Tyrosine Phosphatase SHP-2
Created by Garren Montgomery
Tyrosine phosphatase SHP-2 (pdb ID=2SHP) from Homo sapiens is a ubiquitously expressed cytosolic enzyme involved in cell signaling pathways, especially those that are activated by growth factors, cytokines, and hormones (1). Most cellular signaling pathways are controlled by tyrosine phosphorylation. Protein tyrosine phosphatases work in concert with protein tyrosine kinases to regulate the level of phosphorylation necessary for cell growth, differentiation, and migration (2). Due to its role in the regulation of growth factor and cytokine signaling pathways, mutations of SHP-2 leads to cell growth-related diseases including Leopard Syndrome, Noonan Syndrome, and childhood leukemia (1). SHP-2 has a molecular weight of 120,163.97 Da and an isoelectric point (pI) of 6.88.
SHP-2 consists of two identical subunits, which each contain three functionally distinct domains: two Src homology-2 domains (SH2) and one protein tyrosine phosphatase domain (PTP). The PTP domain consists of residues 220-525 and contains the enzyme’s active site. It is primarily responsible for the dephosphorylation of tyrosine-phosphorylated peptides. The N-terminal SH2 domain (residues 3-104) acts as a conformational switch, either inhibiting or activating the PTP domain. The C-terminal SH2 domain (residues 112-216) does not have a significant role in inhibition of the PTP domain but contributes binding energy and substrate specificity to aid in PTP activation (1).
Consistent with the secondary structure displayed by the protein as a whole, the PTP domain of SHP-2 displays a mixed α/β architecture. The PTP domain consists of nine alpha helices and fourteen beta strands. Ten of these beta strands form an antiparallel beta sheet. At the center of this sheet is strand βM that, along with the helix αG contains the PTP signature motif: VHCSAGIGRTG. This motif is found in residues 457-467 of SHP-2, and contains the nucleophilic Cys-459 and all residues necessary for phosphate binding. Another catalytically important secondary structure element of the PTP domain is the WPD loop, which contains Asp-425, a general acid in catalysis (1). His-458, located directly adjacent to the catalytic cysteine residue, generates a dipole that greatly lowers the pKa of Cys-459 to approximately five (9). Thus, Cys-459 is deprotonated at physiological pH, and the nucleophilicity of its thiolate anion makes it the key catalytic residue during substrate deposphorylation.
When the PTP domain binds a tyrosine-phosphorylated receptor substrate such as Insulin receptor substrate-1 (IRS-1; pdb id=1QQG) at its active site, the phosphate oxygens of the substrate are coordinated by the guanidinium side chain of Arg-465 and several of the backbone amide groups in the PTP signature motif (3). This arginine residue is highly conserved in all PTPs and greatly influences binding affinity for the phosphoryl substrate, as any mutation at this position greatly reduces substrate affinity (9). Peptide binding causes the WPD loop (residues 423-432) to close around the bound phosphotyrosyl substrate, bringing with it Asp-425, the acid catalyst (1). In this catalytic conformation, Cys-459 dephosphorylates the substrate via a nucleophilic attack on its phosphate group. Asp-425 both protonates the phenolate leaving group in the substrate, and promotes the hydrolysis of the cysteinyl phosphate intermediate (4). PTPs are extremely specific for phosphotyrosyl peptides. Non-phosphorylated peptides do not bind. In addition, phosphorylated serine and threonine are excluded from the binding cleft due to the location of Cys-459 nine angstroms below the molecular surface (3). Only a fully extended phosphotyrosyl side chain is long enough to reach this depth; serine and threonine side chains are too short.
Contoured around the PTP domain are the SH2 domains. They consist of a four-stranded beta sheet in the center with an alpha helix on both sides of the sheet (1). Like the PTP active site, SH2 recognizes phosphotyrosyl peptides. The SH2 domains are not catalytic, but are responsible for activation of the PTP domain in the presence of phosphopeptides. The phosphopeptide binding sites are primarily located in alpha helix A (residues 12-21), beta strands B (28-35) and D (49-57), and the loop connecting beta strands B and C (36-42) (5). On both the C- and N-terminal SH2 domains, these binding sites are located on the surface of the protein and interact with solvent in the absence of substrate (1).
Upon binding to SH2, the backbone of a phosphotyrosyl peptide is held in position at the binding site by beta strand D via ionic interactions and hydrogen bonds. The phosphotyrosine side chain extends away from this strand to form an ionic interaction with the invariant arginine residue on beta strand B (residue 32) (5). This arginine residue is critical for SH2 function and its mutation halts peptide binding. The location of Arg-32 also provides stereochemical discrimination for phosphotyrosyl peptides (5). As observed in the PTP active site, only tyrosine side chains are long enough to contact this arginine residue, excluding serine and threonine.
