Tandem Histone Folds in the Structure of the N-Terminal Segment of the Ras Activator Son of Sevenless

Structure, Vol. 11, 1583–1593, December, 2003, 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/j.str.2003.10.015 Tandem Histone Folds in the Structure of the N-Terminal Segment of the Ras Activator Son of Sevenless

Holger Sondermann,1 Stephen M. Soisson,2,4 inactive Ras•GDP to active Ras•GTP in response to Dafna Bar-Sagi,3 and John Kuriyan1,* growth factor stimulation. 1Howard Hughes Medical Institute There are two human isoforms of Sos, Sos1 and Sos2, ,residues (Bar-Sagi 1300ف Department of Molecular and Cell Biology which are both composed of Department of Chemistry 1994; Chardin et al., 1993; Webb et al., 1993). The inter- University of California and action between Sos and empty Ras is restricted to a residue domain that is homologous to the Ras 300ف Physical Biosciences Division Lawrence Berkeley National Laboratory activator in yeast, Cdc25 (a schematic representation Berkeley, California 94720 of the domains of Sos is shown in Figure 1A). Despite 2 Laboratories of Molecular Biophysics the relatively limited region of homology to Cdc25, the residue span of the Sos protein is highly 1300ف The Rockefeller University entire 1230 York Avenue conserved from C. elegans to humans, indicating that New York, New York 10021 the domains that flank the Cdc25 domain are equally 3 Department of Molecular Genetics critical for Sos function. One function of the C-terminal and Microbiology domain (residues 1050–1333) is readily apparent, since The State University of New York at Stony Brook it contains proline-rich motifs that bind to the SH3 do- Stony Brook, New York 11794 mains of adaptor proteins that carry Sos to the membrane (Buday and Downward, 1993; Egan et al., 1993; Gale et al., 1993; Li et al., 1993). The functions of the Summary other regions of Sos have been more difficult to dissect, and the continuing investigations on Sos are throwing The Ras activator Son of Sevenless (Sos) contains a up some surprises. Cdc25 homology domain, responsible for nucleotide One unexpected recent discovery involves the Rem exchange, as well as Dbl/Pleckstrin homology (DH/PH) (Ras exchanger motif, residues 550–750) domain, lo- domains. We have determined the crystal structure cated upstream of the Cdc25 domain. The Rem domain of the N-terminal segment of human Sos1 (residues turns out to be important for enabling an allosteric inter- 1–191) and show that it contains two tandem histone action between Sos and Ras•GTP that stimulates the folds. While the N-terminal domain is monomeric in activity of the Cdc25 domain in an unanticipated positive solution, its structure is surprisingly similar to that feedback mechanism (Margarit et al., 2003). Another of histone dimers, with both subunits of the histone surprise is that the Dbl-homology/Pleckstrin-homology “dimer” being part of the same peptide chain. One (DH/PH) unit of Sos appears to be catalytically inactive. histone fold corresponds to the region of Sos that DH/PH units commonly function as nucleotide exchange is clearly similar in sequence to histones (residues factors for GTPases of the Rac/Rho/Cdc42 family (Zheng, 91–191), whereas the other is formed by residues in 2001), but we have been unable to observe nucleotide Sos (1–90) that are unrelated in sequence to histones. exchange factor activity for the isolated DH/PH unit of Residues that form a contiguous patch on the surface Sos in solution (our unpublished data), consistent with of the histone domain of Sos are conserved from C. the fact that the crystal structure of this unit shows it elegans to humans, suggesting a potential role for this to be in an inactive conformation (Soisson et al. 1998; domain in protein-protein interactions. Worthylake et al., 2000). Sos has been shown to stimu- late Rac in cells (Nimnual et al., 1998), suggesting that Introduction there may be an as yet unexplained switching mechanism that controls the activity of the DH/PH units of Sos, Growth factor receptors activate Sos by triggering its perhaps involving phosphatidyl inositol lipid-mediated relocation from the cytoplasm to the plasma membrane, conformational changes in the PH domain (Chen et al., where Ras is localized (for a review, see Schlessinger, 1997; Das et al., 2000). 2000). Encounters between Ras and Sos at the mem- The Ras-exchange activity of Sos appears to be mod- brane result in the release of Ras-bound nucleotides, ulated by intramolecular interactions that are poorly un- which otherwise dissociate very slowly from Ras (Aron- derstood at present (Corbalan-Garcia et al., 1998; Kim heim et al., 1994). By determining a structure of Ras in et al., 1998; Qian et al., 1998). Truncation of the first 550 complex with Sos, we have shown previously that Sos residues of Sos (including the DH/PH domains) or the works by opening up Ras and stabilizing the nucleotide- C-terminal domain of Sos leads to an increased activity free form (Boriack-Sjodin et al., 1998). Subsequent dis- of the Ras activator (Rem/Cdc25) domain, suggesting sociation of the Ras-Sos complex results in the ex- an autoinhibitory role for the flanking domains of Sos. change of Ras-bound nucleotides with those in solution, Results such as these suggest a complex interplay be- a process that is critical for the rapid conversion of tween the domains of Sos, and we have been interested in obtaining a more complete structural understanding *Correspondence: [email protected] of Sos with the eventual goal of working out the regula- 4 Present address: Merck Research Laboratories, P.O. Box 2000, tory mechanism in detail. ,residues of Sos 200ف RY50-105, Rahway, New Jersey 07065. The function of the N-terminal Structure 1584

