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Structural analysis of autoinhibition in the Ras-specific exchange factor RasGRP1


Jeffrey S. Iwig, Yvonne Vercoulen, Rahul Das, Tiago Barros, Andre Limnander, Yan Che, Jeffrey G. Pelton, David E. Wemmer, Jeroen P. Roose and John Kuriyan


eLife.00813 (local copy)

Abstract / Figures from the paper / Supplemental Material / References



Abstract

RasGRP1 and SOS are Ras-specific nucleotide exchange factors that have distinct roles in lymphocyte development. RasGRP1 is important in some cancers and autoimmune diseases but, in contrast to SOS, its regulatory mechanisms are poorly understood. Activating signals lead to the membrane recruitment of RasGRP1 and Ras engagement, but it is unclear how interactions between RasGRP1 and Ras are suppressed in the absence of such signals. We present a crystal structure of a fragment of RasGRP1 in which the Ras-binding site is blocked by an interdomain linker and the membrane-interaction surface of RasGRP1 is hidden within a dimerization interface that may be stabilized by the C-terminal oligomerization domain. NMR data demonstrate that calcium binding to the regulatory module generates substantial conformational changes that are incompatible with the inactive assembly. These features allow RasGRP1 to be maintained in an inactive state that is poised for activation by calcium and membrane-localization signals.


Figures from the paper

Click on the small image to get a bigger one.

First figure from paper

Figure 1. Control of Ras activity in T cells

A) Ras cycles between an inactive, GDP-bound form and an active GTP-bound form. In T cells, two nucleotide exchange factors, SOS and RasGRP1 enhance the removal of nucleotide from Ras, which is then replaced with GTP. Each exchange factor shares a common catalytic module but is regulated by distinct signaling inputs. SOS activity is enhanced by Ras-GTP, generated by RasGRP1, binding to an allosteric site. The regulatory domains from each exchange factor are distinct and are represented in gray. SOS is recruited to the membrane in part by Grb2, which interacts with phosphotyrosine residues in the adapter LAT.

B) The catalytic core of RasGRP1 includes the REM and Cdc25 domains, which are followed by a regulatory module containing the EF domain, membrane binding C1 domain and a predicted coiled coil. An alternate translational start site is present that leads to a RasGRP1 protein without the first 49 residues. The constructs used in this study are shown.




Second figure from paper

Figure 2. Crystal structure of the RasGRP1 autoinhibited catalytic module

A) A crystal structure of the first four domains of RasGRP1 shows the REM domain (blue) buttressing the helical hairpin of the Cdc25 domain (green). The EF domain (magenta) is sandwiched between one side of the Cdc25 domain and the C1 domain (teal). Two zinc ions in the C1 domain are shown as gray spheres. The Cdc25-EF linker (red) traverses the Ras-binding site on the Cdc25 domain. Linkers that could not be modeled due to poor electron density are shown with dotted lines. The N- and C-termini are indicated by N and C, respectively.

B) The C1 domain mediates formation of a crystallographic dimer. The domains of one monomer are denoted with primes.









Third figure from paper

Figure 3. Comparison of the catalytic modules of SOS and RasGRP1

A) Structures of SOScat in the inactive state (PDB ID:2II0) (left) and active state (PDB ID: 1NVV) (right) bound to Ras (orange) at the active and allosteric sites are shown. The switch 1 and switch 2 elements of Ras are shown in purple.

B) The architecture of the catalytic module of RasGRP1 is similar to that of SOS, indicating that active site Ras will bind to at a similar location in the Cdc25 domain.

C) The helical hairpins of inactive SOS (purple), active SOS (yellow) and RasGRP1 (green) are shown with the core of the RasGRP1 Cdc25 domain (gray) and Ras modeled at the active site of RasGRP1 (orange). The helical hairpin of RasGRP1 is rotated ~25° away from Ras relative to the helical hairpin of SOS in the inactive state. In this conformation, the helical hairpin of SOS occludes the Ras binding site.


Fourth figure from paper

Figure 4. The C-terminal domain mediates oligomerization

A) A parallel, dimeric coiled coil is formed by ~40 residues/monomer near the C-terminus of RasGRP1. The individual monomers within the dimer are colored yellow and light yellow. Two pairs of bridging ionic interactions (dotted ellipses), and a Leu/Ile-rich core stabilize the dimer.

B) The coiled coil may stabilize the autoinhibited catalytic module. The ~140 residues between the C1 domain and the coiled coil (gray) are predicted to be primarily unstructured.











