Computational Docking and Solution X-ray Scattering Predict a Membrane-interacting Role for the Histone Domain of the Ras-activator Son of Sevenless
Holger Sondermann, Bhushan Nagar, Dafna Bar-Sagi, and John Kuriyan
Abstract: The Ras-specific nucleotide exchange factor Son of Sevenless (SOS) is a large multi-domain 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 (SAXS), mutagenesis and calorimetry we show that the histone domain folds into the rest of the SOS and docks onto a helical linker that connects the pleckstrin-homology (PH) and the 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.
Figure 1: Domain organization of SOS. The structures of SOScat (bound to two Ras molecules) and SOSDH-PH-cat are shown schematically.
Figure 2: Isothermal titration calorimetry data for SOSHistone binding to SOSDH-PH-cat.
A. Binding of SOSHistone:R153A to SOSDH-PH-cat. Calorimetric titration for SOSHistone:R153A (500 μM) titrated into SOSDH-PH-cat (50 μM). Derived values for Kd and stoichiometry (N) are shown.
B. Binding of SOSHistone:D140A to SOSDH-PH-cat. Calorimetric titration for SOSHistone:D140A (500 μM) titrated into SOSDH-PH-cat (50 μM).
C. Binding of SOSHistone:R153A to SOSDH-PH-cat:R552A. Calorimetric titration for SOSHistone:R153A (500 μM) titrated into SOSDH-PH-cat:R552A (50 μM). Values for Kd and stoichiometry (N) were not determined in B and C due to weak binding.
Fiugre 3: Docking of SOSHistone onto SOSDH-PH-cat.
A. Crystal Structure of SOSDH-PH-cat. The structure of SOSDH-PH-cat (molecule B in pdb entry 1XD4) is shown with coloring according to the diagram shown in Figure 1 (6). A surface-exposed, strictly conserved arginine (Arg552) located in the helical linker between the PH and Rem domain is shown.
B. Crystal Structure of the histone domain of SOS. The structure of SOSHistone (molecule E in pdb entry 1Q9C) is shown (16). The histone folds are shown in green and yellow.
C. Model of SOSHistone-DH-PH-cat. The structure of SOSHistone was docked onto the structure of SOSDH-PH-cat as described in the text (18). A close-up view of the SOSHistone:SOSDH-PH-cat interface is shown. Domains are shown colored according to the diagram in Figure 1A.
Figure 4: Surface of the docked histone domain. The docked model for SOSHistone-DH-PH-cat is shown, with the histone domain shown in surface representation. The electrostatic potential of the histone domain is mapped onto its molecular surface, with red representing negative and blue positive potential (-5 to +5kBT). Electrostatic potentials were calculated using the program GRASP (27), with an ionic strength corresponding to 100 mM KCl.
Figure 5: Small angle X-ray scattering data for SOSDH-PH-cat and SOSHistone-DH-PH-cat.
A. Scattering profile from solutions of SOSDH-PH-cat and SOSHistone-DH-PH-cat. X-ray scattering curves of SOSDH-PH-cat (green) and SOSHistone-DH-PH-cat (black) are shown. Values for the radius of gyration (Rg) obtained from a Guinier plot using the program PRIMUS (28); (22) are shown. As a comparison, the value of Rg calculated from the crystal structure of SOSDH-PH-cat using the program CRYSOL (24) is also shown.
B. Distance distribution function (P(r)) for SOSDH-PH-cat and SOSHistone-DH-PH-cat. P(r) for SOSDH-PH-cat (green) and SOSHistone-DH-PH-cat (black) calculated from the scattering data shown in (A) are shown. Values for Rg and Dmax computed using the program GNOM are listed (23).
C. Comparison of experimental and calculated scattering curves for SOSDH-PH-cat. The experimental scattering curve from SOSDH-PH-cat (green) and that obtained from the atomic structure (orange), computed using CRYSOL, is shown. Rg calculated using CRYSOL and the goodness of fit (κ) are indicated.
D. Evaluation of docking results using the solution scattering profile from SOSHistone-DH-PH-cat. Three models obtained by docking (ClusPro) of the structure SOSHistone onto SOSDH-PH-cat are shown. Model 1 is identical to the one shown in Fig. 2. Models were analyzed as described in C. s = 2π(1/d), where d is the Bragg spacing.
Figure 6: Model for SOS localization at the membrane.
A. Model for SOS localization at the membrane. The model for SOSHistone-DH-PH-cat is shown oriented at the membrane. The phosphatidylinositol phosphate binding site (with inositol phosphate modeled from the structure of the Dapp1 PH domain (29)) is indicated. Note that the histone domain presents a conspicuous region of positive electrostatic potential on the surface, oriented towards the hypothetical location of the membrane. The Ras binding sites are indicated based on a structural superposition of the SOSHistone-DH-PH-cat model with a ternary Ras:SOScat crystal structure ((5); PDB code 1NVV).
B. Electrostatic potential of SOSHistone-DH-PH-cat. Electrostatic potentials were calculated as described in Figure 4.
C. Superposition of the nucleosome with the SOSHistone-DH-PH-cat. SOSHistone-DH-PH-cat is shown with the electrostatic potential mapped onto its molecular surface. The nucleosome (17) is shown in grey with DNA in green. For the superpositioning, one nucleosomal H3/H4 dimer was aligned with the histone domain of SOS (see main text).