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John Kuriyan - One of the best experts on this subject based on the ideXlab platform.

  • A molecular assembly phase transition and kinetic proofreading modulate Ras activation by SOS.
    Science (New York N.Y.), 2019
    Co-Authors: William Y C Huang, John Kuriyan, Yasushi Kondo, Jean K Chung, Steven Alvarez, Young Kwang Lee, Hiu Yue Monatrice Lam, Kabir H. Biswas, Jay T Groves
    Abstract:

    The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) is a key Ras activator that is autoinhibited in the cytosol and activates upon membrane recruitment. Autoinhibition release involves structural rearrangements of the protein at the membrane and thus introduces a delay between initial recruitment and activation. In this study, we designed a single-molecule assay to resolve the time between initial receptor-mediated membrane recruitment and the initiation of GEF activity of individual SOS molecules on microarrays of Ras-functionalized supported membranes. The rise-and-fall shape of the measured SOS activation time distribution and the long mean time scale to activation (~50 seconds) establish a basis for kinetic proofreading in the receptor-mediated activation of Ras. We further demonstrate that this kinetic proofreading is modulated by the LAT (linker for activation of T cells)-Grb2-SOS phosphotyrosine-driven phase transition at the membrane.

  • the interdependent activation of Son of Sevenless and ras
    Cold Spring Harbor Perspectives in Medicine, 2019
    Co-Authors: Pradeep Bandaru, Yasushi Kondo, John Kuriyan
    Abstract:

    Author(s): Bandaru, Pradeep; Kondo, Yasushi; Kuriyan, John | Abstract: The guanine-nucleotide exchange factor (GEF) Son-of-Sevenless (SOS) plays a critical role in metazoan signaling by converting Ras•GDP (guanosine diphosphate) to Ras•GTP (guanosine triphosphate) in response to tyrosine kinase activation. Structural studies have shown that SOS differs from other Ras-specific GEFs in that SOS is itself activated by Ras•GTP binding to an allosteric site, distal to the site of nucleotide exchange. The activation of SOS involves membrane recruitment and conformational changes, triggered by lipid binding, that open the allosteric binding site for Ras•GTP. This is in contrast to other Ras-specific GEFs, which are activated by second messengers that more directly affect the active site. Allosteric Ras•GTP binding stabilizes SOS at the membrane, where it can turn over other Ras molecules processively, leading to an ultrasensitive response that is distinct from that of other Ras-specific GEFs.

  • The Interdependent Activation of Son-of-Sevenless and Ras
    Cold Spring Harbor perspectives in medicine, 2019
    Co-Authors: Pradeep Bandaru, Yasushi Kondo, John Kuriyan
    Abstract:

    The guanine-nucleotide exchange factor (GEF) Son-of-Sevenless (SOS) plays a critical role in metazoan signaling by converting Ras•GDP (guanosine diphosphate) to Ras•GTP (guanosine triphosphate) in response to tyrosine kinase activation. Structural studies have shown that SOS differs from other Ras-specific GEFs in that SOS is itself activated by Ras•GTP binding to an allosteric site, distal to the site of nucleotide exchange. The activation of SOS involves membrane recruitment and conformational changes, triggered by lipid binding, that open the allosteric binding site for Ras•GTP. This is in contrast to other Ras-specific GEFs, which are activated by second messengers that more directly affect the active site. Allosteric Ras•GTP binding stabilizes SOS at the membrane, where it can turn over other Ras molecules processively, leading to an ultrasensitive response that is distinct from that of other Ras-specific GEFs.