two H3:H4 dimers form a tetrameric platform to which two H2A:H2B dimers are bound. Overall, the histone octamer forms a disc-like structure around which DNA is wrapped (for a review, see Luger and Richmond, 1998). Histone folds have also been found in several proteins involved in transcription, such as the TATA box binding protein (TBP)-associated factors (TAFs) (Baxevanis et al., 1995; Xie et al., 1996; Birck et al., 1998). The histone folds in these structures form heterodimers that are structurally similar to the basic nucleosome unit. The

heterodimer formed by TAFII42:TAFII62 also forms higher order oligomers in solution, and an assembly similar to a histone tetramer is seen in the crystal (Xie et al., 1996). Other TAFs appear to be heterodimeric modules that do not form higher order oligomers (Birck et al., 1998; Kamada et al., 2001). We have determined the crystal structure of the N-terminal domain of Sos (residues 1–191), including the region spanning the predicted histone fold. Our structure reveals that this segment of Sos contains two tandem histone folds, despite the lack of any detectable se- Figure 1. Domain Structure of the Sos Protein quence identity in the first 90 residues to the sequences (A) Boundaries of the domains of Sos. The construct used for struc- of histones or TAFs. The two histone folds interact to ture determination consists of residues 1–191 of human Sos1, de- form a self-capped histone pseudodimer that is similar noted “Histone domain.” PxxP motifs are SH3 domain binding sites. to a histone dimer and the monomeric histone pseudo- DH domains are found in GDP/GTP exchange factors for the Rho family of GTPases (RhoGEFs). PH domains bind to phosphatidyl dimer from the hyperthermophilic prokaryote Methano- inositol phosphate (PIP). pyrus kandleri (Luger et al., 1997; Starich et al., 1996; (B) Partial proteolysis of the histone fold of human Sos. Purified Zhu et al., 1998; Fahrner et al., 2001). Sequence compari- protein spanning residues 1–191 of human Sos1 was incubated in son of Sos proteins from C. elegans to humans reveals a the presence or absence of increasing amounts of the protease strictly conserved patch of mainly hydrophobic residues Subtilisin (from 0 to 5 mg/ml final protease concentration) for 30 that are exposed in the pseudodimer. This region of min on ice. Reactions were stopped with 10 mM PMSF, and protein fragments were analyzed by MALDI-TOF mass spectroscopy (ESI- the histone domain might be used as an interface for MS). Note the slight reduction in the apparent molecular mass of interactions with other domains of Sos or with as yet the band corresponding to the histone fold, which is due to a loss of unidentified binding partners. 5 C-terminal residues of the protein. Controls show the undigested protein and the protease alone at the highest concentration used. Similar results were obtained with Elastase, Trypsin, and Chymo- Results and Discussion trypsin. Crystallization and Structure Determination A stable fragment of the N-terminal histone domain of located immediately upstream of the DH/PH unit, is cur- Sos that corresponds to residues 1–187 of human Sos1 rently unknown. A recent study suggests a role for this was identified using partial proteolysis with several pro- N-terminal domain in inhibiting exchange activity for teases followed by mass spectroscopy (Figure 1B). The Rac1 by binding to the PH domain directly (Jorge et al., first 191 residues of Sos were expressed subsequently 2002). Sequence comparisons have shown that a region in E. coli with a hexahistidine affinity tag. We shall refer within this segment, spanning residues 91–190, has sig- to this segment of Sos as the “histone domain.” The nificant sequence similarity to histones, particularly to protein was purified to homogeneity by standard chro- the core nucleosomal protein H2A (Baxevanis et al., matography, and the affinity tag was removed by prote- 1995; Baxevanis and Landsman, 1998; Sullivan et al., olysis. Crystals of the histone domain of human Sos1 2000). This is a striking observation since Sos is the only were obtained by hanging drop vapor diffusion (space protein with detectable sequence similarity to histones group P212121,aϭ 93.4 A˚ ,bϭ 109.2 A˚ ,cϭ212.4 A˚ , nine that is neither nuclear nor involved in DNA or RNA molecules in the asymmetric unit). Selenomethione-sub- binding. stituted protein crystals diffract X-rays to 3.2 A˚ , and Histone folds are structural elements that form oligo- a dataset was measured at the selenium anomalous mers, as exemplified by the octameric assembly of the scattering peak wavelength. Datasets at a remote wave- nucleosome core particle (Luger et al., 1997). The canon- length and the inflection point were also collected but ical histone fold contains three ␣ helices that are con- were omitted from further analysis due to radiation nected by two loops, with the long central helix ␣2 damage. flanked by two shorter helices ␣1 and ␣3. Two histone The structure was solved by single-wavelength anom- monomers form a tight heterodimer in which the mono- alous dispersion (SAD) phasing using the peak wave- mers interact in a head-to-tail orientation. The nucleo- length exclusively, yielding an experimental map that some core particle is an octamer formed by two each was readily interpretable. Nine molecules (chain names of the H2A, H2B, H3, and H4 monomers. In the octamer, A to I; see Figure 8B for the molecular labels) were Crystal Structure of the Histone Domain of Sos 1585

Table 1. Data Collection, Phasing, and Refinement Statistics Data Collectiona

Space Group P212121 Unit Cell a ϭ 93.40 A˚ ,bϭ 109.20 A˚ ,cϭ 212.40 A˚ ; ␣ϭ␤ϭ␥ϭ90Њ # Molecules/asym. unit 9 X-ray source ALS, 8.2.2b,c Wavelength (A˚ ) ␭ϭ0.9793 (Se peak) Resolution (A˚ ) 99–3.20

I/␴I 20.1 (2.88) Completeness (%) 99.0 (96.9)

Rsym (%) 6.6 (36.5) SAD Phasingd Anomalous scatterer Se (30 of 54 sites) Resolution (A˚ ) 20–3.21 Figure of merit 0.56

