inhibiting SHP-2 function also restrains the WPD loop, further inhibiting phosphatase activity. The auto-inhibited conformation is stabilized in SHP-2 through interactions between the N-terminal SH2 and PTP domains, such as salt bridge formation between Glu-258 and Arg-4, hydrogen bonds between Asn-281 and Glu-69, Ser-502 and Glu-76, Gln-506 and Ala-72, and Asn-58 (5).

         SHP099 is bound at the central tunnel formed at the interface of the three domains of each subunit when the N-terminal domain is blocking the active site of the PTP domain, thus stabilizing the protein in its inactive state and inhibiting its catalytic activity. Two molecules of SHP099 can be bound to each SHP-2 molecule. Hydrogen bonds between SHP099 and SHP-2 at Arg-111 (N-terminal domain), Phe-113 (C-terminus domain), and Glu-250 (PTP domain) and extensive hydrophobic interactions between the dichlorophenyl group of SHP099 and Leu-254, Gln-257, Pro-491, and Gln-495 of the PTP domain of SHP-2 give the small molecule a low dissociation constant of 0.073 μM (K=1.38 x 107 M-1) (1).

           An important aspect of drugs that target specific proteins is their specificity, which prevents off targeting effects. The specificity of SHP099 for SHP-2 was tested against SHP-1 (PDB ID: 2B3O), a tyrosine phosphatase sharing 61% of SHP-2’s amino acid sequence (4). The Dali server uses a sum of pairs method to compare intramolecular distances of tertiary structures. Position Specific Iterated Basic Local Alignment Search Tool (PSI-BLAST) compares the number of gaps between the protein of interest’s amino acid sequence and the comparison protein’s amino acid sequence. PSI-BLAST shows an E-value of 1 x 10-118 and Dali shows a Z-score of 34.6 for the PTP domain of SHP-1 compared to SHP-2. The E value illustrates significant similarity between the primary structures of SHP-1 and SHP-2, because it is over 0.05 (9). Because the Z-score is greater than 2, the two proteins have similar tertiary structure (10). Both SHP-2 and SHP-1 consist of two SHP domains and one PTP domain. SHP-1 and SHP-2 are similar in sequence and structure but select for different substrates, making finding a specific molecule to target just one protein tyrosine phosphatse a challenge. SHP-1 is expressed in hematopoietic cells to inhibit cell signaling, while SHP-2 is expressed widely and activates cell signaling (6).

        The difference in the linker region between the two SH2 domains in SHP-1, compared to SHP-2, creates a significantly larger central tunnel with approximate dimensions of 1,012 Å3 compared to 464 Å3 in SHP-2. The difference is highlighted by residue Arg-109 in SHP-1 compared to Arg-111 in SHP-2. Key interactions between the SH2 domains and SHP099 are lost in the larger central tunnel of SHP-1. Therefore, SHP099 is specific for SHP-2. The effectiveness of the SHP099 molecule in inhibiting SHP-2 activity was examined using statistical analysis and mouse models. The half maximal inhibitory concentration (IC50) of SHP099 for SHP-2 is 0.071 μM. Therefore, SHP-2 has a high affinity for SHP099 and can effectively inhibit it at low concentrations. SHP099 was given to nude mice with malignant tumors and was effective at inhibiting tumor growth with no observed adversary or off-targeting effects (1).

        The drug complex of SHP099 with non-receptor Protein Tyrosine Phosphatase SHP2 is a specific and effective inhibitor of SHP-2 activity (1). SHP-2 does not remove phosphate groups from RTKs when it is bound to SHP099, thus proliferative cell pathways are not activated (2). By keeping the protein in its auto-inhibited state, SHP099 has the potential treat developmental pathologies such as Noonan syndrome and several cancer types, including leukemia, lung, and breast cancer in humans. SHP099 is specific for SHP-2 and does not bind to SHP-1, a protein sharing 61% homology with SHP-2, due to the difference in placement of one arginine residue that is essential for the binding of SHP099.