Journal: bioRxiv

Article Title: Ionizable networks mediate pH-dependent allostery in SH2 signaling proteins

doi: 10.1101/2024.08.21.608875

Figure Lengend Snippet: (a) Schematic for in silico pK a prediction method for proteins with solved structures (see text and methods for details). Briefly, all available structures in the protein database are curated, electrostatic properties are calculated using PROPKA, results are filtered for ionizable residues with physiologically relevant predicted pK a values, and data are visualized in a 3D structure or a 2D residue interaction network. (b) Crystal structure of SHP2 shown in cartoon and surface format (PDB ID:2SHP). Protein tyrosine phosphatase (PTP) domain colored in grey, SH2 domains colored in yellow. (c) Structure of SHP2 (PDB ID:2SHP) with protein tyrosine phosphatase (PTP) domain in grey and SH2 domains in yellow. Residues identified through in silico ionizable network prediction pipeline shown in spheres. Residues with predicted pK a shifts (cyan) cluster with ionizable interactors (magenta) across the phosphatase-SH2 domain interaction interface of SHP2. (d) Table of predicted pK a s for cyan residues identified using in silico ionizable network prediction pipeline on 47 SHP2 structures (mean ± SD). (e) Residue interaction network of residues with predicted pK a shifts (cyan) and their ionizable interactors (magenta). Length of edges reflect the strength of the coulombic interaction, with stronger coulombic interactions having shorter edge lengths (f) Zoom of SHP2 structure at the PTP-SH2 interaction interface. Networked residues from a and b are shown in stick. Residues with predicted pK a shifts in cyan and ionizing interactors in magenta.

Article Snippet: Full-length SHP2 variants were cloned into the pGEX-4TI SHP2 WT plasmid , pGEX-4T1 SHP2 WT was a gift from Ben Neel (Addgene plasmid # 8322 ; http://n2t.net/addgene:8322 ; RRID:Addgene_8322)) and expressed in E. Coli for purification.

Techniques: In Silico, Residue

Journal: bioRxiv

Article Title: Ionizable networks mediate pH-dependent allostery in SH2 signaling proteins

doi: 10.1101/2024.08.21.608875

Figure Lengend Snippet: (a) Wild-type (WT) SHP2 in vitro phosphatase activity curves with increasing concentrations of generic substrate p-Nitrophenyl Phosphate (PNPP) at buffer pH ranging from 6.1 to 8.0. (mean ± SEM; N=3 from ≥2 different protein preparations) (b) Double mutant (H116A/E252A) SHP2 in vitro phosphatase activity, assays performed as in A. (mean ± SEM; N=3 from ≥2 different protein preparations) (c) Plot of k cat vs. pH for WT and double mutant (H116A/E252A) SHP2 activity. Calculated from activity curves in a and b. (mean ± SEM) (d) Single-mutant H116A-SHP2 in vitro phosphatase activity, assays performed as in a. (mean ± SEM; N=3 from ≥2 different protein preparations) (e) Single-mutant E252A-SHP2 in vitro phosphatase activity, assays performed as in a. (mean ± SEM; N=3, from ≥2 different protein preparations) (f) Plot of K cat vs. pH for WT and double mutant (H116A/E252A) SHP2 activity. Calculated from activity curves in a, d and e. (mean ± SEM) (g) Proposed pH-sensing mechanism where SH2 domain (yellow) binding to catalytic domain (grey) is titratable by pH. (h) CpHMD (see methods for details) was performed on SHP2 at pH values from 4.0-10.0 (see supplemental videos). Shown are overlapping views of SHP2 structures at the start of CpHMD simulation (t = 0 ns) and end of simulation (t = 8 ns) for pH values 4.5 (pink), 5.5 (purple), 6.5 (blue) and 8.5 (yellow).

Article Snippet: Full-length SHP2 variants were cloned into the pGEX-4TI SHP2 WT plasmid , pGEX-4T1 SHP2 WT was a gift from Ben Neel (Addgene plasmid # 8322 ; http://n2t.net/addgene:8322 ; RRID:Addgene_8322)) and expressed in E. Coli for purification.