Model Refinemente Resolution (A˚ ) 20–3.21

# reflections Rwork/Rfree 32,451/2,436 f Rwork/Rfree (%) 25.5/29.2 Rmsd bond length (A˚ ) 0.003 Rmsd angles (Њ) 0.8 a Values as defined in SCALEPACK (Otwinowski and Minor, 1997); numbers in parentheses are statistics for the outer resolution shell (3.31–3.20 A˚ ). b Advanced Light Source, Berkeley, beamline 8.2.2. c Bijvoet pairs separated. d Values as defined in SOLVE/RESOLVE (Terwilliger and Berendzen, 1999). e Values as defined in CNS (Bru¨ nger et al., 1998). f No sigma cutoffs.

placed into the electron density and refined using stan- Sequence Conservation and the Overall Nature dard software packages (see Experimental Procedures of the Histone Domain Fold and Table 1). The protomer denoted D has the best The sequence of the histone domain of Sos is well con- defined electron density and is the one used for subse- served from C. elegans to mammals (Figure 2A). The quent analysis. The final model includes residues 6–183 sequence conservation is particularly strong in the sec- for molecule D (see Table 2 and Experimental Proce- ond half of the histone domain, within which the pres- dures). Applying noncrystallographic symmetry restraints, ence of a histone fold had been detected previously the final R value is 25.5% for data between 3.21 and (Baxevanis et al., 1995; Baxevanis and Landsman, 1998). 20 A˚ with an R free of 29.2%. The Ramachandran plot The sequence identity between C. elegans Sos and hu- has 99.3% of the residues lying in the most-favored man Sos within the C-terminal half of the histone domain and additionally allowed regions and no residues in the is 35% (Figure 2A). The overall identity within the first disallowed regions. Pairwise comparisons of the nine 191 residues is 27% for the two sequences. protomers results in rms deviations of 0.08–0.13 A˚ in C␣ The structure of the first 191 residues of human Sos1 positions and 0.33–0.52 A˚ for all atoms. Table 1 shows resembles that of a histone dimer (Figure 2B). In addition a summary of the crystallographic analysis. Table 2 sum- to the expected histone fold in the C-terminal region, marizes the content of the final model of the asymmetric the first half of the protein also contains a histone fold. unit and the relationships between the protomers. The two histone folds in the Sos histone domain resem-

Table 2. Content of the Final Model and Relationship between Protomers Rmsd from Protomer Rmsd from Protomer Db Protomer Residues Internal Deletion Db (C␣ atoms) [A˚ ] (All Atoms) [A˚ ]

Da 6–183 — 0 0 A 6–177 96–103 0.11 0.42 B 7–178 — 0.09 0.41 C 10–177 96–101 0.11 0.43 E 6–183 97–98 0.08 0.33 F 6–183 98–101 0.13 0.43 G 6–179 — 0.10 0.47 H 6–183 100–102 0.13 0.46 I 6–183 — 0.11 0.52 a Reference protomer. b Values as calculated in CNS (Bru¨ nger et al., 1998). Structure 1586

Figure 2. Sequence Alignment and Structure of the Histone Domain (A) Sequence alignment of the histone domain of Sos proteins and nucleosomal core histone H2A. The alignment was generated using ClustalW (Henikoff and Henikoff, 1993). Strictly conserved residues are indicated by red boxes, similar residues by orange and yellow boxes. Secondary structure elements corresponding to the histone fold structure of Sos are depicted as gray boxes for ␣ helices and lines for coil regions. Accession numbers are as follows: human Sos1, Q07889; mouse Sos1, Q62245; human Sos2, Q07890; Drosophila melanogaster Sos, P26675; Anopheles gambiae Sos, XP_319944; Caenorhabditis elegans, NP_504235; human histone H2A, NP_254280. (B) Structure of the monomeric, self-capped tandem histone folds of Sos. Stereo ribbon representation of the structure of molecule D is shown. Secondary structure elements are labeled according to the numbering in Figure 2A. Red traces show the H2A homology region (second Crystal Structure of the Histone Domain of Sos 1587

Figure 3. Interfacial Surfaces between the First and Second Histone Folds of Sos (A) Residues of the second histone fold identified as being part of buried interface are shown as solid surface and colored according to their hydrophobicity (Eisenberg et al., 1984). Hydrophobic residues are shown in green. The solvent-accessible surface of the second histone fold of Sos is shown as a mesh representation. The first histone fold of Sos is shown in a cartoon representation. (B) The solvent-accessible surface of the first histone fold of Sos is shown as mesh, and interfacial residues are shown as solid surface. The second histone fold is shown in a cartoon representation. Coloring is identical to (A). Thumbnails show the orientation of the two views of the structure, separated by a 180Њ rotation around the y axis.