Fifth figure from paper

Figure 5. The Cdc25-EF linker inhibits RasGRP1

A) The RasGRP1 Cdc25 domain (left) is shown with the Cdc25-EF linker (red) and Ras modeled using the Ras-SOS complex (right). Tyr 64 of switch 2 of Ras makes crucial contacts at the base of the SOS helical hairpin.

B) Sequence alignment of the Cdc25-EF linker region of different RasGRP proteins reveals partial (light blue) and complete (yellow) conservation of amino acids important for autoinhibition.

C) The in vitro nucleotide exchange activities of different RasGRP1 proteins (10 μM) were compared with 500 nM Ras in solution. Error bars represent ± standard deviation.

D) FACS measurements were used to compare ERK phosphorylation in cells expressing full length RasGRP1 (wild type) or mutant proteins with two (Linker 2A), three (Linker 3D) or five mutations (Linker 5A) to the Cdc25-EF linker as a function of expression level. R271E is a catalytically dead mutant that is shown for reference. The average levels are shown with error bars that represent ± SEM.




Sixth figure from paper

Figure 6. RasGRP1 forms an autoinhibited dimer

A) The C1 domain of RasGRP1 (middle) is structurally similar to the C1 domain of PKCδ (PDB ID: 1PTR), shown here bound to a diacylglycerol mimic (phorbol ester) (yellow) (left). The membrane-interacting residues of the C1 domain (teal sticks) are buried at a dimer interface in the RasGRP1 structure (right).

B) The dimer interface mediated by the C1 domain buries ~2500 Å2 of accessible surface area. The close-up view of the dimerization surface (right) highlights the importance of the EF2 connector (magenta sticks) as it interacts with the membrane-inserting residues of the C1 domain (teal sticks). C) Dimerization mutants were expressed in Jurkat T cells and P-ERK levels were measured by FACS and compared with wild type RasGRP1. R271E is a catalytically dead mutant that is shown for reference. The average levels are shown with error bars that represent ± SEM.










Seventh figure from paper

Figure 7. RasGRP1 displays relatively weak nucleotide exchange activity

The activities of RasGRFCdc25, RasGRP1cat and SOScat were measured in vitro with A) Ras in solution (500 nM), or with B) Ras-coupled vesicles using mant-dGDP fluorescence. Error bars represent ± standard deviation. C) Phosphorylated ERK (P-ERK) levels were measured using FACS as a function of nucleotide exchange factor concentration for RasGRP1cat and SOScat. Representative dot plots are shown (top) with the gates used for quantitation (bottom). Error bars represent ± SEM.





Seventh figure from paper

Figure 8. Diacylglycerol activates RasGRP1

The activity of RasGRP1CEC at 500 nM and 5 μM was measured with Ras in solution, Ras coupled to vesicles containing phosphatidyl choline (PC) or Ras coupled to vesicles with PC with phosphatidyl serine (PS) and diacylglycerol (DAG).









Seventh figure from paper

Figure 9. The RasGRP1 EF hands adopt a closed conformation

The C-terminal domain of calcium-bound calmodulin (PDB ID: 1CLL) is shown on the left. Helices are denoted by the letters A-D. Comparison to apo calmodulin (PDB ID: 1CFD) (middle) shows that calcium (purple sphere) induces a dramatic rearrangement in the helix orientations for both EF hands. The conformation of the EF domain of RasGRP1 (right) is similar to that of apo calmodulin. The four sidechains that directly contact the Ca2+ are shown in sticks. The numbering refers to the positions in the canonical calcium-binding loops as shown in Figure 10. The glutamate at position 12 is rotated away from the other metal-binding residues in apo calmodulin and RasGRP1. Unlike other EF-hand pairs, RasGRP1 lacks the entering helix in EF2 (helix C), which is replaced by a short, hydrophobic linker (EF2 connector, orange).


Seventh figure from paper

Figure 10. Calcium binds to the EF domain of RasGRP1

A) Sequence alignment of the four human RasGRP proteins shows differences in the residues that directly contact metal ions in canonical EF hands (black boxes) and in the number of residues between the two loops. Numbering refers to human RasGRP1.

B) A representative ITC curve from 700 μM CaCl2 titrated into 50 μM RasGRP1EF at 20 °C is shown with the baseline corrected raw data (top). The integrated heats of interaction are fit to a one set of sites model (bottom) with average fitting parameters listed in Table 3.

C) Relative P-ERK levels are shown for wild type RasGRP1 and proteins with mutations to EF1 after stimulation with ionomycin. The 3DA mutant contains alanine mutations at the 1, 3 and 5 positions in the calcium-binding loop of EF1. The E494A mutant contains an alanine at position 12 in the calcium-binding loop of EF1. Measurements for the mutants were normalized to levels for the same construct without ionomycin and plotted as the fraction of activation compared to WT for the lowest RasGRP1 expression gate. Similar results were obtained at higher expression levels, but are omitted for clarity. The average levels are shown with error bars that represent ± SEM.