Techniques: In Vitro, Activity Assay, Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: Ionizable networks mediate pH-dependent allostery in SH2 signaling proteins

doi: 10.1101/2024.08.21.608875

Figure Lengend Snippet: (a) pHi measurements of MCF10A cells. Cells were treated with 25 μM EIPA + 30 μM S0858 for 1 hour to lower pH to 7.10. To raise pHi, cells were treated with 30 mM ammonium chloride for 1 hour to raise pH to 8.00. Untreated cells had a pHi of 7.45. Scatter plot shows (median ± interquartile range, N =10) (b) Representative immunoblots of SHP2, Gab1, and phospho-SHP2 (pY542) from SHP2 immune complexes (SHP2 IP) or whole cell lysates (Cell Lysate) isolated from MCF10A cells prepared as in A. (c) Quantification of replicate data collected as in B. Data was normalized to control in each biological replicate. Scatter plot shows (mean ± SEM, N=4). I (d) Quantification of Co-IP of SHP2 shown in b. Immunoblot intensities in the treatment conditions were normalized to control in each biological replicate. Scatter plot shows (mean ± SEM, N=7). (e) Representative images of MCF10A cells expressing the SHP2 activity reporter (Grb2 TagBFP) pseudocolored on an intensiometric scale. Images show cells prior to manipulating pHi with nigericin buffer (Pre Nigericin) (see methods for details), 50s - after manipulating pHi, and 900s after manipulating pHi. Scale bars: 25μm (f) Quantification of images as in E. Membrane intensity of SHP2 activity reporter was photobleach-corrected and then normalized to initial intensity over time. Line trace shows from single-cell data (mean ± SEM) (6.7 pH, n=30, 7.4 pH, n=30, 7.8 pH, n=25, control, n=28) collected across N=3 biological replicates. (g) Quantification of endpoint membrane intensities of single cells collected as described in f. Scatter plot shows (median ± interquartile range, N = 5) (h) Representative immunoblot of lysates prepared from MCF10A cells expressing either WT SHP2 or H116A/E252A SHP2 and treated as described in a. Immunoblots show total and pY542-SHP2 under low, control, and high pHi conditions. (i) Quantification of replicate data collected as in h. Scatter plots show (mean ± SEM, N=3). Intensities were normalized to the corresponding control condition. For a and g significance was determined using the Kruskal-Wallis test. For c, d, and i significance was determined using a ratio paired t-test to compare between treatment conditions and a one-sample t-test to compare to control. * p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Full-length SHP2 variants were cloned into the pGEX-4TI SHP2 WT plasmid , pGEX-4T1 SHP2 WT was a gift from Ben Neel (Addgene plasmid # 8322 ; http://n2t.net/addgene:8322 ; RRID:Addgene_8322)) and expressed in E. Coli for purification.

Techniques: Western Blot, Isolation, Control, Co-Immunoprecipitation Assay, Expressing, Activity Assay, Membrane

Journal: bioRxiv

Article Title: Ionizable networks mediate pH-dependent allostery in SH2 signaling proteins

doi: 10.1101/2024.08.21.608875

Figure Lengend Snippet: (a) Schematic of pH-driven activation and inhibition of SHP2. At low pH, the SH2 domain is unbound and SHP2 becomes signaling active with increased phosphorylation of Y542, increased GAB1 binding, and increased Grb2 recruitment. (b) Schematic of pH-driven activation and inhibition of Src. At low pH, the SH2 domain is unbound and c-Src becomes signaling active with increased phosphorylation of Y416, decreased phosphorylation of Y527, and increased membrane recruitment.

Article Snippet: Full-length SHP2 variants were cloned into the pGEX-4TI SHP2 WT plasmid , pGEX-4T1 SHP2 WT was a gift from Ben Neel (Addgene plasmid # 8322 ; http://n2t.net/addgene:8322 ; RRID:Addgene_8322)) and expressed in E. Coli for purification.