ble closely the structures of the core nucleosomal his- thermophilic prokaryote Methanopyrus kandleri (rms tones, H2B and H2A, respectively (Figure 2C). Most struc- deviation of 2.2 A˚ over 95 residues for the C␣ positions). tural features of the core histones are preserved in the Like the histone domain of Sos, this protein also forms histone domain of Sos, including the C-terminal helix a self-capped pseudodimer (Figure 4C). ␣C characteristic of the nucleosomal histone H2B (helix Sequence comparisons using BLAST/PsiBLAST (Alt- ␣4 in the histone domain of Sos). schul et al., 1997) do not reveal any nonnuclear proteins The interactions between the two histone folds are with significant sequence similarity to this region of Sos. dominated by hydrophobic residues, resulting in an inti- Thus, the N-terminal domain of Sos appears to be the mate packing similar to that seen in histone fold dimers first example of a nonnuclear protein that contains his- (Figure 3). Ninety-one of the 191 residues of the histone tone folds. The structural comparisons discussed here domain contribute to the buried interfacial surface. The suggest that the histone fold domain of Sos is an evolu- pseudodimer formation buries about 4175 A˚ 2 of solvent- tionary offshoot of nucleosomal histone heterodimers. accessible surface area. Continuous electron density Given the striking structural correspondence between for the linker connecting the two histone folds is present the first histone fold of Sos and nucleosomal H2B and in four of the nine molecules in the asymmetric unit. between the second histone fold of Sos and nucleoso- Molecules A, C, E, F, and H have weaker electron density mal H2A (Figure 2C), why do sequence comparisons fail for the loop connecting helices ␣4 and ␣5 affecting 2 to detect the presence of the first histone fold of Sos? (molecule E) to 8 (molecule A) residues (see Table 2). That is, both halves of the Sos histone domain have Based on the tight, hydrophobic interaction between the clearly preserved the structure of the nucleosomal his- two histone folds and a clearly defined and continuous tone components, but only the second one has main- polypeptide chain that is virtually identical in all nine tained the similarity at the sequence level. As discussed protomers, domain swapping between subunits in the in the next section, certain surface regions of the second crystal seems unlikely. histone fold are conserved across Sos proteins from The structure of the heterodimeric H2A/H2B nucleo- different species. We speculate that some as yet un- some unit superimposes well with the histone domain known interactions involving Sos preserve elements of of Sos, with an rms deviation in C␣ positions of 1.2 A˚ this interface in a manner that is functionally important, over 144 residues (Figure 4A). Although the sequences thereby constraining the extent of sequence drift. In- of histones H3 and H4 are less closely related to that triguingly, there is overlap between this conserved re- of the Sos histone domain, structural overlay with the gion of Sos and the interfacial regions in histone tetra- H3/H4 dimer is also good (rms deviation in C␣ positions mers, but as discussed below, it is as yet unclear of 1.7 A˚ over 116 residues) (Figure 4B). The next closest whether Sos engages in interactions that correspond to structural neighbor is the histone protein of the hyper- the formation of histone tetramers.

histone fold), and green structural elements depict regions of the N-terminal histone fold of Sos (first histone fold). (C) Cartoon representation of the isolated histone folds and comparison with the nucleosomal core histones H2A, H2B, H3, and H4. The N-terminal and the C-terminal histone folds of Sos are colored as in (B). Nucleosomal histone fold structures were taken from the nucleosome octamer structure (PDB code: 1KX5; Davey et al., 2002). Structure 1588

Figure 4. Comparison of the Histone Domain of Sos with the Structures of Various Histone Proteins Superpositions were generated by TOP3D (Lu, 2000). The histone domain of Sos is shown in gray. The darker gray represents the C-terminal H2A homology region. Rms deviations for the C␣ backbone atoms between structures being compared are indicated. Alignment with the nucleosomal core histone dimers H2A/H2B (A) and H3/H4 (B) are shown. H2A is colored in light green, H2B in dark green. H3 and H4 are shown in orange and red, respectively (PDB code: 1KX5; Davey et al., 2002). (C) Alignment with Methanopyrus kandleri histone (PDB code: 1F1E; Fahrner et al., 2001).

Surface Properties and Conservation that a domain presented by another region of Sos, or An analysis of the surface of the histone pseudodimer by another molecule, would be able to pry open this of Sos shows that the two opposite faces of the domain densely packed intramolecular interface. have distinct properties (Figure 5). In one region, formed The correspondence between the location of the re- mainly by helices ␣6 and ␣7 and to a lesser extent by gion of surface-exposed and conserved residues in the ␣2, residues that are at the surface are highly conserved Sos histone domain and the regions on the nucleosomal between Sos proteins across species (Figures 2A and histone surfaces that are used to form tetramers now 5B). Residues on the other face of the molecule are not raises the possibility that the Sos histone domain may, conserved to the same extent. This pattern of surface as a unit, promote oligomerization. We therefore studied conservation does not extend to the sequences of his- the aggregation properties of the domain using static tone proteins (data not shown), suggesting that this pat- light scattering. tern is specific to Sos. The region containing highly con- Multiangle light scattering measurements coupled served residues overlaps with a groove on the surface with size exclusion chromatography (Wyatt, 1997) was that contains exposed hydrophobic residues (Figure 5C). used to assess the molecular weight and oligomeriza- Strikingly, the conserved and partially hydrophobic tion state of the Sos histone domain in solution. This patch on the surface of the Sos tandem histone fold is analysis demonstrates that the histone domain is essen- mg/ml (0.18 4ف tially monomeric at concentrations of in the vicinity of elements that in the nucleosome core mM), with a molecular weight measured to be 24.6 kDa octamer are utilized to build higher order structures (Fig- (Ϯ1.8%) compared to the calculated weight of 22.4 kDa ure 6). In such an assembly, the analogous surface is (Figure 7A). The sample shows low polydispersity indi- buried in the interface between the H3:H4 dimers and cating a narrow molar mass distribution, without any in the H3:H4/H2A:H2B tetramer (red and yellow surface, indication for an association into higher order oligomers respectively). (Figure 7B). mg/ml; 0.71 16ف) At higher protein concentrations Oligomerization Behavior of the Sos Histone mM), distinct higher molecular weight species can be Domain in Solution detected in addition to the monomeric protein, probably When we first noticed the presence of one histone fold representing oligomers (Figure 7C, gray areas). Molecu- in the N-terminal domain of Sos, we wondered whether lar weight determinations across these peaks are inac- this domain might induce Sos to dimerize, with the single curate due to low signal strength, but can be very histone fold in the domain providing one of the two roughly estimated to be around 135 and 280 kDa, re- partners in a histone-like dimer. The crystal structure spectively. The increase in molecular weight happens that we have now determined suggests that such a in distinct size steps, a behavior one would expect for mechanism is unlikely because the two histone folds oligomerization events. A rough estimate for the dissoci- -A˚ 2 of surface area be- ation constant of such an interaction is in the submillimo 4200ف cap each other, burying tween them, much of it hydrophobic. It seems unlikely lar to millimolar regime. Crystal Structure of the Histone Domain of Sos 1589