Seventh figure from paper

Figure 11. Calcium binding induces a large conformational change in the EF domain

The helices of the EF domains of apo RasGRP1 (left) and calcium-bound RasGRP2 (right) are shown with the Cdc25 domain of RasGRP1. The two EF domains were superimposed using helix B, which contains three hydrophobic residues (spheres) that interact extensively with the Cdc25 domain. Using this frame of reference, the angle between helices A and B changes by ~40° upon calcium binding. The conformational change in the EF domain could disrupt the docking of the Cdc25-EF linker (red). The footprint of the C1 domain is shown with a dotted black line.


Seventh figure from paper

Figure 12. Model of RasGRP1 activation

Inactive RasGRP1 (left) is stabilized by the C1-dimer interface, which sequesters the membrane-interacting surface of the C1 domain, and the active-site blocking Cdc25-EF linker (red). The C-terminal coiled coil stabilizes the dimer, thereby preventing inappropriate Ras activation. The autoinhibited form is activated by multiple signaling inputs that enhance nucleotide exchange activity (right). Diaclyglycerol binding disrupts C1 dimerization, while Ca2+ binding to EF1 causes a conformational change that contributes to C1 reorientation, and the release of the inhibitory segment from the Ras-binding surface. Phosphorylation of the Cdc25 domain could aid in removal of the inhibitory linker.






Supplemental Material


First supplimental figure
Figure 1-figure supplement 1. Domain architecture of RasGRP1 and SOS.

RasGRP1 and SOS contain similar catalytic cores (REM + Cdc25 domains), but differ in the flanking regulatory domains.



First supplimental figure
Figure 2-figure supplement 1. The Cdc25-EF linker occupies the Ras binding site in the Cdc25 domain.
The electron density from a kick omit map without the Cdc25-EF linker is shown in orange mesh at 2.5σ (Fo-Fc).










First supplimental figure
Figure 5-figure supplement 1. Cellular analysis of RasGRP1 nucleotide exchange activity.
(1) Jurkat cells expressing low endogenous levels of RasGRP1 are transfected with plasmid DNA encoding myc-tagged wild type RasGRP1 or RasGRP1 variants. (2) Cells rest for five hours after transfection. (3) Each cell displays a different level of myc-RasGRP1 protein, resulting in a population of cells with a range of expression levels. myc-RasGRP1 will activate Ras, which leads to an increased level of phosphorylated ERK (P-ERK) (4) Fluorochrome-conjugated antibodies are applied to detect myc-tagged RasGRP1 and P-ERK. (5) Fluorescence activated cell sorting (FACS) is used to measure expression levels of P-ERK and myc-tagged RasGRP1 in each cell. Each point in the dot plot represents the P-ERK level of a single cell as a function of the RasGRP1 expression level. (6) Cells are divided in four different groups based on the expression of myc-tagged RasGRP1: untransfected, low, medium (med), and high expression. (7) For each group of cells the geometric mean (mean fluorescence intensity) of the P-ERK level is determined and normalized to the P-ERK level in untransfected cells from the wild type RasGRP1 experiment. The normalized P-ERK levels for each bin are then plotted for each RasGRP1 protein.


First supplimental figure
Figure 6-figure supplement 1. Oligomerization of RasGRP1CEC.
The gel filtration elution profiles of RasGRP1CEC were obtained with 10 mM or 150 mM protein in 25 mM Tris (pH 8.5) 100 mM NaCl 10% glycerol 1 mM TCEP. The elution peak positions for molecular weight standards of indicated size are shown with arrows. RasGRP1CEC forms multiple oligomeric states and the distribution is dependent on the protein concentration.







First supplimental figure
Figure 10-figure supplement 1. Sequence alignment of EF-hand proteins.
RasGRP1 contains significantly fewer amino acids between the two loops of the EF hands as compared to other EF hand-containing proteins. Positions of canonical metal-binding sidechains are highlighted in yellow. Residues in the loops of each EF hand (gray lines) are numbered 1-12.


First supplimental figure
Figure 10-figure supplement 2. RasGRP1EF undergoes a significant conformational change upon calcium binding.
1H - 15N HSQC spectra are shown for 250 μM RasGRP1EF with 5 mM EDTA (top) or 400 μM CaCl2 (bottom). Higher calcium concentrations did not significantly change the spectrum except for a further broadening of resonances. The arrow highlights the single resonance that is diagnostic of a glycine residue at position 6 in calcium-bound EF loops. Spectra were obtained in 25 mM Tris (pH 7.0) 100 mM NaCl 1 mM TCEP 15 mM β-octylglucoside.