  • single molecule processivity of sos cat the catalytic core of the ras gef Son of Sevenless
    Biophysical Journal, 2011
    Co-Authors: Lars Iversen, Jodi Gureasko, John Kuriyan, Hsiunglin Tu, Jeffrey S Iwig, Jay T Groves
    Abstract:

    The Ras GEF Son of Sevenless (SOS) activates the membrane-anchored G-protein Ras by catalyzing the replacement of Ras bound GDP with GTP. We have previously shown that in addition to the catalytic site, SOS has a catalytically inactive allosteric binding site for Ras, which allows SOS to localize and up-concentrate at Ras presenting membranes, dramatically increasing the Ras-GDP turnover rate. Together, the catalytic and allosteric sites form the catalytic core of SOS (SOScat).In the present work we use TIRF fluorescence microscopy to show that in vitro, single SOScat enzymes can be highly processive, remaining surface bound via Ras in the allosteric site, while turning over hundreds to thousands of Ras with the catalytic site.By confining individual SOScat enzymes to micron-scale two-dimensional supported lipid bilayer ‘reaction chambers’ we can monitor arrays of hundreds to thousands of single SOScat enzymes, probing the variability in turnover rate and processivity within the enzyme ensemble. We show that only a small fraction of the SOScat enzymes are processive, and that this fraction is modulated by the nucleotide binding state of Ras, with Ras-GTP promoting a higher fraction of processive SOScat than Ras-GDP.

  • role of the histone domain in the autoinhibition and activation of the ras activator Son of Sevenless
    Proceedings of the National Academy of Sciences of the United States of America, 2010
    Co-Authors: Jodi Gureasko, Dafna Barsagi, Olga Kuchment, Holger Sondermann, Debora L Makino, John Kuriyan
    Abstract:

    Membrane-bound Ras is activated by translocation of the Son of Sevenless (SOS) protein to the plasma membrane. SOS is inactive unless Ras is bound to an allosteric site on SOS, and the Dbl homology (DH) and Pleckstrin homology (PH) domains of SOS (the DH-PH unit) block allosteric Ras binding. We showed previously that the activity of SOS at the membrane increases with the density of PIP2 and the local concentration of Ras-GTP, which synergize to release the DH-PH unit. Here we present a new crystal structure of SOS that contains the N-terminal histone domain in addition to the DH-PH unit and the catalytic unit (SOSHDFC, residues 1–1049). The structure reveals that the histone domain plays a dual role in occluding the allosteric site and in stabilizing the autoinhibitory conformation of the DH-PH unit. Additional insight is provided by kinetic analysis of the activation of membrane-bound Ras by mutant forms of SOS that contain mutations in the histone and the PH domains (E108K, C441Y, and E433K) that are associated with Noonan syndrome, a disease caused by hyperactive Ras signaling. Our results indicate that the histone domain and the DH-PH unit are conformationally coupled, and that the simultaneous engagement of the membrane by a PH domain PIP2-binding interaction and electrostatic interactions between a conserved positively charged patch on the histone domain and the negatively charged membrane coincides with a productive reorientation of SOS at the membrane and increased accessibility of both Ras binding sites on SOS.

Dafna Barsagi - One of the best experts on this subject based on the ideXlab platform.

  • role of the histone domain in the autoinhibition and activation of the ras activator Son of Sevenless
    Proceedings of the National Academy of Sciences of the United States of America, 2010
    Co-Authors: Jodi Gureasko, Dafna Barsagi, Olga Kuchment, Holger Sondermann, Debora L Makino, John Kuriyan
    Abstract:

    Membrane-bound Ras is activated by translocation of the Son of Sevenless (SOS) protein to the plasma membrane. SOS is inactive unless Ras is bound to an allosteric site on SOS, and the Dbl homology (DH) and Pleckstrin homology (PH) domains of SOS (the DH-PH unit) block allosteric Ras binding. We showed previously that the activity of SOS at the membrane increases with the density of PIP2 and the local concentration of Ras-GTP, which synergize to release the DH-PH unit. Here we present a new crystal structure of SOS that contains the N-terminal histone domain in addition to the DH-PH unit and the catalytic unit (SOSHDFC, residues 1–1049). The structure reveals that the histone domain plays a dual role in occluding the allosteric site and in stabilizing the autoinhibitory conformation of the DH-PH unit. Additional insight is provided by kinetic analysis of the activation of membrane-bound Ras by mutant forms of SOS that contain mutations in the histone and the PH domains (E108K, C441Y, and E433K) that are associated with Noonan syndrome, a disease caused by hyperactive Ras signaling. Our results indicate that the histone domain and the DH-PH unit are conformationally coupled, and that the simultaneous engagement of the membrane by a PH domain PIP2-binding interaction and electrostatic interactions between a conserved positively charged patch on the histone domain and the negatively charged membrane coincides with a productive reorientation of SOS at the membrane and increased accessibility of both Ras binding sites on SOS.

