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The GTPase-activating protein p120RasGAP has an evolutionarily conserved “FLVR-unique” SH2 domain

Open AccessPublished:June 15, 2020DOI:https://doi.org/10.1074/jbc.RA120.013976
      The Src homology 2 (SH2) domain has a highly conserved architecture that recognizes linear phosphotyrosine motifs and is present in a wide range of signaling pathways across different evolutionary taxa. A hallmark of SH2 domains is the arginine residue in the conserved FLVR motif that forms a direct salt bridge with bound phosphotyrosine. Here, we solve the X-ray crystal structures of the C-terminal SH2 domain of p120RasGAP (RASA1) in its apo and peptide-bound form. We find that the arginine residue in the FLVR motif does not directly contact pTyr1087 of a bound phosphopeptide derived from p190RhoGAP; rather, it makes an intramolecular salt bridge to an aspartic acid. Unexpectedly, coordination of phosphotyrosine is achieved by a modified binding pocket that appears early in evolution. Using isothermal titration calorimetry, we find that substitution of the FLVR arginine R377A does not cause a significant loss of phosphopeptide binding, but rather a tandem substitution of R398A (SH2 position βD4) and K400A (SH2 position βD6) is required to disrupt the binding. These results indicate a hitherto unrecognized diversity in SH2 domain interactions with phosphotyrosine and classify the C-terminal SH2 domain of p120RasGAP as “FLVR-unique.”
      Src homology 2 (SH2) domains are the best studied pTyr-binding protein scaffolding domains (
      • Liu B.A.
      • Jablonowski K.
      • Raina M.
      • Arcé M.
      • Pawson T.
      • Nash P.D.
      The human and mouse complement of SH2 domain proteins: establishing the boundaries of phosphotyrosine signaling.
      ). They were originally identified within oncogenes of avian RNA viruses and are the second of four homology domains in Src family kinases (
      • Sadowski I.
      • Stone J.C.
      • Pawson T.
      A noncatalytic domain conserved among cytoplasmic protein-tyrosine kinases modifies the kinase function and transforming activity of Fujinami sarcoma virus P130gag-fps.
      ,
      • Pawson T.
      Specificity in signal transduction: from phosphotyrosine–SH2 domain interactions to complex cellular systems.
      ,
      • Nash P.D.
      Why modules matter.
      ) critical for both autoregulation and targeting (
      • Pawson T.
      Specificity in signal transduction: from phosphotyrosine–SH2 domain interactions to complex cellular systems.
      ,
      • Shah N.H.
      • Amacher J.F.
      • Nocka L.M.
      • Kuriyan J.
      The Src module: an ancient scaffold in the evolution of cytoplasmic tyrosine kinases.
      ,
      • Boggon T.J.
      • Eck M.J.
      Structure and regulation of Src family kinases.
      ). Following identification, SH2 domains rapidly revealed their presence in many kinases, phosphatases, small GTPase regulators, adaptor proteins, and transcription factors (
      • Liu B.A.
      • Jablonowski K.
      • Raina M.
      • Arcé M.
      • Pawson T.
      • Nash P.D.
      The human and mouse complement of SH2 domain proteins: establishing the boundaries of phosphotyrosine signaling.
      ,
      • Schlessinger J.
      • Lemmon M.A.
      SH2 and PTB domains in tyrosine kinase signaling.
      ,
      • Liu B.A.
      • Engelmann B.W.
      • Nash P.D.
      The language of SH2 domain interactions defines phosphotyrosine-mediated signal transduction.
      ), and they are now known to integrate signals from the 90 tyrosine kinases to help regulate myriad signaling pathways and cellular processes (
      • Liu B.A.
      • Jablonowski K.
      • Raina M.
      • Arcé M.
      • Pawson T.
      • Nash P.D.
      The human and mouse complement of SH2 domain proteins: establishing the boundaries of phosphotyrosine signaling.
      ,
      • Schlessinger J.
      • Lemmon M.A.
      SH2 and PTB domains in tyrosine kinase signaling.
      ,
      • Liu B.A.
      • Engelmann B.W.
      • Nash P.D.
      The language of SH2 domain interactions defines phosphotyrosine-mediated signal transduction.
      ,
      • Kaneko T.
      • Huang H.
      • Cao X.
      • Li X.
      • Li C.
      • Voss C.
      • Sidhu S.S.
      • Li S.S.
      Superbinder SH2 domains act as antagonists of cell signaling.
      ). In total, over 120 SH2 domains have been identified in the human genome distributed among ∼110 proteins (
      • Liu B.A.
      • Nash P.D.
      Evolution of SH2 domains and phosphotyrosine signalling networks.
      ,
      • Suga H.
      • Torruella G.
      • Burger G.
      • Brown M.W.
      • Ruiz-Trillo I.
      Earliest holozoan expansion of phosphotyrosine signaling.
      ). Almost all of the members of this broad family retain a primary functional role: to bind short linear phosphotyrosine motifs (
      • Songyang Z.
      • Shoelson S.E.
      • Chaudhuri M.
      • Gish G.
      • Pawson T.
      • Haser W.G.
      • King F.
      • Roberts T.
      • Ratnofsky S.
      • Lechleider R.J.
      SH2 domains recognize specific phosphopeptide sequences.
      ,
      • Huang H.
      • Li L.
      • Wu C.
      • Schibli D.
      • Colwill K.
      • Ma S.
      • Li C.
      • Roy P.
      • Ho K.
      • Songyang Z.
      • Pawson T.
      • Gao Y.
      • Li S.S.
      Defining the specificity space of the human SRC homology 2 domain.
      ,
      • Songyang Z.
      • Shoelson S.E.
      • McGlade J.
      • Olivier P.
      • Pawson T.
      • Bustelo X.R.
      • Barbacid M.
      • Sabe H.
      • Hanafusa H.
      • Yi T.
      Specific motifs recognized by the SH2 domains of Csk 3BP2, fps/fes, Grb-2, HCP, SHC, Syk and Vav.
      ). The structural conformation that allows this binding is extremely well-conserved across the family.
      The SH2 domain comprises a central antiparallel seven-stranded β-sheet (βA to βG) sandwiched between two α-helices (αA and αB) (
      • Waksman G.
      • Kominos D.
      • Robertson S.C.
      • Pant N.
      • Baltimore D.
      • Birge R.B.
      • Cowburn D.
      • Hanafusa H.
      • Mayer B.J.
      • Overduin M.
      • Resh M.D.
      • Rios C.B.
      • Silverman L.
      • Kuriyan J.
      Crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides.
      ,
      • Eck M.J.
      • Shoelson S.E.
      • Harrison S.C.
      Recognition of a high-affinity phosphotyrosyl peptide by the Src homology-2 domain of p56lck.
      ,
      • Overduin M.
      • Rios C.B.
      • Mayer B.J.
      • Baltimore D.
      • Cowburn D.
      Three-dimensional solution structure of the src homology 2 domain of c-abl.
      ,
      • Waksman G.
      • Shoelson S.E.
      • Pant N.
      • Cowburn D.
      • Kuriyan J.
      Binding of a high affinity phosphotyrosyl peptide to the Src SH2 domain: crystal structures of the complexed and peptide-free forms.
      ) (nomenclature defined by Eck et al. (
      • Eck M.J.
      • Shoelson S.E.
      • Harrison S.C.
      Recognition of a high-affinity phosphotyrosyl peptide by the Src homology-2 domain of p56lck.
      )). This creates two binding sites—a deep pocket and a shallow cleft—within the ∼100-amino acid globular domain. Linear phosphotyrosine peptides bind perpendicular to the β-sheet in a two-pronged interaction with the pocket and cleft, a mode of binding that is reliably mirrored in cells and in the purified setting (
      • Nash P.D.
      Why modules matter.
      ,
      • Songyang Z.
      • Shoelson S.E.
      • McGlade J.
      • Olivier P.
      • Pawson T.
      • Bustelo X.R.
      • Barbacid M.
      • Sabe H.
      • Hanafusa H.
      • Yi T.
      Specific motifs recognized by the SH2 domains of Csk 3BP2, fps/fes, Grb-2, HCP, SHC, Syk and Vav.
      ). The deep pocket binds the pTyr residue, which is captured by electrostatic interactions with an arginine residue invariant in all pTyr-binding SH2 domains (
      • Kaneko T.
      • Huang H.
      • Cao X.
      • Li X.
      • Li C.
      • Voss C.
      • Sidhu S.S.
      • Li S.S.
      Superbinder SH2 domains act as antagonists of cell signaling.
      ). In contrast, the shallow cleft binds residues C-terminal to the pTyr and varies considerably between SH2 domains allowing specificity determination (
      • Liu B.A.
      • Engelmann B.W.
      • Nash P.D.
      The language of SH2 domain interactions defines phosphotyrosine-mediated signal transduction.
      ,
      • Huang H.
      • Li L.
      • Wu C.
      • Schibli D.
      • Colwill K.
      • Ma S.
      • Li C.
      • Roy P.
      • Ho K.
      • Songyang Z.
      • Pawson T.
      • Gao Y.
      • Li S.S.
      Defining the specificity space of the human SRC homology 2 domain.
      ,
      • Kaneko T.
      • Huang H.
      • Zhao B.
      • Li L.
      • Liu H.
      • Voss C.K.
      • Wu C.
      • Schiller M.R.
      • Li S.S.
      Loops govern SH2 domain specificity by controlling access to binding pockets.
      ,
      • Liu H.
      • Huang H.
      • Voss C.
      • Kaneko T.
      • Qin W.T.
      • Sidhu S.
      • Li S.S.
      Surface loops in a single SH2 domain are capable of encoding the spectrum of specificity of the SH2 family.
      ). This two-pronged interaction also dictates a consistent orientation of the bound phosphopeptide N to C terminus with respect to the SH2 domain (
      • Waksman G.
      • Shoelson S.E.
      • Pant N.
      • Cowburn D.
      • Kuriyan J.
      Binding of a high affinity phosphotyrosyl peptide to the Src SH2 domain: crystal structures of the complexed and peptide-free forms.
      ). In v-Src, the deep pocket arginine residue is part of the Phe172-Leu173-Val174-Arg175 sequence, or “FLVR motif” (
      • Waksman G.
      • Kominos D.
      • Robertson S.C.
      • Pant N.
      • Baltimore D.
      • Birge R.B.
      • Cowburn D.
      • Hanafusa H.
      • Mayer B.J.
      • Overduin M.
      • Resh M.D.
      • Rios C.B.
      • Silverman L.
      • Kuriyan J.
      Crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides.
      ), and arginine at this position (notated as the fifth residue on strand βB, βB5) is conserved in 117 of 120 human SH2 domains (
      • Kaneko T.
      • Huang H.
      • Cao X.
      • Li X.
      • Li C.
      • Voss C.
      • Sidhu S.S.
      • Li S.S.
      Superbinder SH2 domains act as antagonists of cell signaling.
      ). Multiple lines of evidence indicate that the FLVR arginine is required for SH2–pTyr binding. To date, all structural studies of SH2 domain interactions with pTyr have demonstrated direct interactions between the FLVR arginine and pTyr. Further, the binding affinity of unphosphorylated peptides is on the order of 1000-fold weaker than phosphopeptides (
      • Waksman G.
      • Kominos D.
      • Robertson S.C.
      • Pant N.
      • Baltimore D.
      • Birge R.B.
      • Cowburn D.
      • Hanafusa H.
      • Mayer B.J.
      • Overduin M.
      • Resh M.D.
      • Rios C.B.
      • Silverman L.
      • Kuriyan J.
      Crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides.
      ,
      • Waksman G.
      • Shoelson S.E.
      • Pant N.
      • Cowburn D.
      • Kuriyan J.
      Binding of a high affinity phosphotyrosyl peptide to the Src SH2 domain: crystal structures of the complexed and peptide-free forms.
      ). Additionally, the FLVR arginine was found to be the primary contributor of binding free energy (
      • Waksman G.
      • Kumaran S.
      • Lubman O.
      SH2 domains: role, structure and implications for molecular medicine.
      ,
      • Bradshaw J.M.
      • Mitaxov V.
      • Waksman G.
      Investigation of phosphotyrosine recognition by the SH2 domain of the Src kinase.
      ). Consequently, mutations of this residue are invariably used to generate a “dead” SH2 domain (
      • Bibbins K.B.
      • Boeuf H.
      • Varmus H.E.
      Binding of the Src SH2 domain to phosphopeptides is determined by residues in both the SH2 domain and the phosphopeptides.
      ,
      • Mayer B.J.
      • Jackson P.K.
      • Van Etten R.A.
      • Baltimore D.
      Point mutations in the abl SH2 domain coordinately impair phosphotyrosine binding in vitro and transforming activity in vivo.
      ).
      p120RasGAP (RASA1, RasGAP, Ras GTPase-activating protein 1) was one of the first SH2 domain proteins to be identified (
      • Trahey M.
      • McCormick F.
      A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants.
      ,
      • Vogel U.S.
      • Dixon R.A.
      • Schaber M.D.
      • Diehl R.E.
      • Marshall M.S.
      • Scolnick E.M.
      • Sigal I.S.
      • Gibbs J.B.
      Cloning of bovine GAP and its interaction with oncogenic ras p21.
      ,
      • Trahey M.
      • Wong G.
      • Halenbeck R.
      • Rubinfeld B.
      • Martin G.A.
      • Ladner M.
      • Long C.M.
      • Crosier W.J.
      • Watt K.
      • Koths K.
      Molecular cloning of two types of GAP complementary DNA from human placenta.
      ) and was the first GTPase-activating protein (GAP) to be discovered (
      • Trahey M.
      • McCormick F.
      A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants.
      ,
      • Bernards A.
      • Settleman J.
      GAP control: regulating the regulators of small GTPases.
      ,
      • Bos J.L.
      • Rehmann H.
      • Wittinghofer A.
      GEFs and GAPs: critical elements in the control of small G proteins.
      ,
      • McCormick F.
      • Adari H.
      • Trahey M.
      • Halenbeck R.
      • Koths K.
      • Martin G.A.
      • Crosier W.J.
      • Watt K.
      • Rubinfeld B.
      • Wong G.
      Interaction of ras p21 proteins with GTPase activating protein.
      ). It is ubiquitously expressed and required for life, because animals without p120RasGAP have major vascular defects (
      • Henkemeyer M.
      • Rossi D.J.
      • Holmyard D.P.
      • Puri M.C.
      • Mbamalu G.
      • Harpal K.
      • Shih T.S.
      • Jacks T.
      • Pawson T.
      Vascular system defects and neuronal apoptosis in mice lacking ras GTPase-activating protein.
      ,
      • Pamonsinlapatham P.
      • Hadj-Slimane R.
      • Lepelletier Y.
      • Allain B.
      • Toccafondi M.
      • Garbay C.
      • Raynaud F.
      p120-Ras GTPase activating protein (RasGAP): a multi-interacting protein in downstream signaling.
      ). p120RasGAP contains two SH2 domains at its N terminus that sandwich an SH3 domain, followed by PH and C2 domains for membrane recruitment and a RasGAP domain (Fig. 1A) (
      • McCormick F.
      • Adari H.
      • Trahey M.
      • Halenbeck R.
      • Koths K.
      • Martin G.A.
      • Crosier W.J.
      • Watt K.
      • Rubinfeld B.
      • Wong G.
      Interaction of ras p21 proteins with GTPase activating protein.
      ,
      • Adari H.
      • Lowy D.R.
      • Willumsen B.M.
      • Der C.J.
      • McCormick F.
      Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain.
      ,
      • Laskowski R.A.
      PDBsum: summaries and analyses of PDB structures.
      ,
      • Meyer P.A.
      • Socias S.
      • Key J.
      • Ransey E.
      • Tjon E.C.
      • Buschiazzo A.
      • Lei M.
      • Botka C.
      • Withrow J.
      • Neau D.
      • Rajashankar K.
      • Anderson K.S.
      • Baxter R.H.
      • Blacklow S.C.
      • Boggon T.J.
      • et al.
      Data publication with the structural biology data grid supports live analysis.
      ). The SH2 domains are used to directly interact with multiple phosphorylated binding partners including the p190RhoGAPs, the Eph receptor tyrosine kinases, and the Dok scaffolding proteins (
      • Pamonsinlapatham P.
      • Hadj-Slimane R.
      • Lepelletier Y.
      • Allain B.
      • Toccafondi M.
      • Garbay C.
      • Raynaud F.
      p120-Ras GTPase activating protein (RasGAP): a multi-interacting protein in downstream signaling.
      ). These interactions are correlated with altered RasGAP activity and consequently with regulation of Ras signaling (
      • Moran M.F.
      • Polakis P.
      • McCormick F.
      • Pawson T.
      • Ellis C.
      Protein-tyrosine kinases regulate the phosphorylation, protein interactions, subcellular distribution, and activity of p21ras GTPase-activating protein.
      ,
      • Bryant S.S.
      • Briggs S.
      • Smithgall T.E.
      • Martin G.A.
      • McCormick F.
      • Chang J.H.
      • Parsons S.J.
      • Jove R.
      Two SH2 domains of p120 Ras GTPase-activating protein bind synergistically to tyrosine phosphorylated p190 Rho GTPase-activating protein.
      ,
      • Dail M.
      • Richter M.
      • Godement P.
      • Pasquale E.B.
      Eph receptors inactivate R-Ras through different mechanisms to achieve cell repulsion.
      ,
      • Kulkarni S.V.
      • Gish G.
      • van der Geer P.
      • Henkemeyer M.
      • Pawson T.
      Role of p120 Ras-GAP in directed cell movement.
      ,
      • van der Geer P.
      • Henkemeyer M.
      • Jacks T.
      • Pawson T.
      Aberrant Ras regulation and reduced p190 tyrosine phosphorylation in cells lacking p120-Gap.
      ). The p120RasGAP SH2 domains therefore play critical roles in spatial–temporal regulation of Ras signaling by their interactions with this wide array of partners.
      Figure thumbnail gr1
      Figure 1Structure of p120RasGAP C-terminal SH2 domain. A, schematic of p120RasGAP interaction with p190RhoGAP. Domains are depicted, and the region co-crystallized is shown in a dashed box. B, structure of apo p120RasGAP C-SH2 indicating secondary structure elements and locations of the pTyr-binding pocket and specificity cleft. C, structure of p120RasGAP C-SH2 in complex with a synthesized phosphopeptide corresponding to p190RhoGAP-A residues 1086DpYAEPMDA1093. Simulated annealing omit FobsFcalc difference map contoured at 3σ RMSD is shown. D, surface electrostatics for p120RasGAP C-SH2 with p190RhoGAP-A peptide. E, interactions observed between p120RasGAP C-SH2 (green) and p190 phosphopeptide (yellow) defined by PDBsum (
      • Meyer P.A.
      • Socias S.
      • Key J.
      • Ransey E.
      • Tjon E.C.
      • Buschiazzo A.
      • Lei M.
      • Botka C.
      • Withrow J.
      • Neau D.
      • Rajashankar K.
      • Anderson K.S.
      • Baxter R.H.
      • Blacklow S.C.
      • Boggon T.J.
      • et al.
      Data publication with the structural biology data grid supports live analysis.
      ). Hydrogen bonds are shown by red lines; nonbonded elements are shown by dashed lines. F, alignment of secondary structure features for p120RasGAP C-SH2, compared with p120RasGAP N-SH2 (PDB code 6PXC) (
      • van der Geer P.
      • Henkemeyer M.
      • Jacks T.
      • Pawson T.
      Aberrant Ras regulation and reduced p190 tyrosine phosphorylation in cells lacking p120-Gap.
      ), and Src (PDB code 1SPS) (
      • Waksman G.
      • Shoelson S.E.
      • Pant N.
      • Cowburn D.
      • Kuriyan J.
      Binding of a high affinity phosphotyrosyl peptide to the Src SH2 domain: crystal structures of the complexed and peptide-free forms.
      ). β-Strands are highlighted yellow, and α-helices are highlighted orange. Residues that contact p190 phosphopeptide as defined by PDBsum are indicated with bold and underlined text. Residues discussed in the text are indicated.
      The SH2 domains of p120RasGAP are thought to be canonical SH2 domains that mediate direct interactions with pTyr residues using their FLVR arginine residue, which is conserved in both domains. We recently confirmed this to be the case for the N-terminal SH2 domain (
      • Jaber Chehayeb R.
      • Stiegler A.L.
      • Boggon T.J.
      Crystal structures of p120RasGAP N-terminal SH2 domain in its apo form and in complex with a p190RhoGAP phosphotyrosine peptide.
      ); however, in contrast we now demonstrate that the C-terminal SH2 domain of p120RasGAP is a novel SH2 domain. Using X-ray crystallography, we find that the βB5 FLVR arginine does not contact phosphotyrosine pTyr1087 of a bound p190RhoGAP peptide; instead, it makes a salt bridge to residue Asp380. Analysis of 715 aligned SH2 domain structures shows that this position of the FLVR arginine is rare, and point mutagenesis demonstrates that the FLVR arginine is not required for pTyr binding. Instead, pTyr binding is mediated by an alternate array of residues, including an unusual Arg398 at the βD4 position. These findings demonstrate that the C-terminal SH2 domain of p120RasGAP is “FLVR-unique.”

