Crystal structure analysis of human Sirt2 and its ADP-ribose complex

Journal of Structural Biology 182 (2013) 136–143

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Crystal structure analysis of human Sirt2 and its ADP-ribose complex Sébastien Moniot a, Mike Schutkowski b, Clemens Steegborn a,⇑ a b

Department of Biochemistry, University of Bayreuth, 95440 Bayreuth, Germany Department of Enzymology, Martin-Luther University Halle-Wittenberg, 06099 Halle (Saale), Germany

a r t i c l e

i n f o

Article history: Received 7 December 2012 Received in revised form 8 February 2013 Accepted 14 February 2013 Available online 26 February 2013 Keywords: Sirtuins Crystal structure Deacetylase Co-substrate-induced conformational change Aging

a b s t r a c t Sirtuins are NAD+-dependent protein deacetylases that regulate metabolism and aging-related processes. Sirt2 is the only cytoplasmic isoform among the seven mamalian Sirtuins (Sirt1-7) and structural information concerning this isoform is limited. We crystallized Sirt2 in complex with a product analog, ADPribose, and solved this first crystal structure of a Sirt2 ligand complex at 2.3 Å resolution. Additionally, we re-refined the structure of the Sirt2 apoform and analyzed the conformational changes associated with ligand binding to derive insights into the dynamics of the enzyme. Our analyses also provide information on Sirt2 peptide substrate binding and structural states of a Sirt2-specific protein region, and our insights and the novel Sirt2 crystal form provide helpful tools for the development of Sirt2 specific inhibitors. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction Sirtuins form the conserved protein deacetylases (PDAC) class III that regulates metabolism and lifespan in lower organisms (Guarente and Picard, 2005). In mammals, they are associated with a large set of functions such as cellular stress resistance and genomic stability. Among the four PDAC classes, the Sirtuin family is unique in using NAD+ as a co-substrate, rendering Sirtuins metabolic sensors. They are involved in various metabolism and age-related diseases and thus considered attractive therapeutic targets (Bauer et al., 2010; Haigis and Sinclair, 2010). Seven Sirtuin isoforms, Sirt1-7, have been identified in mammals. They differ in their substrate specificities and subcellular localizations. Sirt1, 6 and 7 are nuclear proteins, which, for example, affect chromosome stability and transcription regulation (Michan and Sinclair, 2007; Haigis and Sinclair, 2010). Sirt3, 4 and 5 are located in mitochondria where they regulate metabolic enzymes and stress response mechanisms (Schlicker et al., 2008; Gertz and Steegborn, 2010; Verdin et al., 2010). Sirt2 is the only mainly cytoplasmic isoform (Michishita et al., 2005) but has also been observed in the nucleus (Bae et al., 2004; Inoue et al., 2007) and appears to contain several nuclear export motifs (Wilson et al., 2006; Cen et al., 2011). It deacetylates various targets, such as a-tubulin (North et al., 2003) and the histone H4 (Vaquero et al., 2006), and thereby regulates functions in cell cycle progres⇑ Corresponding author. Address: University of Bayreuth, Dept. Biochemistry, Universitätsstr. 30, 95440 Bayreuth, Germany. Fax: +49 (921) 552432. E-mail address: [email protected] (C. Steegborn). 1047-8477/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jsb.2013.02.012

sion, cellular motility and differentiation (Cen et al., 2011). Sirt2 exists in two splice variants, a 389-residue form normally referred to with residue numbers (also in the present manuscript), and a 352-residue form dominant in brain that lacks an N-terminal extension to the catalytic domain (Dryden et al., 2003; Suzuki and Koike, 2007; Flick and Lüscher, 2012). The strong expression of Sirt2 in brain indicates a central function in this tissue, and it was indeed identified to regulate myelination (Beirowski et al., 2011) and to contribute to pathologies of neurodegenerative diseases such as Parkinson’s and Huntington’s disease (Outeiro et al., 2007; de Oliveira et al., 2012). Sirt2 shares the typical Sirtuin architecture with a conserved catalytic core of 275 residues flanked by N- and C-terminal extensions. So far, little is known about the role and structure of the N- and C-terminal Sirt2 extensions but they seem to control Sirt2 subcellular localization and catalytic activity (Flick and Lüscher, 2012). A nuclear export sequence in the Sirt2 N-terminus (residues 41–51) is responsible for its subcellular localization (North and Verdin, 2007a), and Sirt2 activity can be modulated by phosphorylation on its C-terminal extension (Ser368 and Ser372) (Pandithage et al., 2008) or acetylation (multiple target lysines identified in the C-terminus) (Han et al., 2008). The crystal structure of the catalytic core of human Sirt2 revealed the typical oval-shaped fold composed of two globular subdomains, a Rossmann-fold and a Zinc-binding domain (Finnin et al., 2001). The large groove located at the interface between the two domains contains the binding sites for substrate and co-substrate, acetylated protein and NAD+, as well as the catalytic residues. The commonly accepted sirtuin reaction mechanism involves, as a first