  • allosteric gating of Son of Sevenless activity by the histone domain
    Proceedings of the National Academy of Sciences of the United States of America, 2010
    Co-Authors: Kamlesh K Yadav, Dafna Barsagi
    Abstract:

    Regulated activation of Ras by receptor tyrosine kinases (RTK) constitutes a key transduction step in signaling processes that control an array of fundamental cellular functions including proliferation, differentiation, and survival. The principle mechanism by which Ras is activated down stream of RTKs involves the stimulation of guanine nucleotide exchange by the ubiquitous guanine nucleotide exchange factor Son of Sevenless (Sos). In resting conditions, Sos activity is constrained by intramolecular interactions that maintain the protein in an autoinhibited conformation. Structural, biochemical, and genetic studies have implicated the histone domain (Sos-H), which comprises the most N-terminal region of Sos, in the regulation of Sos autoinhibition. However, the molecular underpinnings of this regulatory function are not well understood. In the present study we demonstrate that Sos-H possesses in vitro and in vivo membrane binding activity that is mediated, in part, by the interactions between a cluster of basic residues and phosphatidic acid. This interaction is required for Sos-dependent activation of Ras following EGF stimulation. The inducible association of Sos-H with membranes contributes to the catalytic activity of Sos by forcing the domain to adopt a conformation that destabilizes the autoinhibitory state. Thus, Sos-H plays a critical role in governing the catalytic output of Sos through the coupling of membrane recruitment to the release of autoinhibition.

  • membrane dependent signal integration by the ras activator Son of Sevenless
    Nature Structural & Molecular Biology, 2008
    Co-Authors: Jodi Gureasko, Dafna Barsagi, William J Galush, Sean Boykevisch, Jay T Groves, Holger Sondermann, John Kuriyan
    Abstract:

    The kinetics of Ras activation by Son of Sevenless (SOS) changes profoundly when Ras is tethered to membranes, instead of being in solution. SOS has two binding sites for Ras, one of which is an allosteric site that is distal to the active site. The activity of the SOS catalytic unit (SOScat) is up to 500-fold higher when Ras is on membranes compared to rates in solution, because the allosteric Ras site anchors SOScat to the membrane. This effect is blocked by the N-terminal segment of SOS, which occludes the allosteric site. We show that SOS responds to the membrane density of Ras molecules, to their state of GTP loading and to the membrane concentration of phosphatidylinositol-4,5-bisphosphate (PIP2), and that the integration of these signals potentiates the release of autoinhibition.

  • computational docking and solution x ray scattering predict a membrane interacting role for the histone domain of the ras activator Son of Sevenless
    Proceedings of the National Academy of Sciences of the United States of America, 2005
    Co-Authors: Holger Sondermann, Dafna Barsagi, B Nagar, John Kuriyan
    Abstract:

    The Ras-specific nucleotide exchange factor Son of Sevenless (SOS) is a large, multidomain protein with complex regulation, including a Ras-dependent allosteric mechanism. The N-terminal segment of SOS, the histone domain, contains two histone folds, which is highly unusual for a cytoplasmic protein. Using a combination of computational docking, small-angle x-ray scattering, mutagenesis, and calorimetry, we show that the histone domain folds into the rest of SOS and docks onto a helical linker that connects the pleckstrin-homology (PH) and Dbl-homology (DH) domains of SOS to the catalytic domain. In this model, a positively charged surface region on the histone domain is positioned so as to provide a fourth potential anchorage site on the membrane for SOS in addition to the PH domain, the allosteric Ras molecule, and the C-terminal adapter-binding site. The histone domain in SOS interacts with the helical linker, using a region of the surface that in nucleosomes is involved in histone tetramerization. Adjacent surface elements on the histone domain that correspond to the DNA-binding surface of nucleosomes form the predicted interaction site with the membrane. The orientation and position of the histone domain in the SOS model implicates it as a potential mediator of membrane-dependent activation signals.