      Results

      p120RasGAP interaction with p190RhoGAP is critical for recruitment of RhoGAP activity to sites of cell adhesion and for suppression of RasGAP activity at these locations (
      • Moran M.F.
      • Polakis P.
      • McCormick F.
      • Pawson T.
      • Ellis C.
      Protein-tyrosine kinases regulate the phosphorylation, protein interactions, subcellular distribution, and activity of p21ras GTPase-activating protein.
      ,
      • Bryant S.S.
      • Briggs S.
      • Smithgall T.E.
      • Martin G.A.
      • McCormick F.
      • Chang J.H.
      • Parsons S.J.
      • Jove R.
      Two SH2 domains of p120 Ras GTPase-activating protein bind synergistically to tyrosine phosphorylated p190 Rho GTPase-activating protein.
      ,
      • Chang J.H.
      • Gill S.
      • Settleman J.
      • Parsons S.J.
      c-Src regulates the simultaneous rearrangement of actin cytoskeleton, p190RhoGAP, and p120RasGAP following epidermal growth factor stimulation.
      ,
      • Sfakianos M.K.
      • Eisman A.
      • Gourley S.L.
      • Bradley W.D.
      • Scheetz A.J.
      • Settleman J.
      • Taylor J.R.
      • Greer C.A.
      • Williamson A.
      • Koleske A.J.
      Inhibition of Rho via Arg and p190RhoGAP in the postnatal mouse hippocampus regulates dendritic spine maturation, synapse and dendrite stability, and behavior.
      ,
      • Bradley W.D.
      • Hernández S.E.
      • Settleman J.
      • Koleske A.J.
      Integrin signaling through Arg activates p190RhoGAP by promoting its binding to p120RasGAP and recruitment to the membrane.
      ). Contacts between p120RasGAP and p190RhoGAP are mediated by the two SH2 domains of p120RasGAP (termed N-SH2 and C-SH2), which bind to phosphorylated tyrosine residues Tyr1087 and Tyr1105 of p190RhoGAP (
      • Hu K.Q.
      • Settleman J.
      Tandem SH2 binding sites mediate the RasGAP-RhoGAP interaction: a conformational mechanism for SH3 domain regulation.
      ,
      • Hernández S.E.
      • Settleman J.
      • Koleske A.J.
      Adhesion-dependent regulation of p190RhoGAP in the developing brain by the Abl-related gene tyrosine kinase.
      ); both pTyr residues reside in the preferred pYXXP sequence recognized by both SH2 domains of p120RasGAP (
      • Huang H.
      • Li L.
      • Wu C.
      • Schibli D.
      • Colwill K.
      • Ma S.
      • Li C.
      • Roy P.
      • Ho K.
      • Songyang Z.
      • Pawson T.
      • Gao Y.
      • Li S.S.
      Defining the specificity space of the human SRC homology 2 domain.
      ,
      • Kaneko T.
      • Huang H.
      • Zhao B.
      • Li L.
      • Liu H.
      • Voss C.K.
      • Wu C.
      • Schiller M.R.
      • Li S.S.
      Loops govern SH2 domain specificity by controlling access to binding pockets.
      ). We previously reported the crystal structure of N-SH2 of p120RasGAP and investigated its interaction with a p190RhoGAP pTyr1105 phosphopeptide, which revealed a canonical SH2 domain structure and pTyr peptide-binding mode with Kd of 0.3 ± 0.1 μm (
      • Jaber Chehayeb R.
      • Stiegler A.L.
      • Boggon T.J.
      Crystal structures of p120RasGAP N-terminal SH2 domain in its apo form and in complex with a p190RhoGAP phosphotyrosine peptide.
      ). In the present study, we aim to determine the structure and pTyr-binding mode of C-SH2 of p120RasGAP. Toward this end, we expressed and purified the isolated C-SH2 domain recombinantly and performed crystallization studies. We obtained crystals of the apo form of p120RasGAP C-SH2 and solved the structure to 1.5 Å resolution. To examine the structural basis of pTyr binding, we also solved a 1.5 Å co-crystal structure of p120RasGAP C-SH2 with a pTyr1087 p190RhoGAP phosphopeptide.
      The structure of the apo form of C-SH2 (Fig. 1B and Table 1) reveals a typical overall SH2 fold that is most similar to the p120RasGAP N-SH2 (PDB code 6PXC) (
      • Jaber Chehayeb R.
      • Stiegler A.L.
      • Boggon T.J.
      Crystal structures of p120RasGAP N-terminal SH2 domain in its apo form and in complex with a p190RhoGAP phosphotyrosine peptide.
      ) with root-mean-squared deviation (RMSD) of ∼1.3 Å over 94 Cα atoms and 31% sequence identity; it is also highly similar to the C-terminal SH2 domain of PLCγ-1 (
      • McKercher M.A.
      • Guan X.
      • Tan Z.
      • Wuttke D.S.
      Multimodal recognition of diverse peptides by the C-terminal SH2 domain of phospholipase C-γ1 protein.
      ) (PDB code 5TQ1; RMSD, ∼1.8 Å over 101 Cα atoms, 27% identity) and Nck2 (
      • Frese S.
      • Schubert W.D.
      • Findeis A.C.
      • Marquardt T.
      • Roske Y.S.
      • Stradal T.E.
      • Heinz D.W.
      The phosphotyrosine peptide binding specificity of Nck1 and Nck2 Src homology 2 domains.
      ) (PDB code 2CIA; RMSD, 1.1 Å over 93 Cα atoms, 25%). The two-pronged pTyr pocket and specificity cleft interaction sites (
      • Eck M.J.
      • Shoelson S.E.
      • Harrison S.C.
      Recognition of a high-affinity phosphotyrosyl peptide by the Src homology-2 domain of p56lck.
      ,
      • Waksman G.
      • Shoelson S.E.
      • Pant N.
      • Cowburn D.
      • Kuriyan J.
      Binding of a high affinity phosphotyrosyl peptide to the Src SH2 domain: crystal structures of the complexed and peptide-free forms.
      ,
      • Bradshaw J.M.
      • Grucza R.A.
      • Ladbury J.E.
      • Waksman G.
      Probing the “two-pronged plug two-holed socket” model for the mechanism of binding of the Src SH2 domain to phosphotyrosyl peptides: a thermodynamic study.
      ) are clearly visible in p120RasGAP C-SH2 (Fig. 1B), with a strongly electropositive deep pocket and a hydrophobic shallow cleft both available to interact with a linear peptide. We then determined the 1.5 Å co-crystal structure of C-SH2 in complex with a synthetic phosphopeptide corresponding to p190RhoGAP residues 1086–1092 (1086DpYAEPMD1092) (Fig. 1C and Table 1). The structure of C-SH2 in complex with phosphopeptide is experimentally similar to the apo form (RMSDs 0.9 Å over 105 Cα atoms and 1.0 Å over 102 Cα atoms for chains A and B, respectively). As expected, the N- to C-terminal orientation of the bound phosphopeptide is consistent with other SH2 domain structures (
      • Waksman G.
      • Shoelson S.E.
      • Pant N.
      • Cowburn D.
      • Kuriyan J.
      Binding of a high affinity phosphotyrosyl peptide to the Src SH2 domain: crystal structures of the complexed and peptide-free forms.
      ) and is dictated by placing pTyr1087 into the deep electrostatic pocket and Pro1090 into the shallow cleft, recapitulating canonical electrostatic and van der Waals interactions (Fig. 1D). The residues of p120RasGAP C-SH2 that contact the phosphopeptide are generally similar to previously studied SH2–phosphopeptide interactions (
      • Eck M.J.
      • Shoelson S.E.
      • Harrison S.C.
      Recognition of a high-affinity phosphotyrosyl peptide by the Src homology-2 domain of p56lck.
      ,
      • Waksman G.
      • Shoelson S.E.
      • Pant N.
      • Cowburn D.
      • Kuriyan J.
      Binding of a high affinity phosphotyrosyl peptide to the Src SH2 domain: crystal structures of the complexed and peptide-free forms.
      ,
      • Bradshaw J.M.
      • Grucza R.A.
      • Ladbury J.E.
      • Waksman G.
      Probing the “two-pronged plug two-holed socket” model for the mechanism of binding of the Src SH2 domain to phosphotyrosyl peptides: a thermodynamic study.
      ) (Fig. 1, E and F). Curiously, however, we observed that the FLVR motif arginine residue, Arg377, is oriented differently to other SH2 domains in both the apo and peptide-bound crystal forms. We thus investigated this major structural difference in detail.
      Table 1Data collection and refinement statistics
      p120RasGAP C-SH2p120RasGAP C-SH2 bound to p190RhoGAP phosphopeptide
      Data collection
          PDB accession code6WAX6WAY
          Wavelength (Å)0.979200.97920
          Resolution range (Å)50–1.50 (1.55–1.50)30–1.50 (1.55–1.50)
          Space groupP 21 21 21C 2 2 21
          Cell dimensions
              a, b, c (Å)52.5, 65.6, 71.663.1, 83.9, 54.2
              α, β, γ (°)90, 90, 9090, 90, 90
          Unique reflections40,19423,209
          Multiplicity11.7 (7.0)21.2 (8.6)
          Completeness (%)100 (99.9)100 (99.8)
          Mean II24.1 (2.0)23.6 (2.0)
          Wilson B factor (Å2)22.618.7
          Rpim (%)2.8 (40.7)3.1 (29.3)
          CC½0.993 (0.689)0.995 (0.806)
          CC*0.998 (0.903)0.999 (0.945)
      Refinement
          Resolution range (Å)48.4–1.50 (1.54–1.50)27.3–1.50 (1.57–1.50)
          Reflections used in refinement40,121 (2590)23,164 (2606)
          Reflections used for Rfree1943 (126)1148 (130)
          Reflections used for Rfree (%)4.844.96
          Rwork (%)17.9 (28.5)16.5 (24.8)
          Rfree (%)19.7 (29.7)19.0 (27.8)
          No. of non-hydrogen atoms20771188
              SH2 domains1800921
              Peptide70
              Water243197
              Other solvent34
          No. protein residues207 (105 chain A, 102 chain B)116 (106 SH2, 10 peptide)
          Residue
              p120RasGAP SH2340–444, 343–444339–444
               p190RhoGAP phosphopeptide1085–1094
          RMSD
              Bond lengths (Å)0.0060.005
              Bond angles (°)0.8720.809
          Ramachandran plot (%)
              Favored98.098.1
              Allowed2.01.9
              Outliers00
          Rotamer outliers (%)1.00
          MolProbity clashscore5.272.08
          Average B factor (Å2)32.627.5
              SH2 domains30.924.4
              Copies A, B29.6, 32.3
              Peptide34.9
              Water42.339.3
      In pTyr-binding SH2 domains, the conserved FLVR arginine at βB5 is required to form the base of the electropositive pocket and to hydrogen bond to pTyr (
      • Waksman G.
      • Kumaran S.
      • Lubman O.
      SH2 domains: role, structure and implications for molecular medicine.
      ,
      • Bradshaw J.M.
      • Mitaxov V.
      • Waksman G.
      Investigation of phosphotyrosine recognition by the SH2 domain of the Src kinase.
      ). Unexpectedly, in the p120RasGAP C-SH2/phosphopeptide co-crystal structure, the FLVR arginine Arg377 does not contact pTyr directly. Instead, Arg377 makes a salt bridge to Asp380 (at position BC1) (Fig. 2A), an interaction that is also observed in the apo C-SH2 structure (Fig. 2B). Therefore, unlike typical SH2 domains, C-SH2 Arg377 does not orient toward the phosphotyrosine-binding site in the absence of pTyr, nor does it undergo conformational change to contact pTyr directly in the presence of phosphopeptide. This unique orientation contrasts with p120RasGAP's N-SH2 domain, which binds phosphotyrosine in a canonical fashion (Fig. 2C) (
      • Jaber Chehayeb R.
      • Stiegler A.L.
      • Boggon T.J.
      Crystal structures of p120RasGAP N-terminal SH2 domain in its apo form and in complex with a p190RhoGAP phosphotyrosine peptide.
      ).
      Figure thumbnail gr2
      Figure 2An unusual conformation for the FLVR arginine of p120RasGAP C-SH2. A, crystal structure of phosphopeptide-bound p120RasGAP C-SH2 showing the FLVR arginine Arg377, Asp380, and Tyr389 and p190RhoGAP pTyr1087. B, crystal structure of apo p120RasGAP C-SH2. C, crystal structure of phosphopeptide-bound p120RasGAP N-SH2 (PDB code 2PXC) (
      • van der Geer P.
      • Henkemeyer M.
      • Jacks T.
      • Pawson T.
      Aberrant Ras regulation and reduced p190 tyrosine phosphorylation in cells lacking p120-Gap.
      ) showing the equivalent residues and phosphotyrosine. D, superposition of FLVR motif arginines from 592 Dali-aligned SH2 domains. E, crystal structure of Src family kinase Fyn (PDB code 4U1P) shows residue BC1 (Glu53) caps helix αB.