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step, the release of nicotinamide from NAD+ and nucleophilic addition of the acetyl oxygen on the 10 -carbon of the nicotinamide ribose (N-ribose) to form a C10 -O-alkylamidate intermediate. The N-ribose 20 -hydroxyl group activated by a conserved catalytic His residue then attacks the O-alkylamidate intermediate carbon to yield a 10 20 -cyclic intermediate that will finally be resolved by a base-activated water molecule to yield the deacetylated lysine and 2’-O-acetyl-ADP-ribose (Sauve, 2010). Analysis of the structure also revealed features specific to Sirt2 such as an insertion in the Rossmann-fold which includes the a12-helix (residues 296– 303)(Finnin et al., 2001). Pharmacological inhibition of Sirt2 appears to be an attractive approach for treatment of neurological diseases (Cen, 2010; de Oliveira et al., 2012) and various types of cancer (Kim et al., 2011; de Oliveira et al., 2012). Several inhibitors for Sirt2 have been described, such as salermide (Lara et al., 2009), CSC13 (Schlicker et al., 2011) and AGK2 (Outeiro et al., 2007), but they are not yet highly potent and/or show limited specificity, especially against Sirt1, or even have not been tested against most isoforms (Cen, 2010; Chen, 2011). Moreover, binding sites and inhibition mechanisms of these compounds are largely unknown, while a complex set of conformational change is known to occur during Sirtuin catalysis, including a closure movement upon substrate binding and different conformational states of a so-called cofactor loop. Thus, the molecular details of ligand binding and associated conformational changes are of great interest (Moniot et al., 2012). Here, we describe the crystal structure of hSirt2 in complex with ADP-ribose (ADPr). We analyze structural details of Sirt2 ligand interactions, and compare conformational changes between apo-Sirt2 and its complex with ADPr as a product analog as well as structures of other Sirtuins and identify general conformational changes in Sirtuins and Sirt2-specific features. 2. Materials and methods 2.1. Protein cloning, expression, and purification of Sirt2 A gene fragment encoding human Sirt2 (residues 34–356) was cloned into pGEX-4T3 (GE Healthcare, Little Chalfont, United Kingdom) using BamH1 and Xho1. Sirt2 was expressed using the autoinduction method in Escherichia coli Codon+ grown 5 h at 37 °C, then 24 h at 25 °C in TB medium supplemented with 0.2% lactose. Harvested cells were resuspended in buffer A (50 mM Tris pH 8.0, 500 mM NaCl, 5 mM BME) disrupted using a microfluidizer (Microfluidic, Newton, USA) and cell debris removed by 45 min centrifugation at 4 °C, 18,000 rpm in a HFA22.50 rotor. The supernatant was supplemented with 10% glycerol and incubated with GST-Buster QF glutathione resin (Amocol, Teltow, Germany) for 1 h at 4 °C. The resin was washed in a column with 10 volumes buffer A and eluted with 50 mM Tris pH 8.0, 500 mM NaCl, 5 mM BME, 20 mM reduced glutathione. The protein was concentrated in an amicon concentrator (Millipore, Billerica, USA) and digested 48 h at 4 °C with thrombin (GE Healthcare) during dialysis against buffer A. The protein was diluted 10 times with 50 mM Tris pH 8.0, 50 mM NaCl, 5 mM BME and further purified by anion exchange chromatography using an HitrapQ FF column (GE Healthcare). GST and Sirt2 could be selectively eluted using a stepwise gradient of 8% and 18% of a 50 mM Tris pH 8.0, 1 M NaCl, 5 mM BME buffer, respectively. Sirt2 was then run on a Superdex200 gel filtration column (GE healthcare) in buffer B (50 mM Tris pH 8.0, 150 mM NaCl, 2 mM TCEP), analyzed by SDS–PAGE, concentrated, and kept at 4 °C.

