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provided by Elsevier - Publisher Connector Current Biology, Vol. 14, 1778–1782, October 5, 2004, 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cub.2004.09.044 Structural Biochemistry of ATP-Driven Dimerization and DNA-Stimulated Activation of SMC

Alfred Lammens, Alexandra Schele, filtration (Figure 1A). Dimer formation was also observed and Karl-Peter Hopfner* for wtSMCcd in the presence of the slowly hydrolysable Gene Center and Institute of Biochemistry ATP analog ATP␥S (data not shown). wtSMCcd did not University of Munich form stable dimers during gel filtration in the presence Feodor-Lynen-Straße 25 of ATP, probably because of more rapid ATP hydrolysis. 81377 Munich We determined the ATP bound crystal structure of Germany pfSMCcd-E1098Q to 2.5 A˚ resolution by a single-wave- length anomalous dispersion experiment (see the Sup- plemental Data available with this article online). Inspec- tion of the crystal packing revealed that an ATP bound Summary SMCcd-SMCcd homodimer with sandwiched Mg2ϩ-ATP molecules assembled along the 2-fold symmetry axis Structural maintenance of chromosome (SMC) pro- of the crystal lattice (Figure 1C). The SMCcd domains teins play a central role in higher-order chromosome are arranged to face each other, creating two composite structure in all kingdoms of life [1–5]. SMC proteins ATPase active sites in the dimer interface. The compos- consist of a long coiled-coil domain that joins an ATP ite active sites contain Walker A/B motifs from one binding cassette (ABC) ATPase domain on one side SMCcd subunit and the signature motif from the oppos- and a dimerization domain on the other side [6]. SMC ing SMCcd subunit. The roots of the two coiled-coil proteins require ATP binding or hydrolysis to promote domains protrude from the same side of the SMCcd cohesion and condensation, which is suggested to dimer surface, well in accordance with electron-micro- proceed via formation of SMC rings or assemblies scopic data of / complexes [6, 14, 15]. [7–11]. To learn more about the role of ATP in the Thus, the structure supports a mechanism in which ATP architecture of SMC proteins, we report crystal struc- binding to and hydrolysis by the SMC ABC domains tures of nucleotide-free and ATP bound P. furiosus control formation and disruption of SMC protein rings SMC ATPase domains. ATP dimerizes two SMC ATP- or assemblies (Figure 1D). ase domains by binding to opposing Walker A and signature motifs, indicating that ATP binding can di- ATP Bound and Dimerization Interface rectly assemble SMC proteins. DNA stimulates ATP Two ATP molecules are tightly sandwiched in the hydrolysis in the engaged SMC ABC domains, sug- SMCcd dimer interface, each bound via ␣ and ␤ phos- gesting that ATP hydrolysis can be allosterically regu- phates to Walker A motifs to one subunit and via the ␥ lated. Structural and mutagenesis data identify an phosphate to the signature motif of the opposing subunit SMC protein conserved-arginine finger that is required (Figures 1B and 2). ATP:protein contacts (1800 A˚ 2 buried for DNA stimulation of the ATPase activity and directly surface) contribute a similar amount of buried surface connects a putative DNA interaction site to ATP. Our area to the ABC-ABC dimer interface as protein:protein results suggest that stimulation of the SMC ATPase contacts (2600 A˚ 2 buried surface) do. Thus, ATP binding activity may be a specific feature to regulate the ATP- and hydrolysis is a major driving force for opening and driven assembly and disassembly of SMC proteins. closing SMC protein rings. The adenine moieties are specifically recognized by three main-chain hydrogen bonds to residues 71 and 72 (Figure 2). The ␣ and ␤ Results and Discussion phosphates are bound by the P loop. ␤ and ␥ phos- phates bind the active-site magnesium ion, which is Structure of the ATP Bound SMC ATPase additionally coordinated by residues S40, D1097 (via a Domain Dimer water molecule), and Q145. From the opposing subunit, To help understand the role of ATP binding and hydroly- ATP is bound by hydrogen bonds of K1064 and K1061 sis by the ATP binding cassette (ABC) domain in promot- to the ribose 2Ј-OH and 3Ј-OH, respectively, and by two ing higher-order structure of structural maintenance of hydrogen bonds from the signature motif S1070-O␥ and chromosome (SMC) proteins, we crystallized the cata- G1072-N to the ATP ␥ phosphate (Figure 2). Besides lytic ABC domain of the Pyrococcus furiosus SMC pro- these ATP-mediated contacts, several protein-protein tein (pfSMCcd) and determined its crystal structure in interactions contribute to the SMCcd dimer. At the cen- the presence of ATP. For crystallization of a stable ATP ter of the dimer interface, the major interaction is medi- bound complex, it was necessary to introduce the ated by the D loop (Figure 2). The conserved DA box, Walker B motif mutation E1098Q, which traps the ATP which has long been recognized as an important motif complex of SMC proteins [12, 13]. SMCcd-E1098Q in SMC proteins [16], forms the central dimerization ele- forms dimers in the presence of ATP in solution, as ment whereby A1101 hydrophobically interacts with judged from the increased hydrodynamic radius in gel A1101 from the opposing subunit. A crystallographically observed water molecule is poised for collinear attack on the scissile P␥-O bond *Correspondence: [email protected] (Figure 2). This likely nucleophile for ATP hydrolysis is SMC ATPase Crystal Structure 1779