First supplimental figure
Figure 10-figure supplement 3. Metal ions bind to the EF1 module of RasGRP1.
Tb3+ binding was measured using FRET between tryptophan and tyrosine residues and bound Tb3+ ions for wild type RasGRP1CEC and RasGRP1CEC with mutations of three Asp residues (positions 1, 3, 5) in EF1 or EF2 to Ala. Affinities for RasGRP1CEC and the EF2 mutant were determined by fitting the data to a single binding site model. Measurements were made in 25 mM Tris (pH 8.0), 100 mM NaCl, 10% glycerol and 1 mM TCEP.







First supplimental figure
Figure 10-figure supplement 4. Circular dichroism of RasGRP1EF.
Both metal free- and Ca2+-RasGRP1EF (A) and RasGRP2EF (B) show minima near 222 nm and 208 nm as expected for a folded, helical protein. A small increase in helicity is observed upon calcium addition for both proteins, which could reflect a stabilization of secondary structure. Spectra were obtained in 10 mM Tris (pH 7.5) 100 mM NaCl.



First supplimental figure
Figure 10-figure supplement 5. Calcium binding to RasGRP2EF induces a significant conformational change.
1H - 15N HSQC spectra are shown for 500 μM RasGRP2EF with 5 mM EDTA (top) or 1.2 mM CaCl2 (bottom). The two arrows highlight the resonances that are diagnostic of glycine residues at position 6 in calcium-bound EF loops. The backbone resonance assignments are labeled.














First supplimental figure
Figure 10-figure supplement 6. RasGRP2EF binds two calcium ions.
A representative ITC curve is shown for titration of 50 μM RasGRP2EF with 1 mM CaCl2. The integrated heats were fit with a one set of sites model. B) The averaged fitting parameters are compared with those of RasGRP1EF.















First supplimental figure
Figure 11-figure supplement 1. NMR analysis of Ca RasGRP2EF.
A) The chemical shift index (top) was calculated using TALOS+ and is shown as a function residue number. Positive values represent an α-helical propensity (cylinders), while negative values are indicative of β-strand (arrows). Estimated order parameters (S2) were derived from the random coil index and are plotted versus residue number. B) A structural ensemble of the 10 lowest energy structures for Ca-RasGRP2EF was determined as described in the materials and methods. Helices are labeled with the letters A-D.











First supplimental figure
Figure 11-figure supplement 2. Comparison of apo RasGRP1EF and Ca RasGRP2EF.
The EF domains of apo RasGRP1 and Ca2+-bound RasGRP2 are shown superimposed using helix B (blue). In this orientation, helix A (green) from Ca2+-bound RasGRP2 is rotated ~40° away from helix B, relative to the conformation of apo RasGRP1.










References


1. Dower NA, et al. (2000) RasGRP is essential for mouse thymocyte differentiation and TCR signaling. Nature immunology 1(4):317-321.

2. Ebinu JO, et al. (2000) RasGRP links T-cell receptor signaling to Ras. Blood 95(10):3199-3203.

3. Kortum RL, et al. (2011) Targeted Sos1 deletion reveals its critical role in early T-cell development. Proceedings of the National Academy of Sciences of the United States of America 108(30):12407-12412.

4. Ahearn IM, Haigis K, Bar-Sagi D, & Philips MR (2012) Regulating the regulator: post-translational modification of RAS. Nature reviews. Molecular cell biology 13(1):39-51.

5. Rajalingam K, Schreck R, Rapp UR, & Albert S (2007) Ras oncogenes and their downstream targets. Biochimica et biophysica acta 1773(8):1177-1195.

6. Vetter IR & Wittinghofer A (2001) The guanine nucleotide-binding switch in three dimensions. Science (New York, N.Y.) 294(5545):1299-1304.

7. Bos JL, Rehmann H, & Wittinghofer A (2007) GEFs and GAPs: critical elements in the control of small G proteins. Cell 129(5):865-877.

8. Cherfils J & Zeghouf M (2013) Regulation of small GTPases by GEFs, GAPs, and GDIs. Physiological reviews 93(1):269-309.

9. Stone JC (2011) Regulation and Function of the RasGRP Family of Ras Activators in Blood Cells. Genes & cancer 2(3):320-334.