Techniques: Activation Assay, Inhibition, Phospho-proteomics, Binding Assay, Membrane

Journal: bioRxiv

Article Title: The pathogenic T42A mutation in SHP2 rewires the interaction specificity of its N-terminal regulatory domain

doi: 10.1101/2023.07.10.548257

Figure Lengend Snippet: ( A ) Domain architecture diagram of SHP2. Relevant mutations and the catalytic cysteine (Cys 459 ) are indicated. ( B ) SHP2 is kept in its auto-inhibited state by interactions between the N-SH2 and PTP domain (PDB: 4DGP). In its active state, the catalytic cysteine is accessible. The structure of SHP2 E76K (PDB: 6CRF) is used to represent the active state. ( C ) The SH2 domains of SHP2 bind to upstream phosphoproteins, such as transmembrane receptors, inducing a conformational change that activates SHP2. ( D ) Disease-associated mutations cluster largely, but not exclusively, on the interdomain interface between the N-SH2 and the PTP domain (PDB: 4DGP). The E76K mutation is a canonical N-SH2/PTP interface mutation. ( E ) Mutations in or near the N-SH2 binding pocket (PDB: 6ROY). ( F ) Mutations in or near the C-SH2 binding pocket (PDB: 6R5G). The well-established specificity-determining regions of the SH2 domains, which dictate +1 to +5 residue preferences, are marked with black dashed lines.

Article Snippet: The SHP2 full-length, wild-type gene used as the template for all SHP2 constructs in this study was cloned from the pGEX-4TI SHP2 WT plasmid, which was a generous gift from Ben Neel (Addgene plasmid #8322) ( ).

Techniques: Mutagenesis, Binding Assay, Residue

Journal: bioRxiv

Article Title: The pathogenic T42A mutation in SHP2 rewires the interaction specificity of its N-terminal regulatory domain

doi: 10.1101/2023.07.10.548257

Figure Lengend Snippet: ( A ) Measured binding affinities of N-SH2 WT against peptides derived from various known SHP2 interactors ( B ) Fold-change in K D for N-SH2 T42A compared to N-SH2 WT , for each of the peptides shown in panel (A). ( C ) Same as (B), but for N-SH2 L43F . ( D ) Same as (B), but for N-SH2 T52S . For ( A )-( D ), N = 3–4 independent protein, peptide, and fluorescent peptide titrations. Source data can be found in .

Article Snippet: The SHP2 full-length, wild-type gene used as the template for all SHP2 constructs in this study was cloned from the pGEX-4TI SHP2 WT plasmid, which was a generous gift from Ben Neel (Addgene plasmid #8322) ( ).

Techniques: Binding Assay, Derivative Assay

Journal: bioRxiv

Article Title: The pathogenic T42A mutation in SHP2 rewires the interaction specificity of its N-terminal regulatory domain

doi: 10.1101/2023.07.10.548257

Figure Lengend Snippet: ( A ) Hydrogen bonding of Thr 42 in SHP2 N-SH2 WT to the phosphoryl group of phosphopeptide ligands in several crystal structures (PDB: 6ROY, 1AYA, 1AYB, 3TL0, 5DF6, 5X94, and 5X7B). ( B ) Representative structure of N-SH2 WT bound to the PD-1 pTyr 223 (ITIM) peptide at the end of one MD simulation. N = 3 simulations of 1 μs each. ( C ) Representative structure of N-SH2 T42A bound to the PD-1 pTyr 223 (ITIM) peptide at the end of one MD simulation. N = 3 simulations of 1 μs each. ( D ) Overlay of the representative states shown in panels B and C. The N-SH2 WT state is in yellow with a dark-gray ligand. The N-SH2 T42A state is in light gray, with a light gray ligand. ( E ) Distribution of distances between the Lys 55 Nζ atom and the phosphotyrosine phosphorus atοm in simulations of the PD-1 pTyr 223 peptide bound to N-SH2 WT (black) or N-SH2 T42A (red). ( F ) Distribution of distances between the Lys 55 Nζ atom and the +2 Glu Cδ atom in simulations of the PD-1 pTyr 223 peptide bound to N-SH2 WT (black) or N-SH2 T42A (red). ( G ) An ion pair between Lys 55 and the +2 Glu residue (Glu 225) in the PD-1 pTyr 223 (ITIM) peptide, frequently observed in N-SH2 T42A simulations. ( H ) Peptide-specific effects of the T42A mutation in the presence and absence of the K55R mutation. N = 2–5 independent titrations.

Article Snippet: The SHP2 full-length, wild-type gene used as the template for all SHP2 constructs in this study was cloned from the pGEX-4TI SHP2 WT plasmid, which was a generous gift from Ben Neel (Addgene plasmid #8322) ( ).

