Structural insights into Noonan/LEOPARD syndrome-related mutants of protein-tyrosine phosphatase SHP2 (PTPN11)
© Qiu et al.; licensee BioMed Central Ltd. 2014
Received: 9 November 2013
Accepted: 6 March 2014
Published: 14 March 2014
The ubiquitous non-receptor protein tyrosine phosphatase SHP2 (encoded by PTPN11) plays a key role in RAS/ERK signaling downstream of most, if not all growth factors, cytokines and integrins, although its major substrates remain controversial. Mutations in PTPN11 lead to several distinct human diseases. Germ-line PTPN11 mutations cause about 50% of Noonan Syndrome (NS), which is among the most common autosomal dominant disorders. LEOPARD Syndrome (LS) is an acronym for its major syndromic manifestations: multiple Lentigines, Electrocardiographic abnormalities, Ocular hypertelorism, Pulmonary stenosis, Abnormalities of genitalia, Retardation of growth, and sensorineural Deafness. Frequently, LS patients have hypertrophic cardiomyopathy, and they might also have an increased risk of neuroblastoma (NS) and acute myeloid leukemia (AML). Consistent with the distinct pathogenesis of NS and LS, different types of PTPN11 mutations cause these disorders.
Although multiple studies have reported the biochemical and biological consequences of NS- and LS-associated PTPN11 mutations, their structural consequences have not been analyzed fully. Here we report the crystal structures of WT SHP2 and five NS/LS-associated SHP2 mutants. These findings enable direct structural comparisons of the local conformational changes caused by each mutation.
Our structural analysis agrees with, and provides additional mechanistic insight into, the previously reported catalytic properties of these mutants. The results of our research provide new information regarding the structure-function relationship of this medically important target, and should serve as a solid foundation for structure-based drug discovery programs.
Mutations in PTPN11 cause several human diseases. Germ-line PTPN11 mutations cause ~50% of Noonan Syndrome (NS), which is among the most common autosomal dominant disorders [7, 8]. Gain-of-function mutations in other RAS-RAF-MEK-ERK pathway members, including SOS1[9, 10], KRAS, NRAS, SHOC2, and RAF1[14, 15], are responsible for most remaining NS cases. With an estimated incidence of 1/2,000 live births , NS is characterized by facial dysmorphism, proportional short stature, cardiac anomalies, and various less penetrant phenotypes, such as webbed neck, deafness, and motor delay. Many (20-50%) NS patients develop some type of myeloproliferative disorder (MPD), which is typically mild and self-limited . Rare NS patients progress to Juvenile Myelomonocytic Leukemia (JMML), which is fatal if not treated by bone marrow transplantation, somatic PTPN11 mutations are the single most common cause of sporadic JMML [7, 18–20]. LEOPARD Syndrome (LS), a much less common autosomal dominant disorder, is almost always caused by PTPN11 mutations, and is related to, but distinguishable from, NS [7, 16, 21]. LEOPARD is an acronym for its major syndromic manifestations: multiple Lentigines, Electrocardiographic abnormalities, Ocular hypertelorism, Pulmonary stenosis, Abnormalities of genitalia, Retardation of growth, and Deafness . These patients often have hypertrophic cardiomyopathy (HCM), and might also have an increased risk of neuroblastoma (NS) and acute myeloid leukemia (AML) [23, 24]. Knock-in mouse models have been generated for NS and LS alleles of Ptpn11 and generally reproduce the phenotypes seen in the cognate human syndromes [25–27].
Consistent with the distinct pathogenesis of NS and LS, different types of PTPN11 mutations cause these disorders. Most NS-associated PTPN11 mutations alter residues that reside at the interface between the N-SH2 and PTP domains , resulting in elevated enzymatic activity and enhanced RAS/ERK activation [27–31]. These data suggest that NS mutations disrupt the intramolecular interaction between the N-SH2 and PTP domains, shifting the equilibrium between the closed and open conformations and lowering the activation threshold for SHP2. By contrast, LS mutations typically affect PTP domain residues, result in markedly decreased catalytic activity, and lower RAS/ERK activation in transient transfection assays [31–33]. Studies of the LS Y279C mouse model also indicate that LS mutants may have dominant negative effects in at least some tissues in vivo. Whereas NS phenotypes arise from enhanced MEK/ERK activation and can be prevented or reversed by MEK inhibition [34–36], LS-associated HCM is caused by enhanced PI3K/AKT/mTORC1 activity and can be reversed by rapamycin .
Although multiple studies have reported the biochemical and biological consequences of NS- and LS-associated PTPN11 mutations, their structural consequences have not been analyzed. Here, we report the X-ray structures of five NS/LS SHP2 mutants and discuss how these mutations affect the interaction between different SHP2 domains and its catalytic activity.