1.5 mM NAD+ and 100 lM acetylated or butyrilated CarbamoylPhosphate Synthase 1 peptide (CPS1; Bz-GVLKEYGV-amide) or 250 lM acetylated or palmytoilated a-tubulin peptide (MPSDKTIG). The reaction was started by adding NAD+ and followed for 30 min through the decrease of absorbance at 340 nm in a microplate spectrophotometer MQX200 (MWG-Biotech, Germany). The background signal was measured under the same conditions omitting the substrate peptide from the reaction. Measurements were repeated at least twice. 2.3. Crystallization and diffraction data collection and processing Prior to crystallization, Sirt2 was incubated 1 h at RT with trypsin (1:140 mass ratio) in buffer B supplemented with 2 mM ADPr. Co-crystallization was achieved using vapor diffusion in sitting drops composed of equal volumes (0.2 lL) of the Sirt2 solution (12.9 mg/mL in buffer B, 2 mM ADPr) and reservoir solution (20% PEG 10,000, 100 mM Ammonium acetate, 100 mM Bis-Tris pH 5.5). Bi-pyramidal crystals that appeared within 5 days at 20 °C were mounted in nylon loops and flash-cooled in liquid nitrogen after a quick soak in cryo-solution consisting of crystallization solution supplemented with 25% glycerol. A complete diffraction dataset was collected at BESSY (Berlin, Germany) BL14.1 (Mueller et al., 2012) at 100 K using a wavelength of 1.278 Å and a single crystal. The dataset was processed and scaled using XDS/XSCALE (Kabsch, 2010)(Table 1). Pseudo-merohedral twinning (twin law k,h,l; and twin fraction 0.65/0.35) was detected using phenix.xtriage from the Phenix suite (Adams et al., 2010) and the set of 5% reflections attributed to the Rfree calculation was thus selected using the phenix.reflection_file_converter program that takes lattice symmetry into account to reduce the bias introduced by twinning. Table 1 Diffraction data and refinement statistics.

Diffraction data statistics Space group Unit cell (Å) Resolution limits (Å) Outermost shell (Å) Rmerge (%) CC(1/2) (%) Redundancy I/r(I) Completeness (%) Refinement statistics Resolution range (Å) Total atoms Water atoms R-factor (%) Rfree (%) r.m.s.d Bond length (Å) Bond angle (°) B factor (Å2) Wilson plot Main-chains atoms Side-chains and water atoms ADP ribose Twin operator Twin domain fractions Procheck H-test Phenix.refine twin Fractions a

Activity assays were performed as described in (Smith et al., 2009). Briefly, the reaction mix contained 1 lM Sirt2 (34–356),

Sirt2ADPr

a

P 212121 77.9 78.0 114.3 64.4–2.3 2.33–2.27 7.4 (60.4) 99.3 (36.9) 2.6 (2.5) 10.8 (2.3) 99.0 (98.2)

a a a a

a a a

19.8–1.6b 9198b 1099b 17.0b 19.9b,c

64.4–2.3 9878 278 15.0 18.8d

0.016b 18b / 25.6 18.7b 24.5b /

0.006 0.9

/

66/34 64/36

44.2 34.6 39.8 30.8 k, h, l

Diffraction data statistics of Finnin et al. (2001) deposited in PDB under ID 1j8f. Refinement statistics for the re-refinement described in the present paper. Rfree set of reflections is identical to the one used for refinement of the previously deposited structure (PDB: 1j8f). d The free set of 5% reflections was randomly selected using the phenix.reflection_file_converter taking into account the twinning operator (Adams et al., 2010). b