The presence of two tandem SH2 domains in SHP-2 affects catalytic activity of the PTP domain. Under basal conditions, when no phosphotyrosyl peptides are present, the PTP domain is largely inactive due to the direct inhibition of its active site by the N-terminal SH2 domain (1). When a phosphopeptide binds one of the two SH2 domains, catalytic activity increases tenfold, while phosphopeptide binding to both SH2 domains stimulates catalysis almost one-hundredfold. The C-terminal SH2 domain forms limited contacts with the N-SH2 and PTP domains, and does not have a direct role in the inhibition of PTP (1). Its primary function when bound to a phosphopeptide is to contribute binding affinity for the N-SH2 and PTP domains by recruiting the SHP-2 to appropriate binding proteins in cells, usually near the cell membrane (3).
The N-terminal SH2 domain is an allosteric regulator of phosphatase activity. It is directly involved in inhibition of the PTP domain in the absence of phosphopeptides. In this auto-inhibited structure, the D’E loop and the D’ and E beta strands of the N-SH2 domain insert deeply into the active site of PTP and sterically block access of phosphotyrosyl peptides (1). All residues in the catalytic cleft expected to interact with a phosphotyrosine substrate instead interact with the N-SH2 domain. Asp-61 and Tyr-62 of the D’E loop mimic the interactions of the phosphotyrosine substrate with the active site. Asp-61 and Gly-60 form hydrogen bonds with the catalytic nucleophile Cys-459, and Tyr-62 forms hydrophobic interactions with Tyr-279, a residue within the phosphotyrosine recognition loop (3). Phosphatase activity is further inhibited by the restraint of the WPD loop in the open, catalytically inactive conformation by the D’E loop of the N-terminal SH2 domain (3). The auto-inhibited conformation is stabilized outside of the catalytic cleft through extensive polar interactions between the N-SH2 and PTP domains. A salt bridge forms between Glu-258 and Arg-4, and hydrogen bonds form between Asn-281 and Glu-69, Ser-502 and Glu-76, Gln-506 and Ala-72, and Asn-58 and Gln-506. When the N-SH2 domain binds a phosphopeptide, however, its affinity for the active site is diminished, these polar interactions are broken, and the active site becomes available for binding of phosphotyrosine ligands (1).
The crystal structure of SHP-2 contains only one ligand, dodecane trimethylamine, which is a detergent molecule used during crystallization that binds to a cavity left by mutation of Phe-513 to Ser-513. The absence of a phenyl ring (Phe-513) gives the ligand sufficient room to interact with the serine residue and other residues in the vicinity. Dodecane trimethylamine has no known function in SHP-2, however (1).
Although the peptide ligand specificity for SHP-2 is not well understood, comparison of its binding specificity to that of SHP-1 (pdb id=1FPR) has revealed some mechanisms by which it discriminates between phophotyrosyl peptide substrates. SHP-1 shares 61% sequence identity with SHP-2. Results of protein Blast and DALI show that the PTP domain of SHP-1 has an E-value of 1 x 10-118 and a Z-score of 34.6, respectively, when compared to the sequence of SHP-2 (8). The E-value represents a high degree of similarity between SHP-1 and SHP-2 in primary structure, and the Z-score, because it is greater than two, indicates similar tertiary structures. SHP-2 and SHP-1 have identical domain architecture consisting of two SH2 domains and a PTP domain (shown by pdb id=2B3O) that is responsible for dephosphorylating tyrosine residues on peptide substrates. Despite folding and sequence similarities, SHP-1 and SHP-2 have very different functions in cells as they select for distinct substrates. SHP-1 is expressed solely in hematopoietic cells and is believed to negatively mediate, or inhibit, cell signaling by dephosphorylating appropriate substrates. SHP-2 is expressed in almost every cell type and tends to positively mediate, or enhance, cell signaling (6).
One explanation for the substrate specificity of these proteins involves beta strand F and the loop connecting beta strand F to beta strand H. This sequence in SHP-1 (residues 354-360) is VEKGRNK, while in SHP-2 it is VERGKSK (residues 360-366). This strand of residues, known as the P4 subpocket, binds the region of the phosphotyrosyl peptide N-terminal to the phosphotyrosine residue. The swapping of lysine for arginine at the third residue of the sequence significantly alters specificity of SHP-2 towards growth factor substrates. The guanidinium group of arginine offers more polar interactions with residues of the substrate in this N-terminal region, and thus SHP-2 is postulated to prefer peptide substrates containing more polar residues within the region that binds to its P4 pocket (7).