10. Aiba Y, et al. (2004) Activation of RasGRP3 by phosphorylation of Thr-133 is required for B cell receptor-mediated Ras activation. Proceedings of the National Academy of Sciences of the United States of America 101(47):16612-16617.

11. Limnander A, et al. (2011) STIM1, PKC-delta and RasGRP set a threshold for proapoptotic Erk signaling during B cell development. Nature immunology 12(5):425-433.

12. Roose JP, Mollenauer M, Gupta VA, Stone J, & Weiss A (2005) A diacylglycerol-protein kinase C-RasGRP1 pathway directs Ras activation upon antigen receptor stimulation of T cells. Molecular and cellular biology 25(11):4426-4441.

13. Coughlin JJ, Stang SL, Dower NA, & Stone JC (2005) RasGRP1 and RasGRP3 regulate B cell proliferation by facilitating B cell receptor-Ras signaling. Journal of immunology (Baltimore, Md. : 1950) 175(11):7179-7184.

14. Brodie C, et al. (2004) PKCdelta associates with and is involved in the phosphorylation of RasGRP3 in response to phorbol esters. Molecular pharmacology 66(1):76-84.

15. Roose JP, Mollenauer M, Ho M, Kurosaki T, & Weiss A (2007) Unusual interplay of two types of Ras activators, RasGRP and SOS, establishes sensitive and robust Ras activation in lymphocytes. Molecular and cellular biology 27(7):2732-2745.

16. Diez FR, et al. (2009) RasGRP1 transgenic mice develop cutaneous squamous cell carcinomas in response to skin wounding: potential role of granulocyte colony-stimulating factor. The American journal of pathology 175(1):392-399.

17. Luke CT, Oki-Idouchi CE, Cline JM, & Lorenzo PS (2007) RasGRP1 overexpression in the epidermis of transgenic mice contributes to tumor progression during multistage skin carcinogenesis. Cancer research 67(21):10190-10197.

18. Oki-Idouchi CE & Lorenzo PS (2007) Transgenic overexpression of RasGRP1 in mouse epidermis results in spontaneous tumors of the skin. Cancer research 67(1):276-280.

19. Yang D, et al. (2011) RasGRP3, a Ras activator, contributes to signaling and the tumorigenic phenotype in human melanoma. Oncogene 30(45):4590-4600.

20. Yang Y, et al. (2002) RasGRP4, a new mast cell-restricted Ras guanine nucleotide-releasing protein with calcium- and diacylglycerol-binding motifs. Identification of defective variants of this signaling protein in asthma, mastocytosis, and mast cell leukemia patients and demonstration of the importance of RasGRP4 in mast cell development and function. The Journal of biological chemistry 277(28):25756-25774.

21. Reuther GW, et al. (2002) RasGRP4 is a novel Ras activator isolated from acute myeloid leukemia. The Journal of biological chemistry 277(34):30508-30514.

22. Lauchle JO, et al. (2009) Response and resistance to MEK inhibition in leukaemias initiated by hyperactive Ras. Nature 461(7262):411-414.

23. Oki T, et al. (2012) Aberrant expression of RasGRP1 cooperates with gain-of-function NOTCH1 mutations in T-cell leukemogenesis. Leukemia 26(5):1038-1045.

24. Klinger MB, Guilbault B, Goulding RE, & Kay RJ (2005) Deregulated expression of RasGRP1 initiates thymic lymphomagenesis independently of T-cell receptors. Oncogene 24(16):2695-2704.

25. Hartzell C, et al. (2013) Dysregulated RasGRP1 Responds to Cytokine Receptor Input in T Cell Leukemogenesis. Science signaling 6(268):ra21.

26. Yang D, et al. (2010) RasGRP3 contributes to formation and maintenance of the prostate cancer phenotype. Cancer research 70(20):7905-7917.

27. Starr TK, Jameson SC, & Hogquist KA (2003) Positive and negative selection of T cells. Annual review of immunology 21:139-176.

28. Swan KA, et al. (1995) Involvement of p21ras distinguishes positive and negative selection in thymocytes. The EMBO journal 14(2):276-285.

29. Layer K, et al. (2003) Autoimmunity as the consequence of a spontaneous mutation in Rasgrp1. Immunity 19(2):243-255.

30. Kortum RL, et al. (2012) Deconstructing Ras signaling in the thymus. Molecular and cellular biology 32(14):2748-2759.

31. Yasuda S, et al. (2007) Defective expression of Ras guanyl nucleotide-releasing protein 1 in a subset of patients with systemic lupus erythematosus. Journal of immunology (Baltimore, Md. : 1950) 179(7):4890-4900.