2.2. Coupled deacetylation assays

Sirt2

c

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2.4. Phasing, refinement and validation of the crystal structures The published structure of human Sirt2 (Finnin et al., 2001) was re-refined against structure factors available at the PDB (PDB: 1j8f) keeping the same Rfree reflections set and Refmac5 (Murshudov et al., 2011) and Coot (Emsley et al., 2010) for manual rebuilding. During the latest stages of refinement, TLS parameters obtained from the TLSMD server (Zucker et al., 2010) were used, and the structure validated using the Molprobity server (Chen et al., 2010). Initial phases for the Sirt2/ADPr complex structure were obtained by Patterson searches using Molrep (Vagin and Teplyakov, 2010) and the re-refined apo-Sirt2 structure as a model with the Rossmann fold and the Zinc-binding domains considered as independent components. The two copies of Sirt2 that compose the asymmetric unit were rebuilt using Coot and refined using the Phenix suite v1.8.1 (Adams et al., 2010), in the last stages with TLS parameters obtained from Phenix. The geometry of the model was validated using Molprobity. Refinement statistics for both models can be found in Table 1. The protein surface electrostatic potentials were analyzed using the APBS plugin from Pymol. All figures were generated with Pymol (The PyMOL Molecular Graphics System, Version 1.5.0.1 Schrödinger, LLC). 2.5. Coordinates Coordinates of the re-refined Sirt2 apoform and coordinates and structure factors of the Sirt2/ADPr complex crystal structures were deposited in the Worldwide Protein Data Bank (PDB) under accession codes 3zgo and 3zgv, respectively. 3. Results 3.1. Re-refined structure of human Sirt2 apoenzyme For studying ligand interaction details and conformational changes of human Sirt2, and to compare them to other isoforms to derive general and Sirt2-specific features, we first analyzed the previously published Sirt2 structure (Finnin et al., 2001) available from the Protein Databank (PDB: 1j8f). Several errors, such as a misattribution of the N-terminal a0-helix to its corresponding monomer, improper geometry for bonds to the included hydrogen atoms, and an apparent sequence mutation lead us to re-refine the structure against the published diffraction data. We further used TLS refinement enabling us to account for different substructure dynamics. The re-refined Sirt2 apo structure had R/Rfree factors of 17.0/19.9% (versus 23.5/26.0 in Finnin et al. (2001); for refinement statistics for the new model see Table 1), and test runs with both models and various refinement options indicate that our corrections and the use of more recent refinement algorithm and TLS parameters contributed about equally to this improvement (Suppl. Table 1). The re-refined model consists of three Sirt2 molecules spanning residues 32–356 (48–54 are missing in chains A and B, and chain C starts at residue 54). The N-terminal a0 helices of chain A and B were re-attributed to the correct monomer of the asymmetric unit with gap distances (chain-break) of 17.6 and 20.2 Å, respectively. This assignment is compatible with the flexible 8/9 residue-long loop (46/47–54) that is not defined by the electron density maps, correcting the >45 Å distances of the previously deposited model that cannot be covered by this loop. Careful analysis of the Fourier difference maps also revealed a likely point mutation at position 239, comparably in all three chains of the model. The electron density in this region strongly suggests the presence of a proline residue rather than the wild-type leucine (Suppl. Fig. 1). Our re-refined model was used as apo-Sirt2 struc-