      To assess whether the salt-bridge engagement of FLVR Arg377 with Asp380 in p120RasGAP C-SH2 is unique among determined SH2 domains structures, we conducted in silico analysis. We find that such an orientation is rare among the 715 aligned SH2 domains identified by the DALI server (
      • Holm L.
      • Rosenstrom P.
      Dali server: conservation mapping in 3D.
      ). At the time of this analysis, there are only two other examples of FLVR arginine residues that form interactions with another residues within the SH2 domain itself: 1) in the STAT6 SH2 domain, the FLVR arginine makes salt-bridge contacts to both pTyr and Asp at position BC1 (
      • Li J.
      • Rodriguez J.P.
      • Niu F.
      • Pu M.
      • Wang J.
      • Hung L.W.
      • Shao Q.
      • Zhu Y.
      • Ding W.
      • Liu Y.
      • Da Y.
      • Yao Z.
      • Yang J.
      • Zhao Y.
      • Wei G.H.
      • et al.
      Structural basis for DNA recognition by STAT6.
      ); and 2) in the VAV SH2 domain when bound to an unphosphorylated tyrosine, the FLVR arginine hydrogen bonds to Gln at position BC1 (
      • Chen C.H.
      • Piraner D.
      • Gorenstein N.M.
      • Geahlen R.L.
      • Beth Post C.
      Differential recognition of syk-binding sites by each of the two phosphotyrosine-binding pockets of the Vav SH2 domain.
      ). The atypical orientation of Arg377 can be observed by superposition of aligned SH2 domains, in which the Arg377 side chain is pointed away from the pTyr-binding site, unlike the positions of a majority of the typical arginine side chains (Fig. 2D). The FLVR Arg of p120RasGAP C-SH2 is therefore unique among structures of SH2 domains.
      The molecular basis of this unique FLVR arginine orientation seems to be dictated by both the salt bridge to Asp380 and by steric interference by Tyr389 (residue βC5) (Fig. 2, B and C). Tyrosine at position βC5 is not observed in any other human SH2 domain and is usually a small amino acid: over the 120 human SH2 domains, residue βC5 is observed as Ser (63 of 120 times including N-SH2 of p120RasGAP), Thr (27 times), Ala (13 times), Cys (10 times), Gly (3 times), Val (2 times), Met (1 time), and Tyr (1 time, p120RasGAP C-SH2) (
      • Liu B.A.
      • Jablonowski K.
      • Raina M.
      • Arcé M.
      • Pawson T.
      • Nash P.D.
      The human and mouse complement of SH2 domain proteins: establishing the boundaries of phosphotyrosine signaling.
      ). Small amino acids at βC5 allow the FLVR arginine to engage the pTyr directly as observed in p120RasGAP N-SH2 bound to a phosphopeptide (Fig. 2C). At position BC1 (equivalent to Asp380 in p120RasGAP C-SH2), an acidic residue is frequently observed (Asp, 6 times and Glu, 38 times), but in many SH2 domains it caps helix αB (e.g. Glu179 in Fyn SH2; Fig. 2E). In contrast, in p120RasGAP C-SH2 a helix capping interaction by BC1 is not possible because the FLVR arginine guanidino group prevents close arrangement of helix αB and strand βB. The position of the FLVR arginine therefore seems to accommodate atypical orientations of other elements of the p120RasGAP C-SH2 domain.
      Upon inspection of the p120RasGAP C-SH2 structure in complex with phosphopeptide, we observe several additional unique features of pTyr binding in addition to the orientation of the FLVR arginine and presence of βC5 tyrosine. Across SH2 domains, three major interactions have been observed between the pTyr and its binding pocket: 1) the salt bridge between the pTyr and the FLVR motif βB5 arginine (Arg207 in N-SH2), 2) binding to a basic residue at either position αA2 (Arg188 in N-SH2) or βD6, and 3) binding to residues of the BC loop (between the βB and βC strands (Ser209 in N-SH2; Figs. 1F and 3A) (
      • Waksman G.
      • Kumaran S.
      • Lubman O.
      SH2 domains: role, structure and implications for molecular medicine.
      ,
      • Jaber Chehayeb R.
      • Stiegler A.L.
      • Boggon T.J.
      Crystal structures of p120RasGAP N-terminal SH2 domain in its apo form and in complex with a p190RhoGAP phosphotyrosine peptide.
      ,
      • Kaneko T.
      • Stogios P.J.
      • Ruan X.
      • Voss C.
      • Evdokimova E.
      • Skarina T.
      • Chung A.
      • Liu X.
      • Li L.
      • Savchenko A.
      • Ensminger A.W.
      • Li S.S.
      Identification and characterization of a large family of superbinding bacterial SH2 domains.
      ). Consistent with these interactions, p120RasGAP C-SH2 uses βD6 (Lys400) and residues in the BC loop (Ser379, Asn381, and Thr382; Fig. 3B). However, in contrast, p120RasGAP C-SH2 uses additional residues βC3 (Ser387) and a basic residue at position βD4 (Arg398) to bind pTyr (Figs. 1, E and F, and 3B). Arg398 at position βD4 is unique to p120RasGAP C-SH2; this residue is most commonly a histidine (82 of 120 human SH2 domains; His229 in N-SH2; Fig. 3A) (
      • Liu B.A.
      • Jablonowski K.
      • Raina M.
      • Arcé M.
      • Pawson T.
      • Nash P.D.
      The human and mouse complement of SH2 domain proteins: establishing the boundaries of phosphotyrosine signaling.
      ). The unusual binding site also manifests in the location of the phosphate—when phosphate atoms are mapped, C-SH2 binds to pTyr in a location divergent to all previously determined SH2 domains (Fig. 3C). The p120RasGAP C-SH2 pTyr-binding pocket therefore diverges from either the Src-like (where pTyr is coordinated by an Arg at position αA2) or SAP-like (where pTyr is coordinated by a basic residue at position βD6) pockets (
      • Kaneko T.
      • Stogios P.J.
      • Ruan X.
      • Voss C.
      • Evdokimova E.
      • Skarina T.
      • Chung A.
      • Liu X.
      • Li L.
      • Savchenko A.
      • Ensminger A.W.
      • Li S.S.
      Identification and characterization of a large family of superbinding bacterial SH2 domains.
      ). Sequence alignment of human SH2 domains suggests that no other SH2 domain harbors dual basic residues at positions βD4 and βD6 (1), potentially indicating an evolution of the site to compensate for the loss of direct FLVR arginine binding. Additionally, the amino–aromatic interaction typically contributed by the arginine at αA2 (15,16) is instead fulfilled by the unique Arg398 at βD4 in C-SH2 (Fig. S1).
      Figure thumbnail gr3
      Figure 3Phosphotyrosine-binding sites of p120RasGAP. A and B, detailed binding site interactions of the phosphotyrosine to p120RasGAP N-SH2 (PDB code 2PXC) (
      • van der Geer P.
      • Henkemeyer M.
      • Jacks T.
      • Pawson T.
      Aberrant Ras regulation and reduced p190 tyrosine phosphorylation in cells lacking p120-Gap.
      ) (A) and p120RasGAP C-SH2 (B). C, comparison of phosphotyrosine bound to p120RasGAP C-SH2 with the location of the phosphate atom in 245 phosphotyrosine-bound SH2 domains identified by the Dali server. Locations of the phosphates are shown as orange spheres. SH2 domains identified and superposed using by the Dali server (246 contain phosphotyrosine; 2BBU not included in analysis). D, conservation of key binding site residues over evolution and in human SH2 domains. pTyr-binding residues are colored red, and the FLVR arginine–Asp380 salt bridges are colored green.
      Many SH2–pTyr interactions have been studied in solution, and the measured binding affinities range in the 10−5 to 10−8m (10 μm to 10 nm), or −7 to −11 kcal/mol range (
      • Ladbury J.E.
      • Lemmon M.A.
      • Zhou M.
      • Green J.
      • Botfield M.C.
      • Schlessinger J.
      Measurement of the binding of tyrosyl phosphopeptides to SH2 domains: a reappraisal.
      ). We therefore used isothermal titration calorimetry (ITC) to measure the affinity of p120RasGAP C-SH2 with pTyr1087 phosphopeptide from p190RhoGAP and find the resultant Kd = 0.15 ± 0.04 μm, approximately −9 kcal/mol, to be within this expected range (Table 2 and Fig. S2). We next used ITC to assess the extent of binding of pTyr1087 phosphopeptide to C-SH2 mutants. For canonical SH2–pTyr interactions, the FLVR arginine is the primary contributor of binding free energy (
      • Waksman G.
      • Kumaran S.
      • Lubman O.
      SH2 domains: role, structure and implications for molecular medicine.
      ,
      • Bradshaw J.M.
      • Mitaxov V.
      • Waksman G.
      Investigation of phosphotyrosine recognition by the SH2 domain of the Src kinase.
      ), and mutation of this residue abrogates binding (
      • Bibbins K.B.
      • Boeuf H.
      • Varmus H.E.
      Binding of the Src SH2 domain to phosphopeptides is determined by residues in both the SH2 domain and the phosphopeptides.
      ,
      • Mayer B.J.
      • Jackson P.K.
      • Van Etten R.A.
      • Baltimore D.
      Point mutations in the abl SH2 domain coordinately impair phosphotyrosine binding in vitro and transforming activity in vivo.
      ). However, the unusual nature of the p120RasGAP C-SH2 suggests that the basis for its interaction with pTyr diverges from canonical SH2–pTyr interactions. We therefore tested whether Arg377 contributes to pTyr binding. Using ITC, we find that R377A mutation leads to slight reduction in affinity from WT Kd = 0.15 ± 0.04 μm to mutant Kd = 0.46 ± 0.11 μm but does not abrogate the interaction (Table 2 and Fig. S2). To our knowledge, this is the first SH2 domain studied to date that does not suffer a major loss of pTyr binding when the FLVR arginine residue is mutated. To further define the residues critical for pTyr binding, we introduced alanine point mutations at other basic residues that directly bind the pTyr: Arg398 at βD4 and Lys400 at βD6. Using ITC, we find that although the single mutants R398A and K400A only modestly impact pTyr peptide binding (Kd values of 0.31 ± 0.20 and 0.81 ± 0.15 μm, respectively), in combination, the double R398A/K400A mutant results in an ∼40-fold loss of binding (Kd of 6.24 ± 4.2 μm; Table 2 and Fig. S2). This indicates that unique among SH2 domains, the p120RasGAP C-SH2 requires multiple point mutations to abrogate pTyr binding.
      Table 2Isothermal titration calorimetry for p120RasGAP C-SH2 with 190RhoGAP pTyr1087 peptide
      p120RasGAP C-SH2 proteinnKdΔHΔSTΔSΔG
      μmkcal/molcal/mol*Kkcal/molkcal/mol
      WT0.91 ± 0.080.16 ± 0.05−14.3 ± 1.6−16.7 ± 6.0−5.0 ± 1.7−9.3 ± 0.2
      R377A0.90 ± 0.080.46 ± 0.11−17.3 ± 1.5−29.1 ± 5.0−8.7 ± 1.3−8.7 ± 0.1
      R398A0.87 ± 0.080.19 ± 0.02−16.9 ± 1.3−26.0 ± 4.0−7.7 ± 1.2−9.2 ± 0.4
      K400A0.92 ± 0.140.81 ± 0.15−12.3 ± 3.0−13.4 ± 8.0−4.0 ± 2.0−8.3 ± 0.1
      R398A/K400A1.10 ± 0.096.24 ± 4.00−13.2 ± 4.0−20.1 ± 14.0−6.0 ± 4.0−7.2 ± 0.5
      To assess the evolution of the p120RasGAP C-SH2 domain, we generated an alignment of 209 p120RasGAP sequences. We find that p120RasGAP is present in mammals, invertebrates, sponges, the choanoflagellates Monosiga brevicollis and Salpingoeca rosetta, the placozoa Trichoplax sp. H2, the orthonectid Intoshia linei, and in the single-celled eukaryote Capsapora owczarzaki (Figs. 1A and 3D and Fig. S3); notably, the domain architecture is preserved throughout phylogeny. We next examined the conservation of the unique phosphotyrosine-binding site. We observe that many of the phosphate-binding residues are well-conserved. However, we observe that a bulky residue at βC5 (Tyr389 in human), which sterically hinders canonical orientation of the FLVR arginine, which is retained in vertebrates and many invertebrates as Tyr or Phe, is a small residue (mostly Ser) in corals and more ancient species (Fig. S3). Similarly, the unique Arg398 at position βD4 is highly conserved in vertebrates and invertebrates but is a His in choanoflagellates and in C. owczarzaki. The evolutionary alterations in the unique pTyr-binding site therefore seem to indicate that p120RasGAP's C-terminal SH2 domain originally had a canonical binding site but that it evolved to its FLVR-unique conformation.