  • structural analysis of autoinhibition in the ras activator Son of Sevenless
    Cell, 2004
    Co-Authors: Holger Sondermann, Dafna Barsagi, S M Soisson, Sean Boykevisch, Shaosong Yang, John Kuriyan
    Abstract:

    Abstract The classical model for the activation of the nucleotide exchange factor Son of Sevenless (SOS) involves its recruitment to the membrane, where it engages Ras. The recent discovery that Ras•GTP is an allosteric activator of SOS indicated that the regulation of SOS is more complex than originally envisaged. We now present crystallographic and biochemical analyses of a construct of SOS that contains the Dbl homology-pleckstrin homology (DH-PH) and catalytic domains and show that the DH-PH unit blocks the allosteric binding site for Ras and suppresses the activity of SOS. SOS is dependent on Ras binding to the allosteric site for both a lower level of activity, which is a result of Ras•GDP binding, and maximal activity, which requires Ras•GTP. The action of the DH-PH unit gates a reciprocal interaction between Ras and SOS, in which Ras converts SOS from low to high activity forms as Ras•GDP is converted to Ras•GTP by SOS.

David D.l. Bowtell - One of the best experts on this subject based on the ideXlab platform.

  • Activation of Ras and its downstream extracellular signal-regulated protein kinases by the CDC25 homology domain of mouse Son-of-Sevenless 1 (mSos1).
    Oncogene, 1998
    Co-Authors: Jae-hoon Kim, David D.l. Bowtell, Tohru Kataoka, Mikako Shirouzu, Shigeyuki Yokoyama
    Abstract:

    A fragment consisting of residues 584-1071 of the mouse Son-of-Sevenless 1 (mSos1) protein was found to be sufficient for stimulation of the guanine nucleotide exchange of Ras in vitro, which defines the CDC25 homology (CDC25H) domain of mSos1. Furthermore, we found that the CDC25H-domain fragment activated the extracellular signal-regulated protein kinases (ERKs), and was mainly membrane localized, when expressed in unstimulated human embryonic kidney 293 cells. Then, we examined the roles of other mSos1 domains in autoinhibition of the CDC25H-domain functions in unstimulated cellular environments. First, longer fragments that have the CDC25H domain and the following proline-rich Grb2-binding domain exhibited negligible membrane localization, and accordingly much lower ERK-activation activities, under serum-starved conditions. On the other hand, the preceding Pleckstrin-homology (PH) domain affects neither the ERK-activation activity nor the membrane-localization activity of the CDC25H domain. By contrast, the cells expressing a fragment containing the Dbl homology (DH) domain in addition to the PH and CDC25H domains exhibited remarkably low ERK activities under serum-starved conditions. This autoinhibitory effect of the DH domain on the CDC25H-domain function was shown to be relieved when cells were stimulated with epidermal growth factor. The DH-domain extension affected neither the in vitro guanine nucleotide exchange activity nor the membrane-localization activity of the CDC25H domain. Therefore, one of the roles of the DH domain is to exert an autoinhibition over the CDC25H-domain function on the cell membrane, in the absence, but not in the presence, of extracellular stimuli.