A wild type (WT) SHP2 expression construct 1–539 (comprising the N + C SH2 and PTP domains) was PCR-amplified from PTPN11 cDNA  with a set of custom-designed primers (see Additional file 1: Description S1). The resultant PCR fragment was cloned into a modified version of the plasmid pET28b (Novagen) that generates a fusion protein with an N-terminal hexahistidine tag. The SHP2 catalytic domain expression construct (a.a. 221–524) was cloned into pGEX4T, which introduces a GST-tag at its N-terminus. Mutations were introduced into these expression constructs by site directed-mutagenesis with specifically designed primers bearing one substitution each (see Additional file 1: Description S1). Pfu Ultra II high fidelity DNA polymerase (Stratagene) was used for PCR, with an extension temperature of 68°C over 10 minutes. To remove any traces of the original cDNA, all reactions were subjected to digestion with DpnI (New England Biolabs) for 1 hour at 37°C. Reaction mixtures were transformed into DH5α cells, and the genetic content of all constructs was verified by Sanger sequencing.
Protein expression & purification
Vectors encoding full length versions of SHP2 mutants were transferred into E. coli BL21(DE3). Cells were grown in Terrific Broth containing kanamycin (50 mg/l) in 1 L Tunair flasks at 37°C to an OD600 of 3–5, after which the temperature was lowered to 16°C, and isopropyl-1-thio-β-D-galactopyranoside (IPTG) was added to 0.2 mM. Expression was allowed to proceed overnight, then cells were harvested by centrifugation, flash-frozen in liquid nitrogen, and stored at −80°C. Due to the low level of expression of the Q506P construct, cells expressing this mutant were washed using the osmotic shock technique  prior to freezing. Unless stated otherwise, all purification procedures were carried out at 4°C. Cells were thawed on ice and resuspended in Binding Buffer (see Additional file 2: Table S1 for detailed buffer components), supplemented with phenylmethylsulfonylfluoride and benzamidine. After disruption by sonication and centrifugation at 60,000 g for 40 min, cell-free extracts were passed through a DE-52 column (2.6 × 7 cm) that had been pre-equilibrated with the same buffer, and loaded by gravity flow onto a Ni-nitrilotriacetic acid (NTA) column (Qiagen, Germantown, MD). The latter column was washed with 20–25 volumes of Wash Buffer A, followed by 20–25 volumes of Wash Buffer B and finally with Elution Buffer. N308D and E139D eluted in Elution Buffer, whereas the other three mutants eluted with the Wash buffers. For the latter proteins, the wash fractions were diluted 15-fold and reloaded on fresh Ni-NTA columns. After washing with 10 column volumes of Binding Buffer, N308D and E139D proteins were eluted in elution buffer. These samples were concentrated using a VIVASpin unit (Sartorius NA, Edgewood, NY), and loaded onto a 2.6 × 60 cm Superdex 200 column (GE Healthcare), equilibrated with Gel Filtration buffer. Elution was performed at a flow rate of 3 ml/min at 8°C, with the SHP2 proteins behaving as apparent monomers. Final protein samples were concentrated to 20–40 mg/ml, divided into 1.5 mg aliquots, flash-frozen and stored at −80°C.
SHP2 catalytic domain mutants were transformed into Escherichia coli strain BL21(DE3). A 25 ml aliquot of an overnight culture from a single colony was added to 500 ml of LB/ampicillin (50 μg/ml) and grown at 37°C to A600 = 0.8. IPTG was added to a final concentration of 0.1 mM, and the bacteria were maintained for 16 h at 25°C with shaking, then centrifuged at 6,000 × g for 10 min. at 4°C. Pellets were resuspended in 12.5 ml of a buffer containing 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1% Triton X-100, 10% glycerol, 5 mM dithiothreitol, 2 μg/ml aprotinin, 10 μg/ml leupeptin, 1 μg/ml antipain, 1 μg/ml pepstatin A, 0.5 mg/ml lysozyme and 1 mg/mL DNase I. Suspensions were incubated on ice for 30 minutes, and then sonicated for 10 seconds on ice. Lysates were centrifuged at 14,000 × g for 30 min. at 4°C, and supernatants were transferred to a fresh 15-ml polypropylene tube containing 0.5 ml of glutathione-Sepharose 4B (GE Healthcare Life Sciences). This suspension was rotated end-over-end overnight at 4°C, and then centrifuged at 1000 × g for 1 min. at 4°C. The supernatants were discarded, and the beads were washed 3 times for 5 min. each at 4°C with 10 ml of wash buffer (25 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1% Triton X-100, 10% glycerol, 5 mM dithiothreitol), and then once with PTP assay buffer (25 mM Hepes, pH 7.5, 100 mM NaCl, 2 mM EDTA and 5 mM dithiothreitol). Bound GST fusion proteins were resuspended 1:1 in PTP assay buffer. A 20 uL aliquot of slurry for each mutant was separated on a 10% SDS-polyacrylamide gel, together with different amounts of BSA. The gel was washed in water for 10 minutes, and stained with Colloidal Coomassie Blue for 1 hour at room temperature. Bands were quantified using a LI-COR Odyssey.