      Discussion

      Among protein domains the SH2 family is extraordinarily well-studied, making it unexpected for major divergence to have gone unidentified and even less expected that major divergence should be identified in one of the founding members. Nonetheless, we have shown that the C-terminal SH2 domain of p120RasGAP, the prototypical RasGAP and one of the earliest identified SH2 domains, is divergent from all studied SH2 domains. The lack of FLVR motif arginine contact with phosphotyrosine is unique among SH2 domains; in fact, even SH2 domains that can bind unphosphorylated partners do so in a FLVR arginine-dependent manner (e.g. SAP can bind Tyr as well as pTyr peptides) (
      • Liu B.A.
      • Engelmann B.W.
      • Nash P.D.
      The language of SH2 domain interactions defines phosphotyrosine-mediated signal transduction.
      ,
      • Poy F.
      • Yaffe M.B.
      • Sayos J.
      • Saxena K.
      • Morra M.
      • Sumegi J.
      • Cantley L.C.
      • Terhorst C.
      • Eck M.J.
      Crystal structures of the XLP protein SAP reveal a class of SH2 domains with extended, phosphotyrosine-independent sequence recognition.
      ). We therefore term the p120RasGAP C-SH2 a FLVR-unique SH2 domain.
      These studies, using the isolated C-SH2 domain and single pTyr peptide, do not rule out the possibility that in the context of full-length proteins, potential domain–domain interactions within the p120RasGAP SH2–SH3–SH2 cassette (Fig. 1A) might alter the pTyr-binding site of C-SH2 to a more conventional FLVR-driven conformation. Alternatively, interdomain contacts might create an alternative unique mode of pTyr binding, similar to the tyrosine kinase ZAP70, which contains two SH2 domains separated by a short helical linker domain and binds two pTyr targets in the T-cell receptor. Specifically, the two ZAP70 SH2 domains come together to create an interface that forms one unique pTyr-binding site, whereas the other more typical pTyr-binding site is contained within a single SH2 domain (
      • Hatada M.H.
      • Lu X.
      • Laird E.R.
      • Green J.
      • Morgenstern J.P.
      • Lou M.
      • Marr C.S.
      • Phillips T.B.
      • Ram M.K.
      • Theriault K.
      Molecular basis for interaction of the protein tyrosine kinase ZAP-70 with the T-cell receptor.
      ). Future studies utilizing longer p120RasGAP constructs and dual pTyr-binding partners will help to examine these possibilities.