  • activation of ras and its downstream extracellular signal regulated protein kinases by the cdc25 homology domain of mouse Son of Sevenless 1 msos1
    Oncogene, 1998
    Co-Authors: Mikako Shirouzu, David D.l. Bowtell, Tohru Kataoka, Shigeyuki Yokoyama
    Abstract:

    Activation of Ras and its downstream extracellular signal-regulated protein kinases by the CDC25 homology domain of mouse Son-of-Sevenless 1 (mSos1)

  • the solution structure of the pleckstrin homology domain of mouse Son of Sevenless 1 msos1
    Journal of Molecular Biology, 1997
    Co-Authors: S Koshiba, David D.l. Bowtell, Mikako Shirouzu, T Kigawa, Shigeyuki Yokoyama
    Abstract:

    Abstract The solution structure of the pleckstrin homology (PH) domain of mouse Son-of-Sevenless 1 (mSos1), a guanine nucleotide exchange factor for Ras, was determined by multidimensional NMR spectroscopy. The structure of the mSos1 PH domain involves the fundamental PH fold, consisting of seven β-strands and one α-helix at the C terminus, as determined for the PH domains of other proteins. By contrast, the mSos1 PH domain showed two major characteristic features. First, the N-terminal region, whose amino acid sequence is highly conserved among Sos proteins, was found to form an α-helix, which interacts with the β-sheet structure of the fundamental PH fold. Second, there is a long unstructured loop between β3 and β4. Furthermore, the mSos1 PH domain was found to bind phosphatidylinositol-4,5-bisphosphate by a centrifugation assay. The addition of inositol-1,4,5-trisphosphate to the mSos1 PH domain induced backbone amide chemical shift changes mainly in the β1/β2 loop and the N- and C-terminal parts of the long β3/β4 loop. This inositol-1,4,5-trisphosphate-binding mode of the mSos1 PH domain is somewhat similar to those of the PH domains of pleckstrin and phospholipase Cδ 1 , and is clearly different from those of other PH domains.

  • mammalian homologues of the drosophila Son of Sevenless gene map to murine chromosomes 17 and 12 and to human chromosomes 2 and 14 respectively
    Genomics, 1993
    Co-Authors: Graham C Webb, Caroline S Fernandez, Nancy A Jenkins, Neal G Copeland, David A Largaespada, David D.l. Bowtell
    Abstract:

    Abstract Activating mutations in the ras genes are commonly found in a wide range of human tumors. We recently cloned two mammalian genes, Son of Sevenless 1 (mSos1) and Son of Sevenless 2 (mSos2) , whose protein products appear to be important positive regulators of ras proteins. Given the proposed role of Sos proteins in ras regulation, and the frequent occurrence of activated ras alleles in tumor cells, we were interested in determining whether the Sos genes may also be activated inappropriately by DNA rearrangement in tumor cells. To investigate this possibility, we have determined the chromosomal locations of both the mouse and the human Sos1 and Sos2 genes, using a combination of genetic linkage analysis and in situ hybridization to chromosomal spreads. We find that the murine Sos1 and Sos2 genes map to chromosomes 17E and 12C3.3-D and their human counterparts to chromosomes 2p21-2p2 and 14q21, respectively. Neither the human nor the mouse Sos loci map close to known mutations or to regions showing consistent karyotypic abnormalities in tumor cells.

  • mammalian homologues of the drosophila Son of Sevenless gene map to murine chromosomes 17 and 12 and to human chromosomes 2 and 14 respectively
    Genomics, 1993
    Co-Authors: Graham C Webb, Caroline S Fernandez, Nancy A Jenkins, Neal G Copeland, David A Largaespada, David D.l. Bowtell
    Abstract:

    Abstract Activating mutations in the ras genes are commonly found in a wide range of human tumors. We recently cloned two mammalian genes, Son of Sevenless 1 (mSos1) and Son of Sevenless 2 (mSos2) , whose protein products appear to be important positive regulators of ras proteins. Given the proposed role of Sos proteins in ras regulation, and the frequent occurrence of activated ras alleles in tumor cells, we were interested in determining whether the Sos genes may also be activated inappropriately by DNA rearrangement in tumor cells. To investigate this possibility, we have determined the chromosomal locations of both the mouse and the human Sos1 and Sos2 genes, using a combination of genetic linkage analysis and in situ hybridization to chromosomal spreads. We find that the murine Sos1 and Sos2 genes map to chromosomes 17E and 12C3.3-D and their human counterparts to chromosomes 2p21-2p2 and 14q21, respectively. Neither the human nor the mouse Sos loci map close to known mutations or to regions showing consistent karyotypic abnormalities in tumor cells.