The large distance (50 angstroms) between phosphotyrosyl binding pockets of the two SH2 domains of SHP-2 may also contribute to ligand specificity. In the autoinhibited conformation, a peptide ligand with two phosphotyrosine residues sufficiently separated to bind both SH2 domains at once will stimulate PTP catalysis one hundredfold. In SHP-2, the 50 angstrom separation between its tandem SH2 binding sites discriminates for substrates that have large separations between phosphotyrosine residues. SHP-2 will bind IRS-1, which possesses phosphotyrosines separated by 49 residues. SHP-1, by contrast, has a much smaller gap between its SH2 domains. It selects for diphosphorylated peptide substrates whose tyrosines are more closely spaced, as in SIRP (23-25 residue separation) (1).
Comparison to another related protein, receptor protein tyrosine phosphatase-alpha (pdb id=1YFO) from Mus musculus gives insight into another mechanism of catalytic inhibition used by the tyrosine phosphatase family. RPTPα shares 43% sequence identity with SHP-2, (protein Blast E = 3 x 10-58) and also displays similar tertiary structure as evidenced by the results of DALI (Z= 37.8) (8). RPTPα is an integral membrane protein phosphatase composed of an extracellular domain that binds ligands, a single transmembrane domain, and two intracellular catalytic domains, D1 and D2 (10). The catalytic domains of RPTPα and the PTP domain of SHP-2 show similar tertiary structures composed of a central group of beta strands surrounded on both sides by alpha helices. The PTP domain of SHP-2 is larger, but the active sites of the two proteins are highly homologous in that both contain the signature motif composed of the amino acids HCXXGXXR(S/T), which includes the nucleophilic cysteine and all residues necessary for phosphate binding (9).
Catalytic autoinhibition of RPTPα occurs when the extracellular domain binds a ligand and inhibits phosphatase activity by causing the two D1 monomers inside the cell to dimerize. The amino-terminal segments of each D1 monomer have helix-turn-helix wedges that are inserted into the active site of the opposing monomer. This interaction is stabilized by van der Waals interactions and hydrogen bonds. Dimerization also forces the WPD loop (containing the catalytic Asp residue) into an open, catalytically inactive conformation (10). This process of steric inhibition is very similar to that displayed by SHP-2, whose N-SH2 domain inserts deep into the catalytic cleft of the PTP domain and also restrains the WPD loop in a similar open conformation (1).
Disregulation of phosphatase activity in SHP-2 can lead to pathology. Helicobacter pylori, a bacterium that infects human gastrointestinal cells, is known to secrete the protein CagA. CagA is injected directly from helicobacter pylori into host cells and undergoes tyrosine phosphorylation at numerous EPIYA motifs throughout its sequence. The phosphorylated tyrosines within these motifs allow a single CagA protein to bind both SH2 domains simultaneously, which not only relieves the steric block of the PTP active site by the N-SH2 domain, but also greatly stimulates PTP activity. As a positive mediator in cell signaling, SHP-2 then continuously activates signaling cascades involved in cell growth, development, and differentiation within intestinal epithelial cells. This finding has lead to the belief that SHP-2 could be a key protein in the development of gastric cancer in those infected with helicobacter pylori (11).
The role of SHP-2 in signaling cascades leading to cancer and other growth problems has prompted research into therapeutic inhibitors of its PTP activity. NSC-87877 (see Images) has been shown to significantly decrease the catalytic activity of SHP-2 by binding to the catalytic cleft of the PTP domain. The B-ring sulfonic acid group of NSC-87877 forms a hydrogen bond with the backbone NH group of the conserved Arg-465 residue, while the A-ring sulfonic acid group forms hydrogen bonds with the side-chain NH2 group of Asn-281 and the NH3 group of Lys-280. The phenyl rings of the structure are stabilized by hydrophobic interactions with the residues in and around the active site of SHP-2. Binding of NSC-87877 to the critical Arg-465 residue inhibits the coordination of the phosphate oxygens on a phosphotyrosyl peptide. Its binding position also blocks the critical Cys-459 nucleophile, making dephosphorylation impossible. Therefore, even when SHP-2 is in its active conformation (when N-SH2 is not bound to the active site), the binding of NSC-87877 inhibits phosphatase activity (12).
Tyrosine phosphatase SHP-2 is responsible for dephosphorylating peptide substrates involved in signaling events promoted by primarily by growth factors and cytokines (1). When the cell is activated by extracellular growth factor ligands, the SH2 domains of SHP-2 route the inactive protein to the plasma membrane, where it is activated by the binding of its SH2 domains to growth factor receptors or substrates that have been phosphorylated at tyrosine residues through the action of tyrosine kinases. Once the allosteric inhibition of the active site is relieved through the binding of phosphopeptides to the N-SH2 domain, the PTP domain is free to catalyze the dephosphorylation of these receptors or substrates and promote signaling downstream from G-proteins such as Ras in the MAP kinase pathway (3). Its close association with the growth factor signaling cascades has made SHP-2 an increasingly studied protein for those interested in drug research to treat growth abnormalities and cancer.