      Classification of p120RasGAP C-SH2

      Canonically, the FLVR arginine is required for interactions with pTyr peptides, contributing over 50% of their free energy of binding to the interaction (
      • Bradshaw J.M.
      • Mitaxov V.
      • Waksman G.
      Investigation of phosphotyrosine recognition by the SH2 domain of the Src kinase.
      ,
      • Ladbury J.E.
      • Arold S.T.
      Energetics of Src homology domain interactions in receptor tyrosine kinase-mediated signaling.
      ), and mutations of this residue result in an effectively “dead” SH2 domain. This residue is highly conserved, and there are only three human SH2 domains that do not contain the FLVR arginine residue (RIN has a His at this position, SH2D5 has a Trp, and Tyk2 has a Met) (
      • Kaneko T.
      • Huang H.
      • Cao X.
      • Li X.
      • Li C.
      • Voss C.
      • Sidhu S.S.
      • Li S.S.
      Superbinder SH2 domains act as antagonists of cell signaling.
      ). For Tyk2, this belies the Janus kinase FERM-SH2 module mediating direct interaction with nonphosphorylated cytoplasmic tails of cytokine receptors (
      • Wallweber H.J.
      • Tam C.
      • Franke Y.
      • Starovasnik M.A.
      • Lupardus P.J.
      Structural basis of recognition of interferon-α receptor by tyrosine kinase 2.
      ,
      • McNally R.
      • Toms A.V.
      • Eck M.J.
      Crystal structure of the FERM-SH2 module of human Jak2.
      ). The lack of diversity at the FLVR arginine position indicates conserved function, which was recently confirmed in bacteria in which a large number of SH2 domains were identified (
      • Kaneko T.
      • Stogios P.J.
      • Ruan X.
      • Voss C.
      • Evdokimova E.
      • Skarina T.
      • Chung A.
      • Liu X.
      • Li L.
      • Savchenko A.
      • Ensminger A.W.
      • Li S.S.
      Identification and characterization of a large family of superbinding bacterial SH2 domains.
      ). Bacterial SH2 domains are classified in two groups, both of which are structurally divergent from eukaryotic SH2 domains, but even these divergent bacterial SH2 domains bind pTyr using the conserved FLVR-arginine (
      • Kaneko T.
      • Stogios P.J.
      • Ruan X.
      • Voss C.
      • Evdokimova E.
      • Skarina T.
      • Chung A.
      • Liu X.
      • Li L.
      • Savchenko A.
      • Ensminger A.W.
      • Li S.S.
      Identification and characterization of a large family of superbinding bacterial SH2 domains.
      ). The C-terminal SH2 of p120RasGAP therefore seems to have evolved uniquely among the SH2 family of proteins.
      The two-pronged pocket and cleft interaction site found in most SH2 domains (
      • Eck M.J.
      • Shoelson S.E.
      • Harrison S.C.
      Recognition of a high-affinity phosphotyrosyl peptide by the Src homology-2 domain of p56lck.
      ,
      • Waksman G.
      • Shoelson S.E.
      • Pant N.
      • Cowburn D.
      • Kuriyan J.
      Binding of a high affinity phosphotyrosyl peptide to the Src SH2 domain: crystal structures of the complexed and peptide-free forms.
      ,
      • Bradshaw J.M.
      • Grucza R.A.
      • Ladbury J.E.
      • Waksman G.
      Probing the “two-pronged plug two-holed socket” model for the mechanism of binding of the Src SH2 domain to phosphotyrosyl peptides: a thermodynamic study.
      ) can be classified into different groups (groups IA to IE, IIA to IID, and III) that have divergent specificities, with the majority displaying a binding preference for the residue at the +2, +3, or +4 position C-terminal to the pTyr (
      • Huang H.
      • Li L.
      • Wu C.
      • Schibli D.
      • Colwill K.
      • Ma S.
      • Li C.
      • Roy P.
      • Ho K.
      • Songyang Z.
      • Pawson T.
      • Gao Y.
      • Li S.S.
      Defining the specificity space of the human SRC homology 2 domain.
      ,
      • Kaneko T.
      • Huang H.
      • Zhao B.
      • Li L.
      • Liu H.
      • Voss C.K.
      • Wu C.
      • Schiller M.R.
      • Li S.S.
      Loops govern SH2 domain specificity by controlling access to binding pockets.
      ) (although P + 1, P > 4, and regions N-terminal to the pTyr can also influence specificity (
      • Hwang P.M.
      • Li C.
      • Morra M.
      • Lillywhite J.
      • Muhandiram D.R.
      • Gertler F.
      • Terhorst C.
      • Kay L.E.
      • Pawson T.
      • Forman-Kay J.D.
      • Li S.C.
      A “three-pronged” binding mechanism for the SAP/SH2D1A SH2 domain: structural basis and relevance to the XLP syndrome.
      ,
      • Tinti M.
      • Kiemer L.
      • Costa S.
      • Miller M.L.
      • Sacco F.
      • Olsen J.V.
      • Carducci M.
      • Paoluzi S.
      • Langone F.
      • Workman C.T.
      • Blom N.
      • Machida K.
      • Thompson C.M.
      • Schutkowski M.
      • Brunak S.
      • et al.
      The SH2 domain interaction landscape.
      ,
      • Virdee S.
      • Macmillan D.
      • Waksman G.
      Semisynthetic Src SH2 domains demonstrate altered phosphopeptide specificity induced by incorporation of unnatural lysine derivatives.
      ,
      • Zadjali F.
      • Pike A.C.
      • Vesterlund M.
      • Sun J.
      • Wu C.
      • Li S.S.
      • Rönnstrand L.
      • Knapp S.
      • Bullock A.N.
      • Flores-Morales A.
      Structural basis for c-KIT inhibition by the suppressor of cytokine signaling 6 (SOCS6) ubiquitin ligase.
      )). The determinants of the specificity cleft are controlled primarily by two loops: one between the E and F β-strands (the EF loop) and one between α-helix B and the β-strand G (the BG loop). These can be thought of as analogous to the complementarity-determining regions, specificity-determining loops of antibodies (
      • Huang H.
      • Li L.
      • Wu C.
      • Schibli D.
      • Colwill K.
      • Ma S.
      • Li C.
      • Roy P.
      • Ho K.
      • Songyang Z.
      • Pawson T.
      • Gao Y.
      • Li S.S.
      Defining the specificity space of the human SRC homology 2 domain.
      ,
      • Kaneko T.
      • Huang H.
      • Zhao B.
      • Li L.
      • Liu H.
      • Voss C.K.
      • Wu C.
      • Schiller M.R.
      • Li S.S.
      Loops govern SH2 domain specificity by controlling access to binding pockets.
      ,
      • Liu H.
      • Huang H.
      • Voss C.
      • Kaneko T.
      • Qin W.T.
      • Sidhu S.
      • Li S.S.
      Surface loops in a single SH2 domain are capable of encoding the spectrum of specificity of the SH2 family.
      ). In this classification scheme, the p120RasGAP C-terminal SH2 domain is classified as a group IB SH2 domain, with specificity for hydrophobic residue at position +3 (13,19). Our crystal structures confirm the interaction site with these loops (Fig. 1).

      Specificity of C-SH2 for phosphotyrosine

      Two determinants of SH2 domain–binding specificity for phosphotyrosine over phosphoserine or phosphothreonine are 1) the depth of the binding pocket and 2) presence of amino–aromatic interactions (
      • Waksman G.
      • Kominos D.
      • Robertson S.C.
      • Pant N.
      • Baltimore D.
      • Birge R.B.
      • Cowburn D.
      • Hanafusa H.
      • Mayer B.J.
      • Overduin M.
      • Resh M.D.
      • Rios C.B.
      • Silverman L.
      • Kuriyan J.
      Crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides.
      ,
      • Eck M.J.
      • Shoelson S.E.
      • Harrison S.C.
      Recognition of a high-affinity phosphotyrosyl peptide by the Src homology-2 domain of p56lck.
      ,
      • Mayer B.J.
      Why two heads are better.
      ,
      • Eck M.J.
      A new flavor in phosphotyrosine recognition.
      ,
      • Yaffe M.B.
      Phosphotyrosine-binding domains in signal transduction.
      ). For p120RasGAP C-SH2 bound to a pTyr-containing peptide, we observe the typical deep, elongated pocket to accommodate the pTyr residue; the groove is lined by Arg398 and Lys400, which cradle the aromatic ring. This amino–aromatic interaction between Arg398 and the pTyr ring is similar to other typical SH2 domains such as Src and Lck (
      • Waksman G.
      • Kominos D.
      • Robertson S.C.
      • Pant N.
      • Baltimore D.
      • Birge R.B.
      • Cowburn D.
      • Hanafusa H.
      • Mayer B.J.
      • Overduin M.
      • Resh M.D.
      • Rios C.B.
      • Silverman L.
      • Kuriyan J.
      Crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides.
      ,
      • Eck M.J.
      • Shoelson S.E.
      • Harrison S.C.
      Recognition of a high-affinity phosphotyrosyl peptide by the Src homology-2 domain of p56lck.
      ); however, Arg at the βD4 position rather than αA2 is unique to C-SH2. Taken together, these observations suggest that p120RasGAP has binding specificity for pTyr residues.

      NMR solution structure of C-SH2

      The NMR solution structure of p120RasGAP C-terminal SH2 domain, residues 340–446, was determined by the RIKEN Structural Genomics/Proteomics Initiative in 2006 and deposited in the Protein Data Bank (code 2GSB). The solution structure contains the 20 lowest energy models. Overall, the superposition with our crystal structures is close (RMSD 1.12 Å over 101 Cα atoms) (Fig. S4A), but in-solution conformational flexibility is observed in the EF loop, which defines +3 specificity (
      • Kaneko T.
      • Huang H.
      • Zhao B.
      • Li L.
      • Liu H.
      • Voss C.K.
      • Wu C.
      • Schiller M.R.
      • Li S.S.
      Loops govern SH2 domain specificity by controlling access to binding pockets.
      ), and in the BC loop, which harbors Asp380. None of the NMR structures exhibit the bidentate salt bridge between Arg377 and Asp380 (residue BC1), and both residues display extensive conformational variability, but their overall orientation is similar to our crystal structures (Fig. S4B). In contrast, Tyr389 is in a broadly similar position to our crystal structures and blocks orientation of the FLVR Arg toward the canonical pTyr-binding position. The unpublished NMR structure therefore supports our crystallographic data that p120RasGAP C-SH2 is a FLVR-unique SH2 domain.

      Functional role of the FLVR-unique C-SH2

      What then might the function of this highly unusual SH2 domain in p120RasGAP be? SH2 domains seem to have helped facilitate the evolutionary increase in complexity of signal transduction pathways and the transition from unicellular to multicellular organisms (as illustrated by a single SH2 domain residing in baker's yeast, Saccharomyces cerevisiae) (
      • Liu B.A.
      • Nash P.D.
      Evolution of SH2 domains and phosphotyrosine signalling networks.
      ). In humans there are ∼120 proteins that contain SH2 domains, but only 10 of these have dual SH2s. For p120RasGAP, the bivalent SH2 domains probably indicate that p120RasGAP has fast rebinding and long dwell times at pTyr-binding partners (
      • Oh D.
      • Ogiue-Ikeda M.
      • Jadwin J.A.
      • Machida K.
      • Mayer B.J.
      • Yu J.
      Fast rebinding increases dwell time of Src homology 2 (SH2)–containing proteins near the plasma membrane.
      ), and cooperation between the p120RasGAP domains is thought to impact signal transduction (
      • Schlessinger J.
      • Lemmon M.A.
      SH2 and PTB domains in tyrosine kinase signaling.
      ) with conformational changes proposed to occur on dual engagement of the SH2 domains with phosphotyrosine partners (
      • Hu K.Q.
      • Settleman J.
      Tandem SH2 binding sites mediate the RasGAP-RhoGAP interaction: a conformational mechanism for SH3 domain regulation.
      ). However, the affinities of SH2 domain interactions with their functional phospho-partners is generally in the moderate 0.1–10 μm range (
      • Ladbury J.E.
      • Arold S.T.
      Energetics of Src homology domain interactions in receptor tyrosine kinase-mediated signaling.
      ,
      • Hause Jr., R.J.
      • Leung K.K.
      • Barkinge J.L.
      • Ciaccio M.F.
      • Chuu C.P.
      • Jones R.B.
      Comprehensive binary interaction mapping of SH2 domains via fluorescence polarization reveals novel functional diversification of ErbB receptors.
      ), allowing ready dissociation and thus preventing masking of the pTyr site and prevention of signal transduction (
      • Haslam N.J.
      • Shields D.C.
      Peptide-binding domains: are limp handshakes safest?.
      ,
      • Mayer B.J.
      Dynamics of receptor tyrosine kinase signaling complexes.
      ). For p120RasGAP, the combination of two tight SH2–pTyr interactions may result in extremely long dwell times that are deleterious for signal transduction. It may be that the altered pTyr-binding site in the C-terminal SH2 domain beneficially impacts p120RasGAP's signaling by tuning recruitment and retention at pTyr partners and thus prevents masking (
      • Haslam N.J.
      • Shields D.C.
      Peptide-binding domains: are limp handshakes safest?.
      ,
      • Mayer B.J.
      Dynamics of receptor tyrosine kinase signaling complexes.
      ) of these sites.

      Broader implications

      Disruption of SH2–pTyr interactions has theoretical clinical potential to regulate diverse signal transduction pathways, and consequently much work was conducted to achieve SH2 domain–specific inhibitors. Clinically useful selectivity has not, however, been achieved in large part because of bioavailability and the molecular similarities between SH2 domains (
      • Waksman G.
      • Kumaran S.
      • Lubman O.
      SH2 domains: role, structure and implications for molecular medicine.
      ). Selective picomolar binders can be designed where SH2 domains diverge as demonstrated for Grb2, which has an unusual specificity pocket that recognizes Asn at residue pY + 2 (
      • Kraskouskaya D.
      • Duodu E.
      • Arpin C.C.
      • Gunning P.T.
      Progress towards the development of SH2 domain inhibitors.
      ,
      • Morlacchi P.
      • Robertson F.M.
      • Klostergaard J.
      • McMurray J.S.
      Targeting SH2 domains in breast cancer.
      ,
      • Shi Z.D.
      • Lee K.
      • Liu H.
      • Zhang M.
      • Roberts L.R.
      • Worthy K.M.
      • Fivash M.J.
      • Fisher R.J.
      • Yang D.
      • Burke Jr., T.R.
      A novel macrocyclic tetrapeptide mimetic that exhibits low-picomolar Grb2 SH2 domain-binding affinity.
      ). Although it remains an outside possibility, nonetheless, it is interesting to speculate that the unique nature of p120RasGAP's C-terminal SH2 domain may represent a novel SH2-inhibitor target with the potential to beneficially alter both Ras and Rho signaling.
      SH2 domains have played an important role in influencing our understanding of how cytosolic signaling occurs. Invariably these domains bind phosphotyrosine partners with their FLVR motif arginine residue acting as the linchpin of the interaction. It is striking, therefore, that one of the first identified SH2 domain proteins should diverge from this paradigm. The functional reasons for this divergence are currently unclear, but the array of bidentate binding partners for p120RasGAP and alterations in p120RasGAP's RasGAP activity on binding to phosphorylated partner proteins may hint at an evolutionary requirement to allow unmasking of the phosphotyrosine sites. The discovery of the unique nature of the C-terminal SH2 domain and how to alter its phosphotyrosine binding may therefore provide guidance to resolving how p120RasGAP functions mechanistically.