Figure 1. Structural Biochemistry of ATP-Driven SMC Dimerization (A) Gel filtration elution profiles of SMCcd-E1098Q with indicated molecular weight standards (in kDa). In the absence of ATP (continuous line), SMCcd elutes as a monomer. In the presence of ATP (dashed line), the majority of SMCcd elutes with the hydrodynamic radius corresponding to a dimer, indicating that ATP promotes engagement of two SMC ABC domains.

(B) SAD and 2Fo Ϫ Fc electron densities (1␴ contour) at the ATP moiety are shown superimposed with the refined model (color-coded sticks). (C) Two orthogonal views of a ribbon model of the ATP bound SMC ATPase dimer with highlighted and annotated secondary structure. One subunit is shown in yellow, the other subunit, representing the 2-fold symmetry-related molecule in the crystal, is shown in green. Two ATP molecules (color-coded sticks with cyan carbons, red oxygens, blue nitrogens, and white phosphor atoms) and two Mg2ϩ ions (magenta spheres) are sandwiched in the dimer interface and are bound to opposing P loop (red) and signature (magenta) motifs. The two coiled-coil domains (indicated by dotted lines) protrude from the same face of the dimer, consistent with electron microscopy studies of multisubunit SMC protein rings. A C-terminal helix (red) reaches across the dimer interface and could be a specific interaction point for other subunits that control assembly of SMC protein rings. (D) Model of the closed SMC ring. Two SMC proteins (yellow/green) form a large ring by stable association via dimerization domains on one side of the coiled coil and ATP-driven association of ABC ATPase domains on the other side of the coiled coil. Additional subunits (orange) interact with the ABC domain and can further control assembly. optimally positioned and activated by hydrogen bonds teins do not possess a “power stroke.” Thus, the role to E1098 (Walker B, mutated to Q) and the backbone of ATP binding and hydrolysis of SMC proteins seems carbonyl oxygen of H1102 from the opposing subunit. to be limited to the formation and disruption of SMC The contributions to positioning of the nucleophile from protein rings or higher-order assemblies. both subunits suggest that ATP hydrolysis only takes Nevertheless, two regions of SMCcd were markedly place in the engaged form of the ABC dimer. These altered by ATP binding. The C-terminal helix rotates findings are supported by mutagenesis data showing away from the ABC domain and binds to the opposing that an S1070R mutation that disengages ABC:ABC in- subunit (Figure 3B). This conformational transition indi- teractions abolishes ATP hydrolysis of SMCcd (see cates that the C-terminal helix is a good candidate for below). the control of SMCcd association and ATPase activity by additional subunits [12]. A second conformational change is observed in a surface loop in the vicinity of Conformational Flexibility and ATP-Induced the ATPase active site (R loop). In the presence of ATP, Structural Switches R59 binds to the ATP ␣ phosphate. In the absence of In many ABC , including ABC transporter and ATP, the R loop is partially disordered and rearranged the DNA repair Rad50, ATP binding induces (Figure 3C). The R loop is located in the center of a a conformational change within the ABC domain; this positively charged surface patch at the concave side of change has been linked to a “power stroke” [17]. To the SMCcd dimer on Lobe I (Figure 3D). An equivalent directly visualize ATP-driven structural changes in the positively charged molecular surface has been impli- SMC ABC domain, we crystallized wtSMCcd in the ab- cated in DNA binding by Rad50 [18]. The ATP-driven sence of ATP and determined the structure to 2.0 A˚ conformational variability and the location in the center resolution (Figure 3A; Supplemental Data). Comparison of a positively charged surface region suggest that the of ATP bound and nucleotide-free SMCcd revealed very R loop is a good candidate for the stimulation of the similar overall conformations, indicating that SMC pro- ATPase activity of SMC proteins by DNA. Current Biology 1780