ture for comparison with the ADPr bound structure (see below) throughout this paper. 3.2. Overall structure of the Sirt2/ADP-ribose complex and comparison to the apoenzyme To analyze substrate/product interaction details, we solved the crystal structure of Sirt2 in complex with ADPr, which covers part of the co-substrate NAD+ and closely resembles the product 20 -Oacetyl-ADP-ribose. Initial crystallization trials with the construct used for the published apo-structure (Finnin et al., 2001) remained unsuccessful, and we therefore used in situ proteolysis with trypsin (see Section 2) to obtain crystals of a Sirt2/ADPr complex. Crystals belonged to space group P212121 and showed partial merohedral twinning (twin fractions 0.65/0.35). The structure was solved through Patterson searches with the apo-structure and refined at 2.3 Å resolution to twinned R/Rfree values of 15.0/18.8% (Table 1). The final model is composed of two copies of the Sirt2 catalytic core spanning residues 55/57–355, each complexing a Zinc cation and binding one clearly defined molecule of ADPr (see below). The quality of the electron density maps allowed rebuilding of the complete structure apart from the loop 139–141 that was not defined in electron density maps and is assumed to be flexible rather than hydrolyzed during trypsinolysis since no positively charged residue (preferred by trypsin) is present in this region. The two monomers that are related to each other through 2-fold non-crystallographic symmetry show no significant differences (rmsd 0.38 Å over 293 Ca atoms), and further descriptions will thus be based on chain A unless explicitly stated. Like other Sirtuins and apo-Sirt2 (Finnin et al., 2001; Sanders et al., 2010; Moniot et al., 2012), the catalytic core of the Sirt2/ADPr complex adopts an elongated fold with 2 subdomains (Fig. 1a): A large domain (residues 53–89, 146–186 and 241–356) that is a variant of the Rossmann-fold (Bellamacina, 1996) and a smaller domain (residues 90–145 and 187–240) known as the Zinc-binding domain. A large groove for accommodating the substrate polypeptide chain at the interface of the two domains is formed by four interconnecting loops (including the flexible loop 139–141) and three loops from the Rossmann-fold domain. Comparison of the Sirt2/ADPr complex structure with apo-Sirt2 reveals major conformational changes. Taking the Rossmann-fold domain as a reference, the Zinc-binding domain undergoes a rotation of 25°, closing the active site groove around the ADPr molecule (Fig. 1b). The Zinc-binding domains of the Sirt2/ADPr complex show slightly higher B factors than the Rossmann-fold domains. This can be explained by a higher flexibility of this domain which is supported by ellipsoidal-shaped density peaks around the zinc atoms in the calculated anomalous maps (Suppl. Fig. 2) and might be related to a looser crystal packing of this subdomain compare to the Rossmann-fold. A second major yet more local conformational difference between apo-Sirt2 and the Sirt2/ADPr complex concerns its a12 helix and the peptide binding channel and is described below. 3.3. The ADP-ribose binding site Positive signals in Fourier difference maps at the co-substrate binding site of Sirt2 clearly showed the presence of bound b-ADPr (Fig. 2a). The ADPr molecule is bound by an extended network of H-bonds directly with residues of the binding site or mediated through water molecules (Fig. 2b; Suppl. Fig. 3). Several sidechains of the active site also contribute to binding through van der Waals interactions, notably Lys287 and Cys324 for the adenine ring and Phe96 and Val266 for the nicotinamidic-ribose (Nribose)(Fig. 2b). This interaction with Phe96 is enabled through the only major local conformational change occurring upon ADPr

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Fig. 1. Overall structures of the ADP-ribose complex and the apo-form of human Sirt2. In both panels, the ADP-ribose (ADPr) complex is displayed in yellow and the apostructure of Sirt2 in petrol. The ADPr is displayed in stick mode. (a) Overall structure of Sirt2 complexed to ADPr. Secondary structure elements were labeled according to Finnin et al. (2001). (b) Rossmann-fold domain of both structures were superposed. Binding of ADPr to Sirt2 induces a 25° rotation of the Zinc-binding domain toward the Rossmann-fold domain, closing the co-substrate binding site around its ligand.

binding, which concerns the co-substrate-binding loop. This L1 loop around Phe96 (residues 90–104) also provides Arg97 and Tyr104 for ADPr binding and undergoes a closure movement over the ADPr molecule (Fig. 2c). This movement also results in a partial unwinding and reorientation of helix a3 (residues 105–110) by an angle of 45° when compared to the apostructure (Fig. 2c). In addition to the rotational domain movement and rearrangement of the L1 loop, ADPr induces local adjustments of the L5 loop (residues 262–268) and especially of the side-chains of Ser263 and Val266 that participate in ADPr binding through H-bonding and van der Waals interactions, respectively (Fig. 2b; Suppl. Fig. 3). The binding site for the adenine moiety, however, appears preformed in the apo-structure and exhibits only minimum alterations. The nicotinamide C-pocket also appears as preformed and the potential binding-site of the carbamide represented by two crystallographic water molecules which superposed with the NAD+ bound to Sir2 from Thermotoga maritima (Sir2Tm) (Hoff et al., 2006)(Fig. 2e). As a result of the Zinc-binding domain rotation, the catalytic His187 side-chain (Nd1 atom) moves in H-bond distance (2.6 Å) to the O3 oxygen atom of the N-ribose moiety. An acetate molecule from the crystallization solution was also found in the active site. It forms a H-bond with the C1 hydroxyl function of the N-ribose and its methyl group forms hydrophobic interactions with the side-chains of Phe119, Ile232 and Phe234 (Fig. 2b). A superposition with structures of peptide-bound Sirtuins (Fig. 2d) shows that the location of this acetate is consistent with the location of the acetyl group of an acetyllysine substrate. In the same manner, its position fits the one described for 20 -O-acetyl-ADP-ribose bound to yeast Hst2 (Zhao et al., 2003). In both cases, the acetate is slightly shifted away from the characteristic binding site, but this is likely due to the presence of a hydroxyl function on the ADPr molecule C1 position that forms a H-bond with the acetate (Fig. 2e, f).