      Materials and methods

      Expression and purification p120RasGAP C-SH2 domain

      cDNA encoding the C-terminal SH2 domain of human p120RasGAP (UniProt ID P20936; residues 340–444) was amplified by PCR and ligated into a modified pET vector, which includes an N-terminal His6 tag and TEV protease recognition site. Two native cysteine residues, Cys372 and Cys402, were mutated to serine by QuikChange mutagenesis (Agilent) to prevent formation of intermolecular disulfide bonds. The following point mutants were also generated by QuikChange mutagenesis: K377A, K398A, K400A, and K398A/K400A. Protein expression of WT and mutant C-SH2 was performed in Rosetta (DE3) cells, which were cultured in 1 liter of Luria broth at 37 °C to an A600 of 0.6–0.8 and then cooled to 18 °C, and protein expression was induced with 0.2 mm isopropyl β-d-thiogalactopyranoside. The cells were harvested by centrifugation, resuspended in lysis buffer (50 mm HEPES, pH 7.3, and 500 mm NaCl), and lysed by freeze-thaw cycles in the presence of lysozyme followed by sonication. Total cellular lysate was clarified by centrifugation at 5000 × g at 4 °C and applied to nickel–nitrilotriacetic acid–agarose resin (Qiagen) for 1 h at 4 °C to capture His6-tagged protein. Beads with bound protein were washed with 20 column volumes of wash buffer containing 50 mm HEPES, pH 7.3, 500 mm NaCl, 20 mm imidazole. The His6 tag was proteolytically removed from the C-SH2 proteins “on bead” overnight at 4 °C by the addition of TEV protease, which itself is also His6-tagged and captured by the nickel–nitrilotriacetic acid resin. The flow-through containing untagged C-SH2 protein was collected and applied to size-exclusion chromatography (Superdex 75; GE Healthcare) in buffer containing 20 mm Tris, pH 7.4, 150 mm NaCl. Finally, C-SH2 proteins were concentrated in a centrifugal filter with a molecular mass cutoff of 3,000 Da (Amicon Ultra, Millipore Sigma). 1 liter of Rosetta (DE3) cell culture resulted in a final purified protein yield of ∼20 mg for WT and up to 10 mg for mutant.

      Peptide synthesis

      A synthetic 7-amino acid peptide of sequence 1086DpYAEPMD1092 native to p190RhoGAP residues 1086–1092 (UniProt Q9NRY4) phosphorylated at Tyr1087 and a 15-amino acid peptide with sequence 1081GFDPSDpYAEPMDAVV1095 corresponding to residues 1081–1095 of p190RhoGAP (UniProt Q9NRY4) with N-terminal acetylation and C-terminal amidation were commercially synthesized (GenScript) and resuspended in sterile-filtered water.

      Crystallization, data collection, structure determination, and refinement

      p120RasGAP C-SH2 WT protein was used in crystallization trials at ∼14 mg/ml. Initial crystal screening was conducted with Index HT kit (Hampton Research) by a TTP Labtech Mosquito in sitting-drop vapor-diffusion trays at room temperature. Single crystals were obtained in Index HT position A3 containing 2.0 m ammonium sulfate, 0.1 m Bis-Tris, pH 6.5, with a 1:1 (v:v) protein:reservoir solution ratio. Optimization of this crystallization condition was achieved in hanging-drop vapor-diffusion plates, with a reservoir buffer containing 2.3 m ammonium sulfate, 0.1 m Bis-Tris, pH 6.5, and ratio of 1:1 (v:v) protein:reservoir solution. Single crystals with approximate dimensions 200 × 200 × 200 mm were harvested from the drop, cryopreserved in reservoir solution supplemented with 20% ethylene glycol, and flash-cooled in liquid nitrogen. X-ray data collection on a single crystal was performed at Northeastern Collaborative Access Team Beamline 24-ID-C at Argonne National Laboratory Advanced Photon Source. X-ray data were processed and scaled in HKL2000 (
      • Otwinowski Z.
      • Minor W.
      Processing of X-ray diffraction data collected in oscillation mode.
      ) in space group P212121 with the unit cell dimensions a = 52.5 Å, b = 65.6 Å, c = 71.6 Å, α = β = γ = 90°, to 1.50 Å resolution. Matthews probability predicted two copies of SH2 in the asymmetric unit, and a molecular replacement solution confirmed the prediction with copies of C-SH2 obtained by Phenix Phaser (
      • McCoy A.J.
      • Grosse-Kunstleve R.W.
      • Adams P.D.
      • Winn M.D.
      • Storoni L.C.
      • Read R.J.
      Phaser crystallographic software.
      ) using the SH2 domain of SAP (PDB code 1D4T) (
      • Poy F.
      • Yaffe M.B.
      • Sayos J.
      • Saxena K.
      • Morra M.
      • Sumegi J.
      • Cantley L.C.
      • Terhorst C.
      • Eck M.J.
      Crystal structures of the XLP protein SAP reveal a class of SH2 domains with extended, phosphotyrosine-independent sequence recognition.
      ) as a search model, yielding the translational function Z score of 12.7. Model building was performed in Phenix Autobuild (
      • Terwilliger T.C.
      • Grosse-Kunstleve R.W.
      • Afonine P.V.
      • Moriarty N.W.
      • Zwart P.H.
      • Hung L.W.
      • Read R.J.
      • Adams P.D.
      Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard.
      ), which successfully built 196 residues and correctly placed 192 in sequence (residues 344–442 of copy A and 350–442 of copy B). Manual model building was performed in Coot (
      • Emsley P.
      • Lohkamp B.
      • Scott W.G.
      • Cowtan K.
      Features and development of Coot.
      ) and refinement in Phenix (
      • Adams P.D.
      • Afonine P.V.
      • Bunkóczi G.
      • Chen V.B.
      • Davis I.W.
      • Echols N.
      • Headd J.J.
      • Hung L.W.
      • Kapral G.J.
      • Grosse-Kunstleve R.W.
      • McCoy A.J.
      • Moriarty N.W.
      • Oeffner R.
      • Read R.J.
      • Richardson D.C.
      • et al.
      PHENIX: a comprehensive Python-based system for macromolecular structure solution.
      ). Two sulfate ions (from the crystallization drop) and six ethylene glycol molecules (from the cryopreservative solution) were also modeled into density. Final refinement statistics are Rwork = 17.9% and Rfree = 19.7%.
      For co-crystallization with the 7-amino acid p190RhoGAP pTyr1087 phosphopeptide, C-SH2 protein at 800 μm (10 mg/ml) was premixed with phosphopeptide concentrations ranging from 4 to 8 mm. Initial crystallization screens were performed with Index HT and SaltRx HT screens (Hampton Research) on a TTP Labtech Mosquito into sitting drops. Optimized co-crystals were obtained with reservoir solution containing 1.2 m sodium citrate tribasic dihydrate, 0.1 m Tris, pH 8.5. Single crystals were cryopreserved in reservoir buffer supplemented with 0.5 m sodium malonate, pH 7.0, before being flash-cooled in liquid nitrogen. X-ray data collection on a single crystal was performed at Northeastern Collaborative Access Team Beamline 24-ID-C at Argonne National Laboratory Advanced Photon Source. X-ray data were processed in HKL2000 (
      • Otwinowski Z.
      • Minor W.
      Processing of X-ray diffraction data collected in oscillation mode.
      ) in space group C2221 with unit cell dimensions a = 63.1 Å, b = 83.9 Å, c = 54.2 Å, α = β = γ = 90° to 1.50 Å resolution and one copy per asymmetric unit. For structure solution, an all-alanine model derived from the NMR structure of p120RasGAP C-SH2 (PDB code 2GSB), truncated to residues 345–442 and B factors set to 20, was used as a search model for molecular replacement. Molecular replacement was performed by Phaser (
      • McCoy A.J.
      • Grosse-Kunstleve R.W.
      • Adams P.D.
      • Winn M.D.
      • Storoni L.C.
      • Read R.J.
      Phaser crystallographic software.
      ), which found a single solution for one copy of C-SH2 with a TFZ score of 10.6. Autobuilding was performed in Phenix (
      • Terwilliger T.C.
      • Grosse-Kunstleve R.W.
      • Afonine P.V.
      • Moriarty N.W.
      • Zwart P.H.
      • Hung L.W.
      • Read R.J.
      • Adams P.D.
      Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard.
      ), which removed model bias and successfully built and placed 100 residues (residues 342–442). Autobuilding also resulted in the building of five residues of the phosphopeptide, corresponding to p190RhoGAP residues 1087–1091. Manual model building was performed in Coot (
      • Emsley P.
      • Lohkamp B.
      • Scott W.G.
      • Cowtan K.
      Features and development of Coot.
      ) and refinement in Phenix (
      • Adams P.D.
      • Afonine P.V.
      • Bunkóczi G.
      • Chen V.B.
      • Davis I.W.
      • Echols N.
      • Headd J.J.
      • Hung L.W.
      • Kapral G.J.
      • Grosse-Kunstleve R.W.
      • McCoy A.J.
      • Moriarty N.W.
      • Oeffner R.
      • Read R.J.
      • Richardson D.C.
      • et al.
      PHENIX: a comprehensive Python-based system for macromolecular structure solution.
      ), yielding final Rwork = 16.5% and Rfree = 19.0%.

      Isothermal titration calorimetry

      Both C-SH2 WT and mutant proteins and pTyr1087 phosphopeptide (15 amino acids) were prepared for ITC by overnight dialysis in a common buffer with a 20 mm Tris, pH 7.4, 150 mm NaCl composition. Slide-A-Lyzer dialysis cassettes were used with a molecular mass cutoff of 3,500 Da were used for protein dialysis, and Micro Float-A-Lyzer dialysis devices with a 100–500-Da cutoff were used for peptide dialysis. The samples were retrieved from their cartridges and spun down for 10 min at 4 °C. The concentrations were measured by Nanodrop (Thermo Fisher). To determine peptide concentration, a phosphotyrosine extinction coefficient of 458.6 m−1 cm−1 at pH 7.4 was used (
      • Zhang Z.Y.
      • Maclean D.
      • Thieme-Sefler A.M.
      • Roeske R.W.
      • Dixon J.E.
      A continuous spectrophotometric and fluorimetric assay for protein tyrosine phosphatase using phosphotyrosine-containing peptides.
      ). For ITC, a Nano-ITC (TA Instruments) was used. 350 μl of protein were injected into the sample cell. Both protein and peptide were degassed for 3 min prior to loading. The sample cell contents were stirred at 350 rpm at 25 °C to achieve continuous mixing. The results were analyzed using Nano-ITC Analyze software.

      Evolutionary analysis

      p120RasGAP sequences were identified using NCBI BLAST and searches of the FlyBase (
      • Thurmond J.
      • Goodman J.L.
      • Strelets V.B.
      • Attrill H.
      • Gramates L.S.
      • Marygold S.J.
      • Matthews B.B.
      • Millburn G.
      • Antonazzo G.
      • Trovisco V.
      • Kaufman T.C.
      • Calvi B.R.
      • FlyBase Consortium
      FlyBase 2.0: the next generation.
      ), WormBase (
      • Harris T.W.
      • Arnaboldi V.
      • Cain S.
      • Chan J.
      • Chen W.J.
      • Cho J.
      • Davis P.
      • Gao S.
      • Grove C.A.
      • Kishore R.
      • Lee R.Y.N.
      • Muller H.M.
      • Nakamura C.
      • Nuin P.
      • Paulini M.
      • et al.
      WormBase: a modern model organism information resource.
      ), and MycoCosm databases (
      • Grigoriev I.V.
      • Nikitin R.
      • Haridas S.
      • Kuo A.
      • Ohm R.
      • Otillar R.
      • Riley R.
      • Salamov A.
      • Zhao X.
      • Korzeniewski F.
      • Smirnova T.
      • Nordberg H.
      • Dubchak I.
      • Shabalov I.
      MycoCosm portal: gearing up for 1000 fungal genomes.
      ). Sequences were aligned using the MAFFT (
      • Katoh K.
      • Standley D.M.
      MAFFT multiple sequence alignment software version 7: improvements in performance and usability.
      ) server and visualized using JalView (
      • Waterhouse A.M.
      • Procter J.B.
      • Martin D.M.
      • Clamp M.
      • Barton G.J.
      Jalview version 2: a multiple sequence alignment editor and analysis workbench.
      ).