nately, the functional significance, if any, of this assembly is unclear. Nevertheless, we describe the nature of the asymmetric unit for completion. The nine molecules in the asymmetric unit can be grouped into three equivalent asymmetric trimers that are similar to each other. The rms deviations in C␣ positions for all 534 residues of the trimer range from 0.6 A˚ (comparing trimers ABC and DEF) to 1.2 A˚ (trimers DEF and GHI) (Figure 8). The main interface between molecules in the trimers involves an interaction between Arg153 of one protomer (e.g., molecule E) and Asp140 and Asp169 of a neigh- boring molecule (e.g., molecule F) (Figure 8A). The argi- nine is not strictly conserved in Sos proteins, since the Drosophila sequence has an alanine at that position (Figure 2A). The interfaces between monomers D and E and monomers E and F which interact via these residues bury about 900 A˚ 2 of surface area and represent the largest interfaces in the molecular assembly. This buried surface is partially overlapping with the conserved, hydrophobic surface region described earlier (Figure 5B). A distinct interface spans molecules D and F within the trimer, with a buried surface of about 500 A˚ 2 between the protomers. The trimeric substructure is an open assembly, and the nine molecules in the asymmetric unit form a super- helical structure that winds through the crystal (Figure 8B). Interactions between trimers in the superhelix are similar to the interactions between molecules within a trimer but are distorted enough to make the trimer the basic unit of the structure. The trimeric and nonameric assembly does not represent any architectural feature seen in the nucleosome or other histone fold structures.

Conclusions The crystallographic analysis reported here reveals the Figure 5. Surface Properties of the Histone Domain of Sos presence of a self-capped histone pseudodimer within residue N-terminal region of Sos. Although the 200ف A) Representation of a histone protomer (molecule D) in two orienta- the) tions. Orientations are preserved in Figures 5B and 5C. function of this histone domain of Sos is uncertain, the (B) Sequence conservation of the histone domain of Sos as depicted in Figure 2 is mapped onto the surface. Conservation from 0%–100% conservation of sequence within the domain, particularly is presented as color gradient from green to gray to red. Identical with respect to surface-exposed residues, suggests a residues are colored in red, similar residues (Ͼ90% conserved) are functional importance that remains to be determined. shown in orange. Since neither DNA binding nor nuclear localization has (C) Hydrophobicity of the histone domain surface according to the been reported for Sos, the function of the histone do- Eisenberg hydrophobicity scale (Eisenberg et al., 1984). Hydropho- main seems to be distinct from that of histones or the bic residues are shown in green, polar and charged residues are in gray and pink, respectively. TAF proteins. While the isolated domain is monomeric in solution, it is possible that the histone domain plays a role in the formation of higher order structures at the sites of receptor clustering. In this regard, the formation Because we have to use high protein concentrations of an open helical structure in the crystal is suggestive. to drive oligomerization of the histone domain of Sos, While the function of this domain remains uncertain, we are uncertain as to whether this represents a physio- the availability of the three-dimensional structure now logically relevant interaction. The conservation of sur- makes possible the dissection, by site-directed muta- face residues does indicate that interactions mediated genesis, of the role of this domain in the intramolecular by this domain are likely to be important. Whether these regulation of Sos. interactions are with some other region of Sos or with another, as yet unknown, protein remains to be deter- Experimental Procedures mined. Protein Expression and Purification Crystal Packing and Noncrystallographic Symmetry cDNA corresponding to residues 1–191 of human SOS1 (Chardin et al., 1993) was cloned into the pPROEX-HT expression plasmid The presence of nine molecules in the asymmetric unit (Invitrogen) yielding an N-terminally hexahistidine-tagged protein. is unusual, and we have examined the crystallographic Escherichia coli cells BL21 (DE3) (Novagen) were transformed by packing extensively for clues as to function. Unfortu- electroporation. Cells were grown in TB medium supplemented with Structure 1590

Figure 6. Structure of the Nucleosome Core Particle (A) The histone octamer (PDB code: 1KX5; Davey et al., 2002) is shown as surface presentation. Histone tails have been removed from the structure for clarity. The H3:H4 dimers are shown in black and red; H2A:H2B dimers are shown in green and yellow. (B) Cartoon presentation of one H3:H4 dimer (black molecules in [A]) in two orientations similar to the one of the histone domain of Sos in Figure 5. Secondary structure elements are indicated. (C) Surface presentations of the H3:H4 dimer are shown in the same orientation as in (B). Colors indicate buried surface areas within the nucleosome octamer using color coding as in (A). Green and yellow areas are interfaces with the H2A:H2B dimers, and red represents the contact surface with the other H3:H4 dimer.

