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Role of the histone domain in the autoinhibition and activation of the Ras activator Son of Sevenless


Jodi Gureasko, Olga Kuchment, Debora Lika Makino, Holger Sondermann, Dafna Bar-Sagi, and John Kuriyan


Published online before print February 4, 2010, doi: 10.1073/pnas.0913915107; PNAS February 23, 2010 vol. 107 no. 8 3430-3435 (local copy)

Abstract / Figures from the paper



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 (SOSHDPC, 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.


Figures from the paper.

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First figure from paper

Figure 1: Structure of SOSHDPC. (A) Domain organization of SOS, with the Noonan syndrome-associated mutations that map to the histone domain, the PH domain and the PH-Rem linker indicated. (B) Crystal structure of SOSHDPC, with coloring according to the diagram in (A).


First figure from paper

Figure 2: Allosteric Ras binding site is occluded by the histone and the DH domains. (A) The structure of autoinhibited SOSHDPC is shown at the membrane. To model Ras at both the active and allosteric Ras binding sites, the structures of SOSHDPC and the ternary Ras∶SOScat∶Ras-GTP complex (3) were aligned through superpositioning of the two respective Rem domains of SOSHDPC and SOScat. Note that the Ras-GTP bound at the allosteric site in the ternary complex overlaps with the histone and the DH domains of SOSHDPC. (B) Close up view of the occlusion of the allosteric site by the histone domain. Only the histone domain from SOSHDPC and the Ras-GTP from the ternary complex are shown for clarity.


First figure from paper

Figure 3: Kinetic analysis of SOS constructs containing mutations in the inhibitory histone domain interface. (A) Detailed view showing the conserved network of interactions between the histone domain, the DH domain, and the PH-Rem linker. Amino acid residues mutated and analyzed in (B) are shown in green, and additional highly conserved residues are indicated in yellow. (B) Nucleotide exchange rates for SOSHDPC(W85A), SOSHDPC(R217A), SOSHDPC(S548R), SOSHDPC(R552G), SOSHPDC, and SOSDPC in the absence and presence of PIP2 in Ras-coupled vesicles are compared (mant-dGDP is exchanged for unlabeled GTP; bulk concentration of Ras and SOS is 1 µM and 100 nM, respectively). SOSHDPC(R217A) and SOSHDPC(W85A) contain mutations that weaken the histone-DH domain interface and the interface between the histone domain and the PH-Rem linker, respectively. SOSHDPC(S548R) and SOSHDPC(R552G) contain amino acid substitutions in the PH-Rem linker that cause Noonan syndrome (21, 22). Note that in contrast to SOSHDPC, the activity of SOSHDPC(W85A), SOSHDPC(R217A), SOSHDPC(S548R), and SOSHDPC(R552G) is increased in a PIP2-dependent manner.


First figure from paper

Figure 4: A membrane-dependent electrostatic switch releases the histone domain. (A) The electrostatic surface potential of the histone domain of autoinhibited SOSHDPC is shown at the membrane, with positive and negative surface potentials colored blue and red, respectively. Electrostatic surface calculations were performed using the APBS Tools plugin for PyMOL (38). SOS is oriented so that both Ras-binding sites are positioned appropriately for connection to the membrane, while also positioning the lipid-interacting face of the PH domain toward the membrane. Note that in the autoinhibited conformation, the negatively charged surface of the histone domain is oriented toward the membrane, whereas the putative membrane-binding surface (19) is oriented roughly orthogonal to it. (B) The simultaneous engagement of the membrane by the histone and the PH domains coincides with a productive reorientation of SOS in the plane of the membrane.


First figure from paper

Figure 5: Synergy between the histone domain and the DH-PH unit is crucial for effective membrane-binding and activation of SOS on membranes. (A) The electrostatic surface potentials of SOSHDPC and constructs of SOSHDPC containing the Noonan syndrome-associated mutation (E108K) in the absence and presence of K121E are compared. Electrostatic surface calculations were performed as described in the legend of Fig. 4. (B) Nucleotide exchange rates for SOSHDPC, SOSHDPC(E108K), SOSHDPC(E108K,K121E), and SOSDPC in the absence and presence of PIP2 in Ras-coupled vesicles are compared (mant-dGDP is exchanged for unlabeled GTP; bulk concentration of Ras and SOS is 1 µM and 100 nM, respectively). Note the ability of the E108K mutation to greatly potentiate the PIP2-dependent activation of SOSHDPC(E108K).


First figure from paper

Figure 6: PIP2-dependent activation of the PH domain-associated Noonan syndrome mutations in SOS. Nucleotide exchange rates for SOSHDPC, SOSHDPC(E433K), SOSHDPC(C441Y), and SOSDPC in the absence and presence of PIP2 in Ras-coupled vesicles are compared (mant-dGDP is exchanged for unlabeled GTP; bulk concentration of Ras and SOS is 1 μM and 100 nM, respectively). Note that the activity of the Noonan syndrome-associated mutations, SOSHDPC(E433K) and SOSHDPC(C441Y), is increased relative to that of SOSHDPC in a PIP2-dependent manner.


First figure from paper

Figure 7: Membrane-association of SOS is enhanced by cooperation of multiple membrane-binding interactions. Membrane-association of SOS is regulated by (i) the coupling to activated receptors, (ii) a PH domain-PIP2 (purple circles) binding interaction, (iii) nonspecific electrostatic interactions between the membrane and a putative membrane-binding surface on the histone domain, and (iv) the engagement of Ras at the allosteric site. The synergistic combination of these membrane-binding events drives the release of autoinhibition and facilitates the productive reorientation of SOS in the plane of the membrane, maximizing the accessibility of both Ras binding sites of SOS at the membrane.