      Data availability

      The coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 6WAX and 6WAY. X-ray diffraction images are available online at SBGrid Data Bank (
      • Waterhouse A.M.
      • Procter J.B.
      • Martin D.M.
      • Clamp M.
      • Barton G.J.
      Jalview version 2: a multiple sequence alignment editor and analysis workbench.
      ): doi: 10.15785/SBGRID/774 (6WAX) and doi: 10.15785/SBGRID/775 (6WAY).

      Acknowledgments

      We thank Mark Lemmon, Benjamin Turk, Byung Hak Ha, and Kimberly Vish for helpful comments and James Murphy for help with the ITC experiments. We also thank the staff at Northeastern Collaborative Access Team Beamline 24-ID-C at the Advanced Photon Source, Argonne National Laboratory.

      Supplementary Material

      Author Profile

      References

        • Liu B.A.
        • Jablonowski K.
        • Raina M.
        • Arcé M.
        • Pawson T.
        • Nash P.D.
        The human and mouse complement of SH2 domain proteins: establishing the boundaries of phosphotyrosine signaling.
        Mol. Cell. 2006; 22 (16793553): 851-868
        • Sadowski I.
        • Stone J.C.
        • Pawson T.
        A noncatalytic domain conserved among cytoplasmic protein-tyrosine kinases modifies the kinase function and transforming activity of Fujinami sarcoma virus P130gag-fps.
        Mol. Cell Biol. 1986; 6 (3025655): 4396-4408
        • Pawson T.
        Specificity in signal transduction: from phosphotyrosine–SH2 domain interactions to complex cellular systems.
        Cell. 2004; 116 (14744431): 191-203
        • Nash P.D.
        Why modules matter.
        FEBS Lett. 2012; 586 (22710154): 2572-2574
        • Shah N.H.
        • Amacher J.F.
        • Nocka L.M.
        • Kuriyan J.
        The Src module: an ancient scaffold in the evolution of cytoplasmic tyrosine kinases.
        Crit. Rev. Biochem. Mol. Biol. 2018; 53 (30183386): 535-563
        • Boggon T.J.
        • Eck M.J.
        Structure and regulation of Src family kinases.
        Oncogene. 2004; 23 (15489910): 7918-7927
        • Schlessinger J.
        • Lemmon M.A.
        SH2 and PTB domains in tyrosine kinase signaling.
        Sci. STKE. 2003; 2003 (12865499): RE12
        • Liu B.A.
        • Engelmann B.W.
        • Nash P.D.
        The language of SH2 domain interactions defines phosphotyrosine-mediated signal transduction.
        FEBS Lett. 2012; 586 (22569091): 2597-2605
        • Kaneko T.
        • Huang H.
        • Cao X.
        • Li X.
        • Li C.
        • Voss C.
        • Sidhu S.S.
        • Li S.S.
        Superbinder SH2 domains act as antagonists of cell signaling.
        Sci. Signal. 2012; 5 (23012655): ra68
        • Liu B.A.
        • Nash P.D.
        Evolution of SH2 domains and phosphotyrosine signalling networks.
        Philos. Trans. R. Soc. Lond. B Biol. Sci. 2012; 367 (22889907): 2556-2573
        • Suga H.
        • Torruella G.
        • Burger G.
        • Brown M.W.
        • Ruiz-Trillo I.
        Earliest holozoan expansion of phosphotyrosine signaling.
        Mol. Biol. Evol. 2014; 31 (24307687): 517-528
        • Songyang Z.
        • Shoelson S.E.
        • Chaudhuri M.
        • Gish G.
        • Pawson T.
        • Haser W.G.
        • King F.
        • Roberts T.
        • Ratnofsky S.
        • Lechleider R.J.
        SH2 domains recognize specific phosphopeptide sequences.
        Cell. 1993; 72 (7680959): 767-778
        • Huang H.
        • Li L.
        • Wu C.
        • Schibli D.
        • Colwill K.
        • Ma S.
        • Li C.
        • Roy P.
        • Ho K.
        • Songyang Z.
        • Pawson T.
        • Gao Y.
        • Li S.S.
        Defining the specificity space of the human SRC homology 2 domain.
        Mol. Cell. Proteomics. 2008; 7 (17956856): 768-784
        • Songyang Z.
        • Shoelson S.E.
        • McGlade J.
        • Olivier P.
        • Pawson T.
        • Bustelo X.R.
        • Barbacid M.
        • Sabe H.
        • Hanafusa H.
        • Yi T.
        Specific motifs recognized by the SH2 domains of Csk 3BP2, fps/fes, Grb-2, HCP, SHC, Syk and Vav.
        Mol. Cell Biol. 1994; 14 (7511210): 2777-2785
        • Waksman G.
        • Kominos D.
        • Robertson S.C.
        • Pant N.
        • Baltimore D.
        • Birge R.B.
        • Cowburn D.
        • Hanafusa H.
        • Mayer B.J.
        • Overduin M.
        • Resh M.D.
        • Rios C.B.
        • Silverman L.
        • Kuriyan J.
        Crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides.
        Nature. 1992; 358 (1379696): 646-653
        • Eck M.J.
        • Shoelson S.E.
        • Harrison S.C.
        Recognition of a high-affinity phosphotyrosyl peptide by the Src homology-2 domain of p56lck.
        Nature. 1993; 362 (7680435): 87-91
        • Overduin M.
        • Rios C.B.
        • Mayer B.J.
        • Baltimore D.
        • Cowburn D.
        Three-dimensional solution structure of the src homology 2 domain of c-abl.
        Cell. 1992; 70 (1505033): 697-704
        • Waksman G.
        • Shoelson S.E.
        • Pant N.
        • Cowburn D.
        • Kuriyan J.
        Binding of a high affinity phosphotyrosyl peptide to the Src SH2 domain: crystal structures of the complexed and peptide-free forms.
        Cell. 1993; 72 (7680960): 779-790
        • Kaneko T.
        • Huang H.
        • Zhao B.
        • Li L.
        • Liu H.
        • Voss C.K.
        • Wu C.
        • Schiller M.R.
        • Li S.S.
        Loops govern SH2 domain specificity by controlling access to binding pockets.
        Sci. Signal. 2010; 3 (20442417): ra34
        • Liu H.
        • Huang H.
        • Voss C.
        • Kaneko T.
        • Qin W.T.
        • Sidhu S.
        • Li S.S.
        Surface loops in a single SH2 domain are capable of encoding the spectrum of specificity of the SH2 family.
        Mol. Cell. Proteomics. 2019; 18 (30482845): 372-382
        • Waksman G.
        • Kumaran S.
        • Lubman O.
        SH2 domains: role, structure and implications for molecular medicine.
        Exp. Rev. Mol. Med. 2004; 6 (14987415): 1-18
        • Bradshaw J.M.
        • Mitaxov V.
        • Waksman G.
        Investigation of phosphotyrosine recognition by the SH2 domain of the Src kinase.
        J. Mol. Biol. 1999; 293 (10543978): 971-985
        • Bibbins K.B.
        • Boeuf H.
        • Varmus H.E.
        Binding of the Src SH2 domain to phosphopeptides is determined by residues in both the SH2 domain and the phosphopeptides.
        Mol. Cell Biol. 1993; 13 (7504171): 7278-7287
        • Mayer B.J.
        • Jackson P.K.
        • Van Etten R.A.
        • Baltimore D.
        Point mutations in the abl SH2 domain coordinately impair phosphotyrosine binding in vitro and transforming activity in vivo.
        Mol. Cell Biol. 1992; 12 (1370711): 609-618
        • Trahey M.
        • McCormick F.
        A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants.
        Science. 1987; 238 (2821624): 542-545
        • Vogel U.S.
        • Dixon R.A.
        • Schaber M.D.
        • Diehl R.E.
        • Marshall M.S.
        • Scolnick E.M.
        • Sigal I.S.
        • Gibbs J.B.
        Cloning of bovine GAP and its interaction with oncogenic ras p21.
        Nature. 1988; 335 (2842690): 90-93
        • Trahey M.
        • Wong G.
        • Halenbeck R.
        • Rubinfeld B.
        • Martin G.A.
        • Ladner M.
        • Long C.M.
        • Crosier W.J.
        • Watt K.
        • Koths K.
        Molecular cloning of two types of GAP complementary DNA from human placenta.
        Science. 1988; 242 (3201259): 1697-1700
        • Bernards A.
        • Settleman J.
        GAP control: regulating the regulators of small GTPases.
        Trends Cell Biol. 2004; 14 (15246431): 377-385
        • Bos J.L.
        • Rehmann H.
        • Wittinghofer A.
        GEFs and GAPs: critical elements in the control of small G proteins.
        Cell. 2007; 129 (17540168): 865-877
        • McCormick F.
        • Adari H.
        • Trahey M.
        • Halenbeck R.
        • Koths K.
        • Martin G.A.
        • Crosier W.J.
        • Watt K.
        • Rubinfeld B.
        • Wong G.
        Interaction of ras p21 proteins with GTPase activating protein.
        Cold Spring Harb. Symp. Quant. Biol. 1988; 53 (2855502): 849-854
        • Henkemeyer M.
        • Rossi D.J.
        • Holmyard D.P.
        • Puri M.C.
        • Mbamalu G.
        • Harpal K.
        • Shih T.S.
        • Jacks T.
        • Pawson T.
        Vascular system defects and neuronal apoptosis in mice lacking ras GTPase-activating protein.
        Nature. 1995; 377 (7477259): 695-701
        • Pamonsinlapatham P.
        • Hadj-Slimane R.
        • Lepelletier Y.
        • Allain B.
        • Toccafondi M.
        • Garbay C.
        • Raynaud F.
        p120-Ras GTPase activating protein (RasGAP): a multi-interacting protein in downstream signaling.
        Biochimie (Paris). 2009; 91 (19022332): 320-328
        • Adari H.
        • Lowy D.R.
        • Willumsen B.M.
        • Der C.J.
        • McCormick F.
        Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain.
        Science. 1988; 240 (2833817): 518-521
        • Laskowski R.A.
        PDBsum: summaries and analyses of PDB structures.
        Nucleic Acids Res. 2001; 29 (11125097): 221-222
        • Moran M.F.
        • Polakis P.
        • McCormick F.
        • Pawson T.
        • Ellis C.
        Protein-tyrosine kinases regulate the phosphorylation, protein interactions, subcellular distribution, and activity of p21ras GTPase-activating protein.
        Mol. Cell Biol. 1991; 11 (2005883): 1804-1812
        • Bryant S.S.
        • Briggs S.
        • Smithgall T.E.
        • Martin G.A.
        • McCormick F.
        • Chang J.H.
        • Parsons S.J.
        • Jove R.
        Two SH2 domains of p120 Ras GTPase-activating protein bind synergistically to tyrosine phosphorylated p190 Rho GTPase-activating protein.
        J. Biol. Chem. 1995; 270 (7629101): 17947-17952
        • Dail M.
        • Richter M.
        • Godement P.
        • Pasquale E.B.
        Eph receptors inactivate R-Ras through different mechanisms to achieve cell repulsion.
        J. Cell Sci. 2006; 119 (16522685): 1244-1254
        • Kulkarni S.V.
        • Gish G.
        • van der Geer P.
        • Henkemeyer M.
        • Pawson T.
        Role of p120 Ras-GAP in directed cell movement.
        J. Cell Biol. 2000; 149 (10769036): 457-470
        • van der Geer P.
        • Henkemeyer M.
        • Jacks T.
        • Pawson T.
        Aberrant Ras regulation and reduced p190 tyrosine phosphorylation in cells lacking p120-Gap.
        Mol. Cell Biol. 1997; 17 (9121432): 1840-1847
        • Jaber Chehayeb R.
        • Stiegler A.L.
        • Boggon T.J.
        Crystal structures of p120RasGAP N-terminal SH2 domain in its apo form and in complex with a p190RhoGAP phosphotyrosine peptide.
        PLoS One. 2019; 14 (31891593): e0226113
        • Chang J.H.
        • Gill S.
        • Settleman J.
        • Parsons S.J.
        c-Src regulates the simultaneous rearrangement of actin cytoskeleton, p190RhoGAP, and p120RasGAP following epidermal growth factor stimulation.
        J. Cell Biol. 1995; 130 (7542246): 355-368
        • Sfakianos M.K.
        • Eisman A.
        • Gourley S.L.
        • Bradley W.D.
        • Scheetz A.J.
        • Settleman J.
        • Taylor J.R.
        • Greer C.A.
        • Williamson A.
        • Koleske A.J.
        Inhibition of Rho via Arg and p190RhoGAP in the postnatal mouse hippocampus regulates dendritic spine maturation, synapse and dendrite stability, and behavior.
        J. Neurosci. 2007; 27 (17928439): 10982-10992
        • Bradley W.D.
        • Hernández S.E.
        • Settleman J.
        • Koleske A.J.