3.4. Substrate peptide binding sites and their interactions with symmetry mates The Sirt2 Rossmann-fold domains are overall very similar in the apo-Sirt2 and Sirt2/ADPr structure (rmsd 0.41 Å for 108 Ca pairs). However, a striking difference can be observed for the a12-helix (residues 296–303) of the apo-structure that is completely unwound in the Sirt2/ADPr complex (Fig. 3a). This region corresponds to an insertion of 15 residues (residues 294–307) in the Rossmannfold characteristic for the Sirt2 isoform (Fig. 3b). This loop adopts the same conformation in both chains of the asymmetric unit and mediates crystal contacts between them by occupying the substrate peptide groove of the partner monomer. Interestingly, the side-chain of residue Leu297 is even pointing into the acetyl-lysine binding site of the other chain and mimicks the binding mode of a substrate acetyl-lysine (Fig. 2d). In the apo-structure, this region is in helical conformation and also mediates contacts leading to ‘‘crystallographic dimers’’, since the a12-helix was described as interacting with the hydrophobic pocket of the large groove at the inter-domain interface of a symmetry mate (Finnin et al., 2001). However, the two crystallographic dimers are overall very different, with the apo-dimer in a head-to-tail association mode while the Sirt2/ADPr complexes are facing each others in the asymmetric unit (Suppl. Fig. 4). In fact, BN-PAGE analysis and size-exclusion chromatography indicate that Sirt2 (34–356) behaves predominantly as a monomer rather than a dimer in solution. Thus, the crystallographic dimers appear to result from crystal packing rather than to reflect a physiologically relevant oligomerization state. Analysis of the electrostatic surfaces of both the apo- and ADPr complexe structures around the peptide-binding site as well as that of the surrounding a10, a11, and a12 helices reveals that they are relatively hydrophobic, which is typical for protein/protein interaction interfaces. The hydrophobic side-chains of a12 res-

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Fig. 2. The active center of the Sirt2/ADP-ribose complex. (a) The ADP-ribose (ADPr) and the acetate of the final model are shown as sticks in a Fourier omit map (2mFo-DFc) contoured at 1.2 sigma (1.12 e/Å3). (b) the ADPr molecule (green) is presented with the residues of the binding site (yellow). Hydrogen bonds are shown as dashed lines. (c) The Zinc-binding domain of the apo-Sirt2 structure (petrol) was superposed to the one of the ADPr complex (yellow). The a3 helix and the co-substrate binding-loop, L1, undergo conformational changes upon ADPr binding. Side-chains of F96, R97, and Y104, which are directly implied in ADPr binding, are displayed as sticks and labeled. (d) The structure of Sirt3 and Sir2Tm in complex with the acetylated AceCS2 (blue; PDB: 3glr) and p53 (green; PDB: 2 h2h) peptides, respectively, were superposed onto the structure of Sirt2/ADPr (chain A in yellow). The a12 loop of the Sirt2/ADPr structure (chain B in purple) mimics a substrate peptide. The acetylated lysines, L297 (a12 loop), the acetate and ADPr molecules are depicted as sticks. (e) The structure of Hst2 in complex with ADPr (PDB: 1q17, pink), or with 20 -O-acetyl ADPr (PDB: 1q1a - corrected, grey) and that of Sir2Tm in complex with NAD+ (PDB: 2h4f; light blue) were superposed on the structure of Sirt2/ADPr. The position of the ‘‘gate-keeper’’ F96 (Sirt2 numbering) is also shown. (f) Close-up view of the NAD+ N-ribose part from the superposition shown in panel (e), with an alkylimidate intermediate bound to human Sirt3 (PDB: 3glt) added in light green. The apparent movement of the N-ribose along the reaction axis is indicated with an arrow, and the position occupied by O4 (NAD+), C1 (alkylimidate), or C2 (ADPr or 20 -O-acetyl-ADPr) is circled.