Figure 2. ATP Bound Active Site Stereo view of the ATP bound active site, shown as ball-and-stick model with the color code of Figure 1C. Only one out of two sym- metrically related composite active sites in the dimer interface is shown. One subunit is depicted in yellow, the other in green. Key side chains are labeled (see text for details), and notable hydrogen bonds are shown as dashed lines. The major dimerization con- tacts are hydrogen bonds to the ATP ␥ phos- phate from the signature motif and to the ri- bose 2Ј- and 3Ј-OH from K1064 and K1061, respectively. Additional contacts are contri- buted from the SMC conserved DA box (A1101), which forms a hydrophobic interac- tion core at the center of the dimerization interface. A water molecule (red sphere) is positioned for collinear attack on the ␥ phos- phate (arrow) by E1098 (mutated to Q in the crystal structure) and the backbone carbonyl of H1102, suggesting that ATP hydrolysis re- quires the fully engaged ABC dimer.

DNA-Stimulated ATPase Activity and eukaryotic multisubunit SMC protein complexes, Recent data suggest that ATP hydrolysis by SMC protein suggesting that this DNA-stimulated ATPase activity di- complexes is involved in the stable association of cohe- rectly acts on the ABC domains in full SMC complexes sion with DNA in vivo and for the generation of DNA as well [19, 20]. Mutations in the Walker A motif (K39A) gyres by condensin in vitro [8, 9, 11]. To test whether and the signature motif (S1070R) abolished ATP hydroly- DNA can directly activate the ATPase activity of SMCcd, sis. The latter has been shown to prevent ATP-induced we performed thin-layer chromatography assays of ABC domain engagement [21]. This indicates that ATP wild-type and mutant SMCcd in the absence or in the hydrolysis specifically occurs in the SMCcd dimer, con- presence of a dsDNA oligonucleotide. We found that sistent with the structural results indicating that both the ATPase activity of P. furiosus SMCcd is slow in the subunits position the putative attacking water molecule absence of DNA but strongly stimulated in the presence (Figure 2). The Walker B mutation (E1098Q) strongly of dsDNA (Figure 4A). A similar DNA-dependent stimula- reduces ATPase and DNA-stimulated ATPase activity. tion of ATPase activity was observed for both bacterial The residual activity is consistent with our observation

Figure 3. ATP-Induced Conformational Changes

(A) MAD and 2Fo Ϫ Fc electron densities (1␴ contour) of a portion of the nucleotide free SMCcd crystal structure along with the re- fined model (color-coded sticks). (B) Superposition of the backbone traces of nucleotide-free (green) and ATP bound (yel- low) SMCcd shows that only minor intrado- main conformational changes are induced by ATP. The largest conformational changes are observed for the R loop, which is implicated in DNA-stimulated control of ATP hydrolysis, and the C helix, which rotates away to avoid steric clash with the opposing subunit (not shown) and to participate in ABC-ABC inter- action. Thus, the predominant role of ATP is probably to control engagement/disengage- ment of two SMC ABC domains. (C) Detailed view of the R loop (red) of super- imposed ATP bound (yellow) and nucleotide- free (gray) SMCcd (shown as backbone worms). R59 (arginine finger) directly hydro- gen bonds to the ATP ␣ phosphate (ball-and- stick model). R59 could participate in ATP hydrolysis by compensating the negative charge on the transition state phosphates. (D) Solvent-accessible surface of the ATP bound SMCcd dimer with electrostatic potential (ϩ7 kT/eϪ [blue] to Ϫ7 kT/eϪ [red]). The central region of the composite DNA is formed by the R loop, which is involved in DNA-stimulated activation of ATP hydrolysis. The circled areas represent the location of the coiled-coil domains. SMC ATPase Crystal Structure 1781