        Integrin signaling through Arg activates p190RhoGAP by promoting its binding to p120RasGAP and recruitment to the membrane.
        Mol. Biol. Cell. 2006; 17 (16971514): 4827-4836
        • Hu K.Q.
        • Settleman J.
        Tandem SH2 binding sites mediate the RasGAP-RhoGAP interaction: a conformational mechanism for SH3 domain regulation.
        EMBO J. 1997; 16 (9034330): 473-483
        • Hernández S.E.
        • Settleman J.
        • Koleske A.J.
        Adhesion-dependent regulation of p190RhoGAP in the developing brain by the Abl-related gene tyrosine kinase.
        Curr. Biol. 2004; 14 (1508284): 691-696
        • McKercher M.A.
        • Guan X.
        • Tan Z.
        • Wuttke D.S.
        Multimodal recognition of diverse peptides by the C-terminal SH2 domain of phospholipase C-γ1 protein.
        Biochemistry. 2017; 56 (28376302): 2225-2237
        • Frese S.
        • Schubert W.D.
        • Findeis A.C.
        • Marquardt T.
        • Roske Y.S.
        • Stradal T.E.
        • Heinz D.W.
        The phosphotyrosine peptide binding specificity of Nck1 and Nck2 Src homology 2 domains.
        J. Biol. Chem. 2006; 281 (16636066): 18236-18245
        • Bradshaw J.M.
        • Grucza R.A.
        • Ladbury J.E.
        • Waksman G.
        Probing the “two-pronged plug two-holed socket” model for the mechanism of binding of the Src SH2 domain to phosphotyrosyl peptides: a thermodynamic study.
        Biochemistry. 1998; 37 (9636054): 9083-9090
        • Holm L.
        • Rosenstrom P.
        Dali server: conservation mapping in 3D.
        Nucleic Acids Res. 2010; 38 (20457744): W545-W549
        • Li J.
        • Rodriguez J.P.
        • Niu F.
        • Pu M.
        • Wang J.
        • Hung L.W.
        • Shao Q.
        • Zhu Y.
        • Ding W.
        • Liu Y.
        • Da Y.
        • Yao Z.
        • Yang J.
        • Zhao Y.
        • Wei G.H.
        • et al.
        Structural basis for DNA recognition by STAT6.
        Proc. Natl. Acad. Sci. U.S.A. 2016; 113 (27803324): 13015-13020
        • Chen C.H.
        • Piraner D.
        • Gorenstein N.M.
        • Geahlen R.L.
        • Beth Post C.
        Differential recognition of syk-binding sites by each of the two phosphotyrosine-binding pockets of the Vav SH2 domain.
        Biopolymers. 2013; 99 (23955592): 897-907
        • Kaneko T.
        • Stogios P.J.
        • Ruan X.
        • Voss C.
        • Evdokimova E.
        • Skarina T.
        • Chung A.
        • Liu X.
        • Li L.
        • Savchenko A.
        • Ensminger A.W.
        • Li S.S.
        Identification and characterization of a large family of superbinding bacterial SH2 domains.
        Nat. Commun. 2018; 9 (30382091): 4549
        • Ladbury J.E.
        • Lemmon M.A.
        • Zhou M.
        • Green J.
        • Botfield M.C.
        • Schlessinger J.
        Measurement of the binding of tyrosyl phosphopeptides to SH2 domains: a reappraisal.
        Proc. Natl. Acad. Sci. U.S.A. 1995; 92 (7536927): 3199-3203
        • Poy F.
        • Yaffe M.B.
        • Sayos J.
        • Saxena K.
        • Morra M.
        • Sumegi J.
        • Cantley L.C.
        • Terhorst C.
        • Eck M.J.
        Crystal structures of the XLP protein SAP reveal a class of SH2 domains with extended, phosphotyrosine-independent sequence recognition.
        Mol. Cell. 1999; 4 (10549287): 555-561
        • Hatada M.H.
        • Lu X.
        • Laird E.R.
        • Green J.
        • Morgenstern J.P.
        • Lou M.
        • Marr C.S.
        • Phillips T.B.
        • Ram M.K.
        • Theriault K.
        Molecular basis for interaction of the protein tyrosine kinase ZAP-70 with the T-cell receptor.
        Nature. 1995; 377 (7659156): 32-38
        • Ladbury J.E.
        • Arold S.T.
        Energetics of Src homology domain interactions in receptor tyrosine kinase-mediated signaling.
        Methods Enzymol. 2011; 488 (21195228): 147-183
        • Wallweber H.J.
        • Tam C.
        • Franke Y.
        • Starovasnik M.A.
        • Lupardus P.J.
        Structural basis of recognition of interferon-α receptor by tyrosine kinase 2.
        Nat. Struct. Mol. Biol. 2014; 21 (24704786): 443-448
        • McNally R.
        • Toms A.V.
        • Eck M.J.
        Crystal structure of the FERM-SH2 module of human Jak2.
        PLoS One. 2016; 11 (27227461): e0156218
        • Hwang P.M.
        • Li C.
        • Morra M.
        • Lillywhite J.
        • Muhandiram D.R.
        • Gertler F.
        • Terhorst C.
        • Kay L.E.
        • Pawson T.
        • Forman-Kay J.D.
        • Li S.C.
        A “three-pronged” binding mechanism for the SAP/SH2D1A SH2 domain: structural basis and relevance to the XLP syndrome.
        EMBO J. 2002; 21 (11823424): 314-323
        • Tinti M.
        • Kiemer L.
        • Costa S.
        • Miller M.L.
        • Sacco F.
        • Olsen J.V.
        • Carducci M.
        • Paoluzi S.
        • Langone F.
        • Workman C.T.
        • Blom N.
        • Machida K.
        • Thompson C.M.
        • Schutkowski M.
        • Brunak S.
        • et al.
        The SH2 domain interaction landscape.
        Cell Rep. 2013; 3 (23545499): 1293-1305
        • Virdee S.
        • Macmillan D.
        • Waksman G.
        Semisynthetic Src SH2 domains demonstrate altered phosphopeptide specificity induced by incorporation of unnatural lysine derivatives.
        Chem. Biol. 2010; 17 (20338519): 274-284
        • Zadjali F.
        • Pike A.C.
        • Vesterlund M.
        • Sun J.
        • Wu C.
        • Li S.S.
        • Rönnstrand L.
        • Knapp S.
        • Bullock A.N.
        • Flores-Morales A.
        Structural basis for c-KIT inhibition by the suppressor of cytokine signaling 6 (SOCS6) ubiquitin ligase.
        J. Biol. Chem. 2011; 286 (21030588): 480-490
        • Mayer B.J.
        Why two heads are better.
        Structure. 1995; 3 (8590006): 977-980
        • Eck M.J.
        A new flavor in phosphotyrosine recognition.
        Structure. 1995; 3 (7545066): 421-424
        • Yaffe M.B.
        Phosphotyrosine-binding domains in signal transduction.
        Nat. Rev. Mol. Cell. Biol. 2002; 3 (11994738): 177-186
        • Oh D.
        • Ogiue-Ikeda M.
        • Jadwin J.A.
        • Machida K.
        • Mayer B.J.
        • Yu J.
        Fast rebinding increases dwell time of Src homology 2 (SH2)–containing proteins near the plasma membrane.
        Proc. Natl. Acad. Sci. U.S.A. 2012; 109 (22886086): 14024-14029
        • Hause Jr., R.J.
        • Leung K.K.
        • Barkinge J.L.
        • Ciaccio M.F.
        • Chuu C.P.
        • Jones R.B.
        Comprehensive binary interaction mapping of SH2 domains via fluorescence polarization reveals novel functional diversification of ErbB receptors.
        PLoS One. 2012; 7 (22973453): e44471
        • Haslam N.J.
        • Shields D.C.
        Peptide-binding domains: are limp handshakes safest?.
        Sci. Signal. 2012; 5 (23012652): pe40
        • Mayer B.J.
        Dynamics of receptor tyrosine kinase signaling complexes.
        FEBS Lett. 2012; 586 (22584051): 2575-2579
        • Kraskouskaya D.
        • Duodu E.
        • Arpin C.C.
        • Gunning P.T.
        Progress towards the development of SH2 domain inhibitors.
        Chem. Soc. Rev. 2013; 42 (23396540): 3337-3370
        • Morlacchi P.
        • Robertson F.M.
        • Klostergaard J.
        • McMurray J.S.
        Targeting SH2 domains in breast cancer.
        Fut. Med. Chem. 2014; 6 (25495984): 1909-1926
        • Shi Z.D.
        • Lee K.
        • Liu H.
        • Zhang M.
        • Roberts L.R.
        • Worthy K.M.
        • Fivash M.J.
        • Fisher R.J.
        • Yang D.
        • Burke Jr., T.R.
        A novel macrocyclic tetrapeptide mimetic that exhibits low-picomolar Grb2 SH2 domain-binding affinity.
        Biochem. Biophys. Res. Commun. 2003; 310 (14521921): 378-383
        • Otwinowski Z.
        • Minor W.
        Processing of X-ray diffraction data collected in oscillation mode.
        Method Enzymol. 1997; 276 (27799103): 307-326
        • McCoy A.J.
        • Grosse-Kunstleve R.W.
        • Adams P.D.
        • Winn M.D.
        • Storoni L.C.
        • Read R.J.
        Phaser crystallographic software.
        J. Appl. Crystallogr. 2007; 40 (19461840): 658-674
        • Terwilliger T.C.
        • Grosse-Kunstleve R.W.
        • Afonine P.V.
        • Moriarty N.W.
        • Zwart P.H.
        • Hung L.W.
        • Read R.J.
        • Adams P.D.
        Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard.
        Acta Crystallogr. D Biol. Crystallogr. 2008; 64 (18094468): 61-69
        • Emsley P.
        • Lohkamp B.
        • Scott W.G.
        • Cowtan K.
        Features and development of Coot.
        Acta Crystallogr. D Biol. Crystallogr. 2010; 66 (20383002): 486-501
        • Adams P.D.
        • Afonine P.V.
        • Bunkóczi G.
        • Chen V.B.
        • Davis I.W.
        • Echols N.
        • Headd J.J.
        • Hung L.W.
        • Kapral G.J.
        • Grosse-Kunstleve R.W.
        • McCoy A.J.
        • Moriarty N.W.
        • Oeffner R.
        • Read R.J.
        • Richardson D.C.
        • et al.
        PHENIX: a comprehensive Python-based system for macromolecular structure solution.
        Acta Crystallogr. D Biol. Crystallogr. 2010; 66 (20124702): 213-221
        • Zhang Z.Y.
        • Maclean D.
        • Thieme-Sefler A.M.
        • Roeske R.W.
        • Dixon J.E.
        A continuous spectrophotometric and fluorimetric assay for protein tyrosine phosphatase using phosphotyrosine-containing peptides.
        Anal. Biochem. 1993; 211 (7686722): 7-15
        • Thurmond J.
        • Goodman J.L.
        • Strelets V.B.
        • Attrill H.
        • Gramates L.S.
        • Marygold S.J.
        • Matthews B.B.
        • Millburn G.
        • Antonazzo G.
        • Trovisco V.
        • Kaufman T.C.
        • Calvi B.R.
        • FlyBase Consortium
        FlyBase 2.0: the next generation.
        Nucleic Acids Res. 2019; 47 (30364959): D759-D765
        • Harris T.W.
        • Arnaboldi V.
        • Cain S.
        • Chan J.
        • Chen W.J.
        • Cho J.
        • Davis P.
        • Gao S.
        • Grove C.A.
        • Kishore R.
        • Lee R.Y.N.
        • Muller H.M.
        • Nakamura C.
        • Nuin P.
        • Paulini M.
        • et al.
        WormBase: a modern model organism information resource.
        Nucleic Acids Res. 2019; 48 (31642470): D762-D767
        • Grigoriev I.V.
        • Nikitin R.
        • Haridas S.
        • Kuo A.
        • Ohm R.
        • Otillar R.
        • Riley R.
        • Salamov A.
        • Zhao X.
        • Korzeniewski F.
        • Smirnova T.
        • Nordberg H.
        • Dubchak I.
        • Shabalov I.
        MycoCosm portal: gearing up for 1000 fungal genomes.
        Nucleic Acids Res. 2014; 42 (24297253): D699-D704
        • Katoh K.
        • Standley D.M.
        MAFFT multiple sequence alignment software version 7: improvements in performance and usability.
        Mol. Biol. Evol. 2013; 30 (23329690): 772-780
        • Waterhouse A.M.
        • Procter J.B.
        • Martin D.M.
        • Clamp M.
        • Barton G.J.
        Jalview version 2: a multiple sequence alignment editor and analysis workbench.
        Bioinformatics. 2009; 25 (19151095): 1189-1191
        • Meyer P.A.
        • Socias S.
        • Key J.
        • Ransey E.
        • Tjon E.C.
        • Buschiazzo A.
        • Lei M.
        • Botka C.
        • Withrow J.
        • Neau D.
        • Rajashankar K.
        • Anderson K.S.
        • Baxter R.H.
        • Blacklow S.C.
        • Boggon T.J.
        • et al.
        Data publication with the structural biology data grid supports live analysis.
        Nat. Commun. 2016; 7 (26947396): 10882