32. Pan W, et al. (2010) MicroRNA-21 and microRNA-148a contribute to DNA hypomethylation in lupus CD4+ T cells by directly and indirectly targeting DNA methyltransferase 1. Journal of immunology (Baltimore, Md. : 1950) 184(12):6773-6781.

33. Plagnol V, et al. (2011) Genome-wide association analysis of autoantibody positivity in type 1 diabetes cases. PLoS genetics 7(8):e1002216.

34. Qu HQ, et al. (2009) Association of RASGRP1 with type 1 diabetes is revealed by combined follow-up of two genome-wide studies. Journal of medical genetics 46(8):553-554.

35. Boriack-Sjodin PA, Margarit SM, Bar-Sagi D, & Kuriyan J (1998) The structural basis of the activation of Ras by Sos. Nature 394(6691):337-343.

36. Gureasko J, et al. (2008) Membrane-dependent signal integration by the Ras activator Son of sevenless. Nature structural & molecular biology 15(5):452-461.

37. Gureasko J, et al. (2010) 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 107(8):3430-3435.

38. Sondermann H, et al. (2004) Structural analysis of autoinhibition in the Ras activator Son of sevenless. Cell 119(3):393-405.

39. Rehmann H, et al. (2008) Structure of Epac2 in complex with a cyclic AMP analogue and RAP1B. Nature 455(7209):124-127.

40. Rehmann H, Das J, Knipscheer P, Wittinghofer A, & Bos JL (2006) Structure of the cyclic-AMP-responsive exchange factor Epac2 in its auto-inhibited state. Nature 439(7076):625-628.

41. Margarit SM, et al. (2003) Structural evidence for feedback activation by Ras.GTP of the Ras-specific nucleotide exchange factor SOS. Cell 112(5):685-695.

42. Boykevisch S, et al. (2006) Regulation of ras signaling dynamics by Sos-mediated positive feedback. Current biology : CB 16(21):2173-2179.

43. Prasad A, et al. (2009) Origin of the sharp boundary that discriminates positive and negative selection of thymocytes. Proceedings of the National Academy of Sciences of the United States of America 106(2):528-533.

44. Das J, et al. (2009) Digital signaling and hysteresis characterize ras activation in lymphoid cells. Cell 136(2):337-351.

45. Daniels MA, et al. (2006) Thymic selection threshold defined by compartmentalization of Ras/MAPK signalling. Nature 444(7120):724-729.

46. Zahedi B, et al. (2011) Phosphoinositide 3-kinase regulates plasma membrane targeting of the Ras-specific exchange factor RasGRP1. The Journal of biological chemistry 286(14):12712-12723.

47. Beaulieu N, et al. (2007) Regulation of RasGRP1 by B cell antigen receptor requires cooperativity between three domains controlling translocation to the plasma membrane. Molecular biology of the cell 18(8):3156-3168.

48. Ebinu JO, et al. (1998) RasGRP, a Ras guanyl nucleotide- releasing protein with calcium- and diacylglycerol-binding motifs. Science (New York, N.Y.) 280(5366):1082-1086.

49. Fuller DM, et al. (2012) Regulation of RasGRP1 function in T cell development and activation by its unique tail domain. PloS one 7(6):e38796.

50. Lorenzo PS, Beheshti M, Pettit GR, Stone JC, & Blumberg PM (2000) The guanine nucleotide exchange factor RasGRP is a high -affinity target for diacylglycerol and phorbol esters. Molecular pharmacology 57(5):840-846.

51. Tazmini G, et al. (2009) Membrane localization of RasGRP1 is controlled by an EF-hand, and by the GEF domain. Biochimica et biophysica acta 1793(3):447-461.

52. Freedman TS, et al. (2006) 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 103(45):16692-16697.

53. Zhang G, Kazanietz MG, Blumberg PM, & Hurley JH (1995) Crystal structure of the cys2 activator-binding domain of protein kinase C delta in complex with phorbol ester. Cell 81(6):917-924.

54. Lupas A, Van Dyke M, & Stock J (1991) Predicting coiled coils from protein sequences. Science (New York, N.Y.) 252(5009):1162-1164.

55. O'Shea EK, Klemm JD, Kim PS, & Alber T (1991) X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil. Science (New York, N.Y.) 254(5031):539-544.

56. Hall BE, Yang SS, Boriack-Sjodin PA, Kuriyan J, & Bar-Sagi D (2001) Structure-based mutagenesis reveals distinct functions for Ras switch 1 and switch 2 in Sos-catalyzed guanine nucleotide exchange. The Journal of biological chemistry 276(29):27629-27637.