100 mg/l Ampicillin at 37ЊC. The temperature was reduced to 18ЊC phosphate buffered saline (PBS) and 20 ␮g protein in a final volume at a cell density corresponding to an absorbance of 1.0 at 600 nm, of 50 ␮l. Subtilisin was added in final concentrations ranging from and protein production was induced with 1 mM IPTG. Protein was 0.005 to 5 mg/ml. Reactions were incubated for 30 min on ice. expressed for 12–16 hr. A selenomethionine-substituted derivative Proteolyis was stopped by addition of 10 mM PMSF (final concentra- was produced in the Escherichia coli strain B834 (DE3) growing in tion). Reactions were analyzed by SDS-PAGE followed by Coomas- M9 minimal medium supplemented with 40 mg/l amino acids, except sie staining and by MALDI-TOF mass spectroscopy (MALDI- for methionine, and containing 25 mg/l seleno-L-methionine under TOF-MS). the growth conditions described above. Cells were collected by centrifugation for 1 hr at 4000 ϫ g, resus- Crystallization, X-Ray Data Collection, pended in NiNTA buffer A (25 mM Tris-Cl [pH 8.2], 500 mM NaCl, and Structure Solution 20 mM imidazole, and 5 mM ␤-mercaptoethanol) and frozen in liquid Crystals were obtained by hanging drop vapor diffusion by mixing nitrogen. Cell suspensions were thawed in a water bath at 25ЊC. equal volumes of protein (5–20 mg/ml) and reservoir solution (4%– After cell lysis using a French press (EmulsiFlex-C5, Avestin), cell 12% PEG3350, 0.2 M L-proline, 0.1 M magnesium acetate, and 5% debris was removed by ultracentrifugation at 125,000 ϫ g for 1 hr ethanol). Native and selenomethionine-derivatized protein yielded at 4ЊC. Clear lysates were loaded onto NiNTA fast flow resin (Qiagen) the same crystal form under similar conditions. Crystals appeared equilibrated in NiNTA buffer A by gravity flow. The resin was washed within 1 day at 20ЊC with typical dimensions of 0.3 mm ϫ 0.2 mm ϫ with 20 column volumes of NiNTA buffer A, and proteins were eluted 0.1 mm. The crystals were transferred to reservoir solution plus 20% with NiNTA buffer B (NiNTA buffer A supplemented with 500 mM glycerol for 5 min. Crystals were frozen in propane and kept at 100K imidazole). Buffers were exchanged using a Fast Desalting column during data collection. (Amersham-Pharmarcia) into TEV buffer (25 mM Tris-Cl [pH 8.0], 50 Crystallographic statistics for data collection are shown in Table 1. mM NaCl, and 5 mM ␤-mercaptoethanol). Hexahistidine tags were Data sets were collected using synchrotron radiation (ALS, Berkeley, cleaved by incubation with Tobacco etch virus (TEV) protease over- beamline 8.2.2). Data reduction was carried out with the software night at 4ЊC. Uncleaved protein, protease, and free tags were re- package DENZO and SCALEPACK (Otwinowski and Minor, 1997). moved by a second NiNTA column. The flow-through was collected The space group was determined to be P212121 with a ϭ 93.4 A˚ , and loaded onto a Superdex200 column (Amersham-Pharmacia) b ϭ 109.2 A˚ ,cϭ 212.4 A˚ . The asymmetric unit consists of nine equilibrated in gel filtration buffer (25 mM Tris-Cl [pH 7.5], 50 mM molecules. NaCl, and 2 mM DTT). Fractions containing protein were pooled Phases were calculated using single anomalous dispersion (SAD) and concentrated on a Centricon ultrafiltration device (Millipore) to with a data set collected at the peak wavelength for selenium and a final concentration of approximately 40 mg/ml. Protein aliquots using the software package SOLVE/RESOLVE (Terwilliger and Be- were frozen in liquid nitrogen and stored at Ϫ80ЊC. rendzen, 1999). A model of the protomer was built manually into an electron density map of high quality (molecule D). This model was Limited Proteolysis used to generate the asymmetric unit manually. Refinement using Proteins were subjected to partial proteolysis by incubation with the RAVE software package (Kleywegt and Read, 1997), CNS increasing amounts of the protease subtilisin. Reactions contained (Bru¨ nger et al., 1998), and O (Jones et al., 1991) yielded a model Crystal Structure of the Histone Domain of Sos 1591

tein (180–710 ␮M) was injected onto a Superdex 200 HR10/30 size exclusion chromatography column (Amersham-Pharmacia) equilibrated in MALS buffer (25 mM Tris-HCl [pH 7.5], 100 mM NaCl, and 1 mM DTT). The chromatography system was coupled to an 18- angle light scattering detector (DAWN EOS) and refractive index detector (Optilab DSP) (Wyatt Technology). Data were collected every 0.5 s at a flow rate of 0.4 ml/min. Data analysis was carried out using the program ASTRA, yielding the molar mass and mass distribution (polydispersity) of the sample.

Acknowledgments

We are grateful to Thomas Earnest, Gerry McDermott, and the scien- tists at beamline 8.2.2, Advanced Light Source (ALS), Berkeley, for assistance with synchrotron data collection, and to James Holton for advice on data collection. Beamline 8.2.2 at the ALS is supported by the Howard Hughes Medical Institute, and the ALS is supported by the U.S. Department of Energy’s Office of Basic Energy Sciences. We thank Marsha Henderson, Oren Rosenberg, Michael Hahn, Greg Bowman, and Bhushan Nagar for valuable assistance and discus- sions, and Natalia Rodionova for mass spectrometry. H.S. is supported by the Leukemia & Lymphoma Society. S.M.S. was supported by the Damon Runyon-Walter Winchell Cancer Research Founda- tion. D.B.-S. is supported by the NIH.