To determine kinetic parameters, fixed amounts of purified GST-WT or -mutant SHP2 catalytic domains (1.6 pmol of WT and N308D, 115pmol of Y279C and 16.3 pmol of Q506P) were incubated with variable concentrations of substrate peptide (R-R-L-I-E-D-A-E-pY-A-A-R-G, Millipore #12-217; Kit #12-217) in PTP assay buffer in a total volume of 50 uL. Reactions were carried out for 10 minutes at 25°C, and phosphate release was quantified by adding Malachite Green (Millipore #17-125) to the supernatants, measuring absorbance at 620 nm, and comparing values to a standard curve generated with varying amounts of KH2PO4. All reactions fell within the linear range. Phosphatase activity is expressed in pmol Pi released/min/pmol enzyme.
Mutant SHP2 proteins were crystallized under conditions similar to those reported previously . In order to obtain the best diffracting crystals, 0.1 M LiCl was added to the literature crystallization conditions for D61G, 5% glycerol for N308D, and 10% glycerol and 0.3 M cycohexyl-methyl-β-D-maltoside for Q506P. The other two mutant proteins and the WT protein were crystallized under literature conditions with optimized precipitant concentrations. Crystals appeared overnight, and reached their full size of about 300 × 300 × 30 microns in one week at room temperature. The stacked plate crystals were separated and flash frozen in liquid nitrogen, using paratone-N oil (Hampton Research Inc.) as a cryo-protectant.
Data were collected at 100 K with a wavelength of 1.0 Å on the Industrial Macromolecular Crystallography Association (IMCA-CAT) beam line at the Advanced Photon Source (Argonne National Laboratory, IL USA). The data were indexed, integrated, and scaled with XDS and XSCALE.
Summary of crystallographic data and refinement statistics
Outermost resolution shell, (Å)
Unit cell parameters
Molecules per asymmetric unit
Average I/σ (I)
R merge , (%)
Refinement and structure statistics
R work , (%)
R free , (%)
RMSD from ideal geometry
Bond lengths, (Å)
Bond angles, (°)
Numbers of atoms
Water oxygen atoms
Results and discussion
We determined the crystal structures of WT SHP2 (residues 1–539), as well as five mutants (D61G, E139D, Y279C, N308D, and Q506P), chosen to represent the spectrum of disease-associated PTPN11 mutations. Mutants D61G, E139D, and N308D are found in NS, Y279C is a canonical LS mutation [16, 21, 42], and Q506P has been reported in both disorders , although it is unclear whether this reflects misdiagnosis or true bi-potentiality of this allele. The D61G mutation affects the N-SH2 domain, E139D lies within the C-SH2 domain and the other three mutations alter the PTP domain (Figure 1b). The enzymatic properties of the full-length versions of these mutants (including the C-terminal tail, which is missing in our crystallization constructs) were characterized previously by our group [29, 33] (Additional file 3: Figure S1), and range from strongly activated (D61G), to mildly activated (N308D), to catalytically impaired (Y279C). Q506P shows altered specificity for some substrates .
Catalytic activities of the indicated SHP2 catalytic domain (221–524) GST fusion proteins were measured using the Malachite Green assay in the presence of different concentrations of PTP-1B peptide R-R-L-I-E-D-A-E-pY-A-A-R-G. Km and kcat calculated by: 1/V = (Km/Vmax) / [pY] + 1/ Vmax
kcat/ Km(s−1 mM−1)
SHP2 is regulated by a molecular switch mechanism that controls its catalytic activity. Upon binding to a tyrosine-phosphorylated binding partner for its SH2 domains, the N-terminal SH2 domain is released from the PTP domain, activating the enzyme. This elegant mechanism ensures that PTP activity is delivered to the right place in the cell at the right time. Remarkably, germ line mutations that disrupt this regulatory machinery in different ways result in distinct disease syndromes. The crystal structures of “true” WT SHP2 and five NS/LS-associated SHP2 mutants reported herein provide direct comparisons of the local conformational changes caused by each mutation. Our structural observations are in agreement with, and can provide mechanistic insight into, the previously reported catalytic properties of these mutants. For example, mutation of D61G in the N-SH2 domain significantly impacts SHP2 activity because this residue is located at the N-SH2/PTP domain interface and its alteration weakens key interactions between the two domains. On the other hand, our data suggest that the C-SH2 domain mutation E139D might interfere with SHP2 binding to tryrosine-phosphorylated ligands. The other three mutants, Y279C, N308D and Q506P, are located in PTP domain, and the local conformational changes induced by each mutation provide insight into their abnormal catalytic properties. The results of our research provide structural insights into this medically important target and could aid in future structure-based drug discovery programs.
Availability of supporting data
The coordinates and diffraction data for SHP2 wild type and mutant crystal structures are available in Protein Data Bank (http://www.rcsb.org/pdb).
These studies were supported by the Ontario Research and Development Challenge Fund (99-SEP-0512) and R37 CA49152 (to BGN). BGN and EFP are Canada Research Chairs, Tier 1, and work in their laboratories is partially supported by the Ontario Ministry of Health and Long Term Care and The Princess Margaret Cancer Foundation. Use of the IMCA-CAT beamline 17-ID at the Advanced Photon Source was supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with the Hauptman-Woodward Medical Research Institute. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. We thank Aiping Dong for providing technical support and the Structural Genomics Consortium, University of Toronto, for the use of their X-ray facilities.
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