57. Ahmadian MR, Wittinghofer A, & Herrmann C (2002) Fluorescence methods in the study of small GTP-binding proteins. Methods in molecular biology (Clifton, N.J.) 189:45-63.

58. Hurley JH, Newton AC, Parker PJ, Blumberg PM, & Nishizuka Y (1997) Taxonomy and function of C1 protein kinase C homology domains. Protein science : a publication of the Protein Society 6(2):477-480.

59. Canagarajah B, et al. (2004) Structural mechanism for lipid activation of the Rac-specific GAP, beta2-chimaerin. Cell 119(3):407-418.

60. Leonard TA, Rozycki B, Saidi LF, Hummer G, & Hurley JH (2011) Crystal structure and allosteric activation of protein kinase C betaII. Cell 144(1):55-66.

61. Gifford JL, Walsh MP, & Vogel HJ (2007) Structures and metal-ion-binding properties of the Ca2+-binding helix-loop-helix EF-hand motifs. The Biochemical journal 405(2):199-221.

62. Ikura M, Minowa O, & Hikichi K (1985) Hydrogen bonding in the carboxyl-terminal half-fragment 78-148 of calmodulin as studied by two-dimensional nuclear magnetic resonance. Biochemistry 24(16):4264-4269.

63. Yang W, et al. (2003) Rational design of a calcium-binding protein. J Am Chem Soc 125(20):6165-6171.

64. Le Clainche L, et al. (2003) Engineering new metal specificity in EF-hand peptides. Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry 8(3):334-340.

65. Schwaller B (2010) Cytosolic Ca2+ buffers. Cold Spring Harbor perspectives in biology 2(11):a004051.

66. Usachev YM, Marchenko SM, & Sage SO (1995) Cytosolic calcium concentration in resting and stimulated endothelium of excised intact rat aorta. The Journal of physiology 489 ( Pt 2):309-317.

67. Zhang M, Tanaka T, & Ikura M (1995) Calcium-induced conformational transition revealed by the solution structure of apo calmodulin. Nature structural biology 2(9):758-767.

68. Ames JB, Dizhoor AM, Ikura M, Palczewski K, & Stryer L (1999) Three-dimensional structure of guanylyl cyclase activating protein-2, a calcium-sensitive modulator of photoreceptor guanylyl cyclases. The Journal of biological chemistry 274(27):19329-19337.

69. Aravind P, et al. (2008) Regulatory and structural EF-hand motifs of neuronal calcium sensor-1: Mg 2+ modulates Ca 2+ binding, Ca 2+ -induced conformational changes, and equilibrium unfolding transitions. Journal of molecular biology 376(4):1100-1115.

70. Veeraraghavan S, et al. (2002) Structural independence of the two EF-hand domains of caltractin. The Journal of biological chemistry 277(32):28564-28571.

71. Petri ET, et al. (2010) Structure of the EF-hand domain of polycystin-2 suggests a mechanism for Ca2+-dependent regulation of polycystin-2 channel activity. Proceedings of the National Academy of Sciences of the United States of America 107(20):9176-9181.

72. DiNitto JP, et al. (2007) Structural basis and mechanism of autoregulation in 3-phosphoinositide-dependent Grp1 family Arf GTPase exchange factors. Molecular cell 28(4):569-583.

73. Wang W, et al. (1995) The Grb2 binding domain of mSos1 is not required for downstream signal transduction. Nature genetics 10(3):294-300.

74. Aronheim A, et al. (1994) Membrane targeting of the nucleotide exchange factor Sos is sufficient for activating the Ras signaling pathway. Cell 78(6):949-961.

75. Golec DP, Dower NA, Stone JC, & Baldwin TA (2013) RasGRP1, but not RasGRP3, is required for efficient thymic beta-selection and ERK activation downstream of CXCR4. PloS one 8(1):e53300.

76. Zhu M, Fuller DM, & Zhang W (2012) The role of Ras guanine nucleotide releasing protein 4 in Fc epsilonRI-mediated signaling, mast cell function, and T cell development. The Journal of biological chemistry 287(11):8135-8143.

77. Mikkers H, et al. (2002) High-throughput retroviral tagging to identify components of specific signaling pathways in cancer. Nature genetics 32(1):153-159.

78. Akagi K, Suzuki T, Stephens RM, Jenkins NA, & Copeland NG (2004) RTCGD: retroviral tagged cancer gene database. Nucleic acids research 32(Database issue):D523-527.

79. Suzuki T, et al. (2002) New genes involved in cancer identified by retroviral tagging. Nature genetics 32(1):166-174.

80. Findlay GM, et al. (2013) Interaction domains of sos1/grb2 are finely tuned for cooperative control of embryonic stem cell fate. Cell 152(5):1008-1020.