Received: August 8, 2003 Revised: August 27, 2003 Accepted: August 30, 2003 Published: December 2, 2003

References

Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D.J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Aronheim, A., Engelberg, D., Li, N., al-Alawi, N., Schlessinger, J., and Karin, M. (1994). Membrane targeting of the nucleotide exchange factor Sos is sufficient for activating the Ras signaling path- way. Cell 78, 949–961. Bar-Sagi, D. (1994). The Sos (Son of Sevenless) protein. Trends Endocrinol. Metab. 5, 165–169. Baxevanis, A.D., and Landsman, D. (1998). Histone Sequence Data- Figure 7. Multiangle Light Scattering Analysis of the Histone Do- base: new histone fold family members. Nucleic Acids Res. 26, main of Sos in Solution 372–375. (A) Purified protein (4 mg/ml) was analyzed by coupled gel filtration/ Baxevanis, A.D., Arents, G., Moudrianakis, E.N., and Landsman, D. multiangle light scattering. The mobile phase consists of 25 mM (1995). A variety of DNA-binding and multimeric proteins contain Tris-HCL (pH 7.5), 100 mM NaCl, and 2 mM DTT. The yellow area the histone fold motif. Nucleic Acids Res. 23, 2685–2691. highlights the analyzed peak. The primary signal (in volts) is plotted Birck, C., Poch, O., Romier, C., Ruff, M., Mengus, G., Lavigne, A.C., against the elution volume. Red traces show the signal of one of Davidson, I., and Moras, D. (1998). Human TAF(II)28 and TAF(II)18 Њ the light scattering detectors (at 90 to the incident beam). In blue, interact through a histone fold encoded by atypical evolutionary the signal from the refractive index detector is shown. conserved motifs also found in the SPT3 family. Cell 94, 239–249. (B) Molecular weights determined by light scattering and concentra- Boriack-Sjodin, P.A., Margarit, S.M., Bar-Sagi, D., and Kuriyan, J. tion determination from each data slice (0.5 s increments) are plotted (1998). The structural basis of the activation of Ras by Sos. Nature against the elution time. Protein concentration was measured by 394, 337–343. change of refractive index as shown in the solid line. (C) Purified protein at 16 mg/ml was subjected to light scattering Bru¨ nger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., as described in (A). The monomeric peak is highlighted in yellow, Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., and higher molecular weight peaks are shown in gray. Pannu, N.S., et al. (1998). Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921. for the protomer. During refinement, noncrystallographic symmetry Buday, L., and Downward, J. (1993). Epidermal growth factor regu- restraints were applied to each of the nine protomers in the asym- lates p21ras through the formation of a complex of receptor, Grb2 metric unit. In the last step, each monomer of the nonameric assem- adapter protein, and Sos nucleotide exchange factor. Cell 73, bly was investigated, and regions of higher flexibility which were 611–620. not visible in the electron density maps were removed from the final Chardin, P., Camonis, J.H., Gale, N.W., van Aelst, L., Schlessinger, model. Table 2 summarizes the content of the final model and the J., Wigler, M.H., and Bar-Sagi, D. (1993). Human Sos1: a guanine relationship of the protomers. nucleotide exchange factor for Ras that binds to GRB2. Science 260, 1338–1343. Multiangle Light Scattering/Size Exclusion Chromatography Chen, R.H., Corbalan-Garcia, S., and Bar-Sagi, D. (1997). The role Purified protein was characterized by multiangle light scattering of the PH domain in the signal-dependent membrane targeting of (MALS) following size exclusion chromatography (Wyatt, 1997). Pro- Sos. EMBO J. 16, 1351–1359. Structure 1592

Figure 8. Noncrystallographic Symmetry in the Crystals of the Sos Histone Domain (A) The central trimer (molecules D, E, and F) is shown. Surface area buried at the three interfaces was calculated using CNS. (B) Assembly in the unit cell. Three trimers form an open helical nonamer. Colors indicate equivalent protomers within the nonameric assembly. All C termini point to the out- side of the assembly, as depicted by black surface presentation. C termini for molecules B, D, F, and H are in the back of the assembly, but are surface exposed.