Figure 4. Structural and Mechanist Role of the Arginine Finger (A) Thin-layer chromatography ATPase activity assays in the absence (white) and presence (black) of DNA show that wtSMCcd has DNA- stimulated ATPase activity. Walker A (K39A) and Walker B (E1098Q) mutations abolish or strongly reduce ATPase activity, respectively. The signature motif mutant S1070R abolishes ATPase activity, suggesting that ATP hydroly- sis requires SMCcd:SMCcd engagement. The R loop mutation (R59A) does not affect the basal ATPase activity but abolishes DNA stimulation. Data represent means of tripli- cate assays (error bars). (B) ATP binding assays (nitrocellulose filter binding) of wild-type and mutant SMCcd. All mutations except Walker A motif K39A show qualitatively unperturbed ATP binding. This indicates that ATPase defects are predomi- nantly associated with ATP hydrolysis rather than ATP binding. Experiments represent triplicates (error bars) with subtracted low- background ATP binding. (C) Relative stimulation of the ATPase activity of SMCcd (white) and SMCcd:ScpA (black) by DNA. Experiments were performed as in Figure 4A. The arginine finger mutation R59A specifically abolishes DNA stimulation of the SMCcd:ScpA complex as well. (D) Sequence alignment showing that the arginine finger (highlighted) in the R loop is highly conserved among SMC proteins. This conservation suggests that the role of the arginine finger in coupling ATP hydrolysis to DNA binding is a more general element of SMC proteins. The following abbreviations were used: Pf, Pyrococcus furiosus; Tm, Thermatoga maritima; Bs, Bacillus subtilis; hu, human; and Sc, . that crystals of ATP:SMCcd-E1098Q dissolved after a The arginine finger is among the most conserved resi- few days, indicating that the observed crystal structure dues in SMC proteins (Figure 4D). This exceptional con- nevertheless shows an active site that is capable of ATP servation indicates that the interaction of the arginine hydrolysis. finger with ATP is a key mechanism in the molecular function of SMC proteins. The structure and mechanistic Conserved Arginine Finger role of arginine fingers on the DNA-stimulated activation Because the R loop showed a substantial rearrangement of the SMCcd ATPase activity shows a remarkable anal- in response to ATP binding, we tested the effect of ogy to the allosteric activation of small by the arginine finger (R59) on the DNA-stimulated ATPase GTPase-activating proteins (GAPs). GAPs insert into the activity. We mutated R59 to alanine and performed ATP- active site of G proteins an arginine finger that both ase activity assays in the presence and absence of DNA. binds GTP and stimulates GTP hydrolysis [22]. R59 most R59A does not affect the basal ATPase activity of likely counterbalances the developing negative charge SMCcd, but it completely abolishes its stimulation by during ATP hydrolysis. The location of the R loop within DNA (Figure 4A). To test whether R59A and other mu- the positively charged molecular surface of SMCcd sug- tants still bind ATP, we performed filter binding assays gests that DNA binding could directly modify the confor- (Figure 4B). wtSMCcd, as well as SMCcd-R59A, E1098Q, mational properties of the R loop; this would lead to a and S1070R, retained ATP binding. On the other hand, stable binding of R59 to ATP. the Walker A mutation SMCcd-K39A is defective in ATP binding. This is consistent with a model stating that Mechanistic Implications and Conclusions R59A abolishes only the DNA stimulation of the ATPase Recent results indicate that ATP binding to SMC pro- activity and that S1070R specifically prevents SMCcd teins is a major driving force in condensation of DNA dimer formation but does not interfere with ATP binding by E. coli MukBEF in vitro. ATP binding was also found to Walker A motifs. We also tested the effect of the to be important for cohesin ring assembly in vivo [7, 9, non-SMC subunit ScpA on the role of R59 in the DNA- 11]. As suggested by the crystal structure, these higher- stimulated ATPase activity. ScpA has no significant ef- order structures are probably mediated by stable ATP fect on the ATPase activity of pfSMCcd (data not shown) bound SMCcd-SMCcd dimers (Figure 1B). The poor although it moderately reduces ATPase activity of Bacil- basal ATPase activity, further regulated by non-SMC lus subtilis SMC [12]. In any case, the ScpA:SMCcd subunits, could result in fairly stable SMC complex/DNA complex also showed pronounced DNA-stimulated structures in both cohesion and condensation [12]. Nev- ATPase activity, which was specifically abolished by ertheless, these structures must be eventually disas- R59A (Figure 4C). Thus, the DNA stimulation of the ATP- sembled for various reasons; for example, to allow trans- ase activity of SMCcd appears to be independent of its port of DNA into cohesin rings or to decondensate DNA association with ScpA. after cell division. Activation of SMC protein ATPase Current Biology 1782

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SMC1: Supplemental Data An essential yeast gene encoding a putative head-rod-tail pro- Detailed Experimental Procedures and a supplemental table are tein is required for nuclear division and defines a new ubiquitous available at www.current-biology.com/cgi/content/full/14/19/1778/ . J. Cell Biol. 123, 1635–1648. DC1/. 17. Hopfner, K.P., and Tainer, J.A. (2003). Rad50/SMC proteins and ABC transporters: Unifying concepts from high-resolution struc- Acknowledgments tures. Curr. Opin. Struct. Biol. 13, 249–255. 18. Hopfner, K.P., Karcher, A., Craig, L., Woo, T.T., Carney, J.P., We thank Frank Uhlmann for communicating unpublished observa- and Tainer, J.A. (2001). Structural biochemistry and interaction tions. We thank Jan Lo¨ we and Kim Nasmyth for exchanging manu- architecture of the DNA double-strand break repair Mre11 scripts prior to publication. We thank Toni Meinhart and the staffs nuclease and Rad50-ATPase. 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