Shigeyuki Yokoyama - One of the best experts on this subject based on the ideXlab platform.

  • Activation of Ras and its downstream extracellular signal-regulated protein kinases by the CDC25 homology domain of mouse Son-of-Sevenless 1 (mSos1).
    Oncogene, 1998
    Co-Authors: Jae-hoon Kim, David D.l. Bowtell, Tohru Kataoka, Mikako Shirouzu, Shigeyuki Yokoyama
    Abstract:

    A fragment consisting of residues 584-1071 of the mouse Son-of-Sevenless 1 (mSos1) protein was found to be sufficient for stimulation of the guanine nucleotide exchange of Ras in vitro, which defines the CDC25 homology (CDC25H) domain of mSos1. Furthermore, we found that the CDC25H-domain fragment activated the extracellular signal-regulated protein kinases (ERKs), and was mainly membrane localized, when expressed in unstimulated human embryonic kidney 293 cells. Then, we examined the roles of other mSos1 domains in autoinhibition of the CDC25H-domain functions in unstimulated cellular environments. First, longer fragments that have the CDC25H domain and the following proline-rich Grb2-binding domain exhibited negligible membrane localization, and accordingly much lower ERK-activation activities, under serum-starved conditions. On the other hand, the preceding Pleckstrin-homology (PH) domain affects neither the ERK-activation activity nor the membrane-localization activity of the CDC25H domain. By contrast, the cells expressing a fragment containing the Dbl homology (DH) domain in addition to the PH and CDC25H domains exhibited remarkably low ERK activities under serum-starved conditions. This autoinhibitory effect of the DH domain on the CDC25H-domain function was shown to be relieved when cells were stimulated with epidermal growth factor. The DH-domain extension affected neither the in vitro guanine nucleotide exchange activity nor the membrane-localization activity of the CDC25H domain. Therefore, one of the roles of the DH domain is to exert an autoinhibition over the CDC25H-domain function on the cell membrane, in the absence, but not in the presence, of extracellular stimuli.

  • activation of ras and its downstream extracellular signal regulated protein kinases by the cdc25 homology domain of mouse Son of Sevenless 1 msos1
    Oncogene, 1998
    Co-Authors: Mikako Shirouzu, David D.l. Bowtell, Tohru Kataoka, Shigeyuki Yokoyama
    Abstract:

    Activation of Ras and its downstream extracellular signal-regulated protein kinases by the CDC25 homology domain of mouse Son-of-Sevenless 1 (mSos1)

  • the solution structure of the pleckstrin homology domain of mouse Son of Sevenless 1 msos1
    Journal of Molecular Biology, 1997
    Co-Authors: S Koshiba, David D.l. Bowtell, Mikako Shirouzu, T Kigawa, Shigeyuki Yokoyama
    Abstract:

    Abstract The solution structure of the pleckstrin homology (PH) domain of mouse Son-of-Sevenless 1 (mSos1), a guanine nucleotide exchange factor for Ras, was determined by multidimensional NMR spectroscopy. The structure of the mSos1 PH domain involves the fundamental PH fold, consisting of seven β-strands and one α-helix at the C terminus, as determined for the PH domains of other proteins. By contrast, the mSos1 PH domain showed two major characteristic features. First, the N-terminal region, whose amino acid sequence is highly conserved among Sos proteins, was found to form an α-helix, which interacts with the β-sheet structure of the fundamental PH fold. Second, there is a long unstructured loop between β3 and β4. Furthermore, the mSos1 PH domain was found to bind phosphatidylinositol-4,5-bisphosphate by a centrifugation assay. The addition of inositol-1,4,5-trisphosphate to the mSos1 PH domain induced backbone amide chemical shift changes mainly in the β1/β2 loop and the N- and C-terminal parts of the long β3/β4 loop. This inositol-1,4,5-trisphosphate-binding mode of the mSos1 PH domain is somewhat similar to those of the PH domains of pleckstrin and phospholipase Cδ 1 , and is clearly different from those of other PH domains.