Techniques: Phospho-proteomics, Residue, Mutagenesis

Journal: bioRxiv

Article Title: The pathogenic T42A mutation in SHP2 rewires the interaction specificity of its N-terminal regulatory domain

doi: 10.1101/2023.07.10.548257

Figure Lengend Snippet: ( A ) SHP2 activation is measured by incubation with phosphopeptide ligands, followed by monitoring dephosphorylation of the small-molecule substrate DiFMUP to generate fluorescent DiFMU. ( B ) Representative activation curves for SHP2 WT . N = 3–17 independent titrations of protein, peptide, and DiFMUP. ( C ) Correlation between the EC 50 of SHP2 WT activation by phosphopeptides and the K D of those phosphopeptides for the N-SH2 WT domain. For EC 50 values in ( B )-( C ), N = 3 independent titrations of protein, peptide, and DiFMUP.( D ) Correlation between activation EC 50 values for SHP2 WT and SHP2 R138Q . For SHP2 R138Q EC 50 values, N = 3–5 independent titrations of protein, peptide, and DiFMUP. ( E ) Comparison of SHP2 WT and SHP2 T42A activation curves for the PD-1 pTyr248 peptide. N = 3–4 independent titrations of protein, peptide, and DiFMUP. ( F ) Comparison of SHP2 WT and SHP2 T42A activation curves for the Imhof-9 peptide. N = 6–17 independent titrations of protein, peptide, and DiFMUP. ( G ) Bubble plot juxtaposing the EC 50 values for activation of SHP2 WT and SHP2 T42A by nine peptides, alongside the fold-change in K D for binding of those peptides to N-SH2 WT vs N-SH2 T42A . The dotted line indicates where EC 50 for SHP2 WT equals EC 50 for SHP2 T42A . Peptides with a large fold-change in binding affinity (larger bubble) have a large fold-change in EC 50 values for SHP2 T42A over SHP2 WT (distance from dotted line). All EC 50 values can be found in .

Article Snippet: The SHP2 full-length, wild-type gene used as the template for all SHP2 constructs in this study was cloned from the pGEX-4TI SHP2 WT plasmid, which was a generous gift from Ben Neel (Addgene plasmid #8322) ( ).

Techniques: Activation Assay, Incubation, Phospho-proteomics, De-Phosphorylation Assay, Comparison, Binding Assay

Journal: bioRxiv

Article Title: The pathogenic T42A mutation in SHP2 rewires the interaction specificity of its N-terminal regulatory domain

doi: 10.1101/2023.07.10.548257

Figure Lengend Snippet: ( A ) Scheme depicting the co-immunoprecipitation experiments with SHP2 and either Gab1, Gab2, or PD-1 in HEK293 cells. SHP2 co-immunoprecipitation results with ( B ) Gab1, ( C ) Gab2, and ( D ) PD-1. For ( B ), ( C ), ( D ), N = 2, 3, and 4 independent cell transfections, respectively. Co-immunoprecipitation of Gab1/Gab2 was detected using an α-FLAG antibody and PD-1 was detected using a PD-1-specific antibody. Co-immunoprecipitation levels of each protein in T42A samples relative to wild-type are normalized for expression level and shown as bar graphs. ( E ) Schematic depiction of EGF stimulation and phospho-Erk signaling experiments in the presence of co-expressed SHP2 and either Gab1 or Gab2. ( F ) Comparison of phospho-Erk levels in response to EGF stimulation in cells expressing Gab1 and either SHP2 WT or SHP2 T42A . N = 4 independent cell transfections and separate stimulations. A paired, one-tailed t-test was used to test for significance. ( G ) Comparison of phospho-Erk levels in response to EGF stimulation in cells expressing Gab2 and either SHP2 WT or SHP2 T42A . N = 3 independent cell transfections and separate stimulations. A paired, one-tailed t-test was used to test for significance. For panels (F) and (G), the bar graphs below the blots indicate phospho-Erk levels, normalized to total Erk levels, relative to the highest p-Erk signal in SHP2 WT time course (2 minutes).

Article Snippet: The SHP2 full-length, wild-type gene used as the template for all SHP2 constructs in this study was cloned from the pGEX-4TI SHP2 WT plasmid, which was a generous gift from Ben Neel (Addgene plasmid #8322) ( ).

Techniques: Immunoprecipitation, Transfection, Expressing, Comparison, One-tailed Test