Corbalan-Garcia, S., Margarit, S.M., Galron, D., Yang, S.S., and Bar- Lu, G. (2000). TOP: a new method for protein structure comparisons Sagi, D. (1998). Regulation of Sos activity by intramolecular interac- and similarity searches. J. Appl. Crystallogr. 33, 176–183. tions. Mol. Cell. Biol. 18, 880–886. Luger, K., and Richmond, T.J. (1998). DNA binding within the nucleo- Das, B., Shu, X., Day, G.J., Han, J., Krishna, U.M., Falck, J.R., and some core. Curr. Opin. Struct. Biol. 8, 33–40. Broek, D. (2000). Control of intramolecular interactions between the Luger, K., Mader, A.W., Richmond, R.K., Sargent, D.F., and Rich- pleckstrin homology and Dbl homology domains of Vav and Sos1 mond, T.J. (1997). Crystal structure of the nucleosome core particle regulates Rac binding. J. Biol. Chem. 275, 15074–15081. at 2.8 A resolution. Nature 389, 251–260. Davey, C.A., Sargent, D.F., Luger, K., Maeder, A.W., and Richmond, T.J. (2002). Solvent mediated interactions in the structure of the Margarit, S.M., Sondermann, H., Hall, B.E., Nagar, B., Hoelz, A., nucleosome core particle at 1.9 a resolution. J. Mol. Biol. 319, 1097– Pirruccello, M., Bar-Sagi, D., and Kuriyan, J. (2003). Structural evi- 1113. dence for feedback activation by Ras.GTP of the Ras-specific nucleotide exchange factor SOS. Cell 112, 685–695. Egan, S.E., Giddings, B.W., Brooks, M.W., Buday, L., Sizeland, A.M., and Weinberg, R.A. (1993). Association of Sos Ras exchange protein Nimnual, A.S., Yatsula, B.A., and Bar-Sagi, D. (1998). Coupling of with Grb2 is implicated in tyrosine kinase signal transduction and Ras and Rac guanosine triphosphatases through the Ras exchanger transformation. Nature 363, 45–51. Sos. Science 279, 560–563. Eisenberg, D., Schwarz, E., Komaromy, M., and Wall, R. (1984). Otwinowski, Z., and Minor, W. (1997). Processing of X-ray diffraction Analysis of membrane and surface protein sequences with the hy- data collected in oscillation mode. Methods Enzymol. 276, 307–326. drophobic moment plot. J. Mol. Biol. 179, 125–142. Qian, X., Vass, W.C., Papageorge, A.G., Anborgh, P.H., and Lowy, Fahrner, R.L., Cascio, D., Lake, J.A., and Slesarev, A. (2001). An D.R. (1998). N terminus of Sos1 Ras exchange factor: critical roles ancestral nuclear protein assembly: crystal structure of the Metha- for the Dbl and pleckstrin homology domains. Mol. Cell. Biol. 18, nopyrus kandleri histone. Protein Sci. 10, 2002–2007. 771–778. Gale, N.W., Kaplan, S., Lowenstein, E.J., Schlessinger, J., and Bar- Schlessinger, J. (2000). Cell signaling by receptor tyrosine kinases. Sagi, D. (1993). Grb2 mediates the EGF-dependent activation of Cell 103, 211–225. guanine nucleotide exchange on Ras. Nature 363, 88–92. Henikoff, S., and Henikoff, J.G. (1993). Performance evaluation of Soisson, S.M., Nimnual, A.S., Uy, M., Bar-Sagi, D., and Kuriyan, J. amino acid substitution matrices. Proteins 17, 49–61. (1998). Crystal structure of the Dbl and pleckstrin homology domains from the human Son of sevenless protein. Cell 95, 259–268. Jones, T.A., Zou, J.Y., Cowan, S.W., and Kjeldgaard (1991). Im- proved methods for building protein models in electron density Starich, M.R., Sandman, K., Reeve, J.N., and Summers, M.F. (1996). maps and the location of errors in these models. Acta Crystallogr. NMR structure of HMfB from the hyperthermophile, Methanother- A 47 (Pt 2), 110–119. mus fervidus, confirms that this archaeal protein is a histone. J. Jorge, R., Zarich, N., Oliva, J.L., Azanedo, M., Martinez, N., de la Mol. Biol. 255, 187–203. Cruz, X., and Rojas, J.M. (2002). HSos1 contains a new amino- Sullivan, S.A., Aravind, L., Makalowska, I., Baxevanis, A.D., and terminal regulatory motif with specific binding affinity for its plecks- Landsman, D. (2000). The histone database: a comprehensive WWW trin homology domain. J. Biol. Chem. 277, 44171–44179. resource for histones and histone fold-containing proteins. Nucleic Kamada, K., Shu, F., Chen, H., Malik, S., Stelzer, G., Roeder, R.G., Acids Res. 28, 320–322. Meisterernst, M., and Burley, S.K. (2001). Crystal structure of nega- Terwilliger, T.C., and Berendzen, J. (1999). Automated MAD and MIR tive cofactor 2 recognizing the TBP-DNA transcription complex. Cell structure solution. Acta Crystallogr. D Biol. Crystallogr. 55, 849–861. 106, 71–81. Webb, G.C., Jenkins, N.A., Largaespada, D.A., Copeland, N.G., Fer- Kim, J.H., Shirouzu, M., Kataoka, T., Bowtell, D., and Yokoyama, S. nandez, C.S., and Bowtell, D.D. (1993). Mammalian homologues of (1998). Activation of Ras and its downstream extracellular signal- the Drosophila Son of sevenless gene map to murine chromosomes regulated protein kinases by the CDC25 homology domain of mouse 17 and 12 and to human chromosomes 2 and 14, respectively. Son-of-sevenless 1 (mSos1). Oncogene 16, 2597–2607. Genomics 18, 14–19. Kleywegt, G.J., and Read, R.J. (1997). Not your average density. Structure 5, 1557–1569. Worthylake, D.K., Rossman, K.L., and Sondek, J. (2000). Crystal structure of Rac1 in complex with the guanine nucleotide exchange Li, N., Batzer, A., Daly, R., Yajnik, V., Skolnik, E., Chardin, P., Bar- region of Tiam1. Nature 408, 682–688. Sagi, D., Margolis, B., and Schlessinger, J. (1993). Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine Wyatt, P.J. (1997). Multiangle light scattering: the basic tool for kinases to Ras signalling. Nature 363, 85–88. macromolecular charakterization. Instrum. Sci. Technol. 25, 1–18. Crystal Structure of the Histone Domain of Sos 1593

Xie, X., Kokubo, T., Cohen, S.L., Mirza, U.A., Hoffmann, A., Chait, B.T., Roeder, R.G., Nakatani, Y., and Burley, S.K. (1996). Structural similarity between TAFs and the heterotetrameric core of the histone octamer. Nature 380, 316–322. Zheng, Y. (2001). Dbl family guanine nucleotide exchange factors. Trends Biochem. Sci. 26, 724–732. Zhu, W., Sandman, K., Lee, G.E., Reeve, J.N., and Summers, M.F. (1998). NMR structure and comparison of the archaeal histone HFoB from the mesophile Methanobacterium formicicum with HMfB from the hyperthermophile Methanothermus fervidus. Biochemistry 37, 10573–10580.

Accession Numbers

Atomic coordinates and structure factors have been deposited in the RCSB Protein Data Bank under ID code 1Q9C.