Holger Sondermann - One of the best experts on this subject based on the ideXlab platform.

  • role of the histone domain in the autoinhibition and activation of the ras activator Son of Sevenless
    Proceedings of the National Academy of Sciences of the United States of America, 2010
    Co-Authors: Jodi Gureasko, Dafna Barsagi, Olga Kuchment, Holger Sondermann, Debora L Makino, John Kuriyan
    Abstract:

    Membrane-bound Ras is activated by translocation of the Son of Sevenless (SOS) protein to the plasma membrane. SOS is inactive unless Ras is bound to an allosteric site on SOS, and the Dbl homology (DH) and Pleckstrin homology (PH) domains of SOS (the DH-PH unit) block allosteric Ras binding. We showed previously that the activity of SOS at the membrane increases with the density of PIP2 and the local concentration of Ras-GTP, which synergize to release the DH-PH unit. Here we present a new crystal structure of SOS that contains the N-terminal histone domain in addition to the DH-PH unit and the catalytic unit (SOSHDFC, residues 1–1049). The structure reveals that the histone domain plays a dual role in occluding the allosteric site and in stabilizing the autoinhibitory conformation of the DH-PH unit. Additional insight is provided by kinetic analysis of the activation of membrane-bound Ras by mutant forms of SOS that contain mutations in the histone and the PH domains (E108K, C441Y, and E433K) that are associated with Noonan syndrome, a disease caused by hyperactive Ras signaling. Our results indicate that the histone domain and the DH-PH unit are conformationally coupled, and that the simultaneous engagement of the membrane by a PH domain PIP2-binding interaction and electrostatic interactions between a conserved positively charged patch on the histone domain and the negatively charged membrane coincides with a productive reorientation of SOS at the membrane and increased accessibility of both Ras binding sites on SOS.

  • differences in flexibility underlie functional differences in the ras activators Son of Sevenless and ras guanine nucleotide releasing factor 1
    Structure, 2009
    Co-Authors: Tanya S. Freedman, Gregory D. Friedland, Tanja Kortemme, Olga Kuchment, Holger Sondermann, John Kuriyan
    Abstract:

    The Ras-specific nucleotide exchange factor Son of Sevenless (Sos) is inactive without Ras bound to a distal allosteric site. In contrast, the catalytic domain of Ras guanine nucleotide releasing factor 1 (RasGRF1) is active intrinsically. By substituting residues from RasGRF1 into Sos, we have generated mutants of Sos with basal activity, partially relieved of their dependence on allosteric activation. We have performed molecular dynamics simulations showing how Ras binding to the allosteric site leads to a bias toward the active conformation of Sos. The trajectories show that Sos fluctuates between active and inactive conformations in the absence of Ras and that the activating mutations favor conformations of Sos that are more permissive to Ras binding at the catalytic site. In contrast, unliganded RasGRF1 fluctuates primarily among active conformations. Our results support the premise that the catalytic domain of Sos has evolved an allosteric activation mechanism that extends beyond the simple process of membrane recruitment.