81. Tartaglia M, et al. (2007) Gain-of-function SOS1 mutations cause a distinctive form of Noonan syndrome. Nature genetics 39(1):75-79.

82. Roberts AE, et al. (2007) Germline gain-of-function mutations in SOS1 cause Noonan syndrome. Nature genetics 39(1):70-74.

83. Kabsch W (2010) XDS. Acta crystallographica. Section D, Biological crystallography 66(Pt 2):125-132.

84. Winn MD, et al. (2011) Overview of the CCP4 suite and current developments. Acta crystallographica. Section D, Biological crystallography 67(Pt 4):235-242.

85. Adams PD, et al. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta crystallographica. Section D, Biological crystallography 66(Pt 2):213-221.

86. McCoy AJ, et al. (2007) Phaser crystallographic software. Journal of applied crystallography 40(Pt 4):658-674.

87. Praznikar J, Afonine PV, Guncar G, Adams PD, & Turk D (2009) Averaged kick maps: less noise, more signal... and probably less bias. Acta crystallographica. Section D, Biological crystallography 65(Pt 9):921-931.

88. Day CL & Alber T (2000) Crystal structure of the amino-terminal coiled-coil domain of the APC tumor suppressor. Journal of molecular biology 301(1):147-156.

89. Krutzik PO & Nolan GP (2006) Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nature methods 3(5):361-368.

90. Delaglio F, et al. (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. Journal of biomolecular NMR 6(3):277-293.

91. Goddard TD & Kneller DG (SPARKY 3. (University of California, San Francisco).

92. Wishart DS, et al. (1995) 1H, 13C and 15N chemical shift referencing in biomolecular NMR. Journal of biomolecular NMR 6(2):135-140.

93. Kay LE, Ikura M, Tschudin R, & Bax A (2011) Three-dimensional triple-resonance NMR Spectroscopy of isotopically enriched proteins. 1990. Journal of magnetic resonance (San Diego, Calif. : 1997) 213(2):423-441.

94. Grzesiek S, Anglister J, & Bax A (1993) Correlation of Backbone Amide and Aliphatic Side-Chain Resonances in 13C/15N-Enriched Proteins by Isotropic Mixing of 13C Magnetization. Journal of Magnetic Resonance, Series B 101:114-119.

95. Hansen MR, Mueller L, & Pardi A (1998) Tunable alignment of macromolecules by filamentous phage yields dipolar coupling interactions. Nature structural biology 5(12):1065-1074.

96. Ottiger M, Delaglio F, & Bax A (1998) Measurement of J and dipolar couplings from simplified two-dimensional NMR spectra. Journal of magnetic resonance (San Diego, Calif. : 1997) 131(2):373-378.

97. Yang D, Tolman JR, Goto NK, & Kay LE (1998) An HNCO-based Pulse Scheme for the Measurement of 13Calpha-1Halpha One-bond Dipolar couplings in 15N, 13C Labeled Proteins. Journal of biomolecular NMR 12(2):325-332.

98. Shen Y, Delaglio F, Cornilescu G, & Bax A (2009) TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. Journal of biomolecular NMR 44(4):213-223.

99. Berjanskii MV, Neal S, & Wishart DS (2006) PREDITOR: a web server for predicting protein torsion angle restraints. Nucleic acids research 34(Web Server issue):W63-69.

100. Guntert P, Billeter M, Ohlenschlager O, Brown LR, & Wuthrich K (1998) Conformational analysis of protein and nucleic acid fragments with the new grid search algorithm FOUND. Journal of biomolecular NMR 12(4):543-548.

101. Brunger AT (2007) Version 1.2 of the Crystallography and NMR system. Nature protocols 2(11):2728-2733.

102. Zweckstetter M & Bax A (2000) Prediction of Sterically Induced Alignment in a Dilute Liquid Crystalline Phase: Aid to Protein Structure Determination by NMR. Journal of the American Chemical Society 122:3791-3792.

103. Clore GM, Gronenborn AM, & Bax A (1998) A robust method for determining the magnitude of the fully asymmetric alignment tensor of oriented macromolecules in the absence of structural information. Journal of magnetic resonance (San Diego, Calif. : 1997) 133(1):216-221.

104. Laskowski RA, Rullmannn JA, MacArthur MW, Kaptein R, & Thornton JM (1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. Journal of biomolecular NMR 8(4):477-486.

105. Bhattacharya A, Tejero R, & Montelione GT (2007) Evaluating protein structures determined by structural genomics consortia. Proteins 66(4):778-795.