  • membrane dependent signal integration by the ras activator Son of Sevenless
    Nature Structural & Molecular Biology, 2008
    Co-Authors: Jodi Gureasko, Dafna Barsagi, William J Galush, Sean Boykevisch, Jay T Groves, Holger Sondermann, John Kuriyan
    Abstract:

    The kinetics of Ras activation by Son of Sevenless (SOS) changes profoundly when Ras is tethered to membranes, instead of being in solution. SOS has two binding sites for Ras, one of which is an allosteric site that is distal to the active site. The activity of the SOS catalytic unit (SOScat) is up to 500-fold higher when Ras is on membranes compared to rates in solution, because the allosteric Ras site anchors SOScat to the membrane. This effect is blocked by the N-terminal segment of SOS, which occludes the allosteric site. We show that SOS responds to the membrane density of Ras molecules, to their state of GTP loading and to the membrane concentration of phosphatidylinositol-4,5-bisphosphate (PIP2), and that the integration of these signals potentiates the release of autoinhibition.

  • A Ras-induced conformational switch in the Ras activator Son of Sevenless
    Proceedings of the National Academy of Sciences of the United States of America, 2006
    Co-Authors: Tanya S. Freedman, Gregory D. Friedland, Tanja Kortemme, Dafna Bar-sagi, Holger Sondermann, Susan Marqusee, John Kuriyan
    Abstract:

    The Ras-specific guanine nucleotide-exchange factors Son of Sevenless (Sos) and Ras guanine nucleotide-releasing factor 1 (RasGRF1) transduce extracellular stimuli into Ras activation by catalyzing the exchange of Ras-bound GDP for GTP. A truncated form of RasGRF1 containing only the core catalytic Cdc25 domain is sufficient for stimulating Ras nucleotide exchange, whereas the isolated Cdc25 domain of Sos is inactive. At a site distal to the catalytic site, nucleotide-bound Ras binds to Sos, making contacts with the Cdc25 domain and with a Ras exchanger motif (Rem) domain. This allosteric Ras binding stimulates nucleotide exchange by Sos, but the mechanism by which this stimulation occurs has not been defined. We present a crystal structure of the Rem and Cdc25 domains of Sos determined at 2.0-A resolution in the absence of Ras. Differences between this structure and that of Sos bound to two Ras molecules show that allosteric activation of Sos by Ras occurs through a rotation of the Rem domain that is coupled to a rotation of a helical hairpin at the Sos catalytic site. This motion relieves steric occlusion of the catalytic site, allowing substrate Ras binding and nucleotide exchange. A structure of the isolated RasGRF1 Cdc25 domain determined at 2.2-A resolution, combined with computational analyses, suggests that the Cdc25 domain of RasGRF1 is able to maintain an active conformation in isolation because the helical hairpin has strengthened interactions with the Cdc25 domain core. These results indicate that RasGRF1 lacks the allosteric activation switch that is crucial for Sos activity.

  • computational docking and solution x ray scattering predict a membrane interacting role for the histone domain of the ras activator Son of Sevenless
    Proceedings of the National Academy of Sciences of the United States of America, 2005
    Co-Authors: Holger Sondermann, Dafna Barsagi, B Nagar, John Kuriyan
    Abstract:

    The Ras-specific nucleotide exchange factor Son of Sevenless (SOS) is a large, multidomain protein with complex regulation, including a Ras-dependent allosteric mechanism. The N-terminal segment of SOS, the histone domain, contains two histone folds, which is highly unusual for a cytoplasmic protein. Using a combination of computational docking, small-angle x-ray scattering, mutagenesis, and calorimetry, we show that the histone domain folds into the rest of SOS and docks onto a helical linker that connects the pleckstrin-homology (PH) and Dbl-homology (DH) domains of SOS to the catalytic domain. In this model, a positively charged surface region on the histone domain is positioned so as to provide a fourth potential anchorage site on the membrane for SOS in addition to the PH domain, the allosteric Ras molecule, and the C-terminal adapter-binding site. The histone domain in SOS interacts with the helical linker, using a region of the surface that in nucleosomes is involved in histone tetramerization. Adjacent surface elements on the histone domain that correspond to the DNA-binding surface of nucleosomes form the predicted interaction site with the membrane. The orientation and position of the histone domain in the SOS model implicates it as a potential mediator of membrane-dependent activation signals.