idues interact with the ‘‘active-site’’ hydrophobic pocket of a symmetry mate molecule in the apo-structure (Finnin et al., 2001) and the interaction of the a12 loop with the partner monomer in the ADPr complex is also mainly based on van der Waals interactions, which might indicate a general function of this protein element in protein/protein interactions. In both Sirt2 structures, the B-factors of residues 296–303 (a12 and neighboring) are significantly lower than the average B value of the model, supporting two stable conformations of this region as an helix or as a loop. Taken together, it seems that Sirt2 is a monomeric molecule with its active-site face displaying a high propensity to interact with proteins and its Sirt2specific insertion being conformationally adaptable. The different interaction modes of this Sirt2 region observed in different crystal contacts might suggest a function in binding varying Sirt2 ligands, possibly different substrate proteins. The loop 296–303 in the Sirt2/ADPr complex interacts with the peptide substrate binding site of another monomer, with Leu297 mimicking the substrate acetyl-lysine (Fig. 2d). The shortest distance between Leu297 and ADPr is barely over 4 Å. As in previous structures of Sirtuin complexes with substrate peptides, the interaction between binding groove and interacting polypeptide resembles an intermolecular b-sheet. The main-chain of residues 295– 298 superposes well with that of Acetyl-coA synthetase 2 (AceCS2) or p53 peptide bound to Sirt3 and Sir2Tm, respectively, and form

four hydrogen bonds in a b-sheet-like pattern (Fig. 3c). The sidechain of Leu297 points in the same direction as the acetylated lysines (Fig. 2d). Van der Waals interactions exist notably between Leu297⁄ (⁄ refers to the partner monomer) and Val233/Phe235/ Val266, Pro295⁄ and Phe244, and Met299⁄ and Pro268. The 295– 298 loop thus acts as a substrate peptide mimetic. However more conformationally constrained than a free peptide, the C-terminal part of the loop (from residue 298 on) starts to deviate form the typical binding mode. Two H-bonds exist between the main-chain atoms of Leu303 of the peptide-like loop and side-chains of a11helix residues, Ser271 and Lys275. The side-chain of Leu303 is also ‘‘inserted’’ into a hydrophobic patch constituted by residues Phe243, Phe244, and Met247 of the a10 helix and Leu272 and Lys275 of a11. A comparison of Sirt3 in complex with substrate-peptide and in complex with substrate-peptide and ADPr, respectively, show that the conformational rearrangement of the co-substrate binding loop induced upon co-substrate binding leads to the opening of a channel from the N-ribose which was proposed as the exit/entry for nicotinamide (Jin et al., 2009). A similar channel is observed in the complex between Sirt2 and ADPr, independent of the different conformation of the ‘‘gate-keeper’’ phenylalanine (Phe96 and Phe157, respectively, in Sirt2 and Sirt3), which is only partially closing this channel in the Sirt2/ADPr complex (Fig. 4a). This proposed exit

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served in the structure of Plasmodium falciparum Sir2A (Pf Sir2A) that was recently co-crystallized with a myristoylated H3K9 substrate peptide and shown to hydrolyze long chain acyl groups from lysine residues more efficiently than acetyl modifications (Zhu et al., 2011). However, testing Sirt2 against butyrilated and palmitoylated substrate peptide showed activity, but significantly weaker than its deacetylase activity (Fig. 4b). Thus, this large channel is accessible to substrate acyl groups but appears not to shift the substrate specificity to long chain acyl groups, in contrast to PfSir2A. The channel thus might indeed function as exit for nicotinamide, but definitive conclusions will have to await further experimental tests.

4. Discussion

Fig. 3. Unwinding of Sirt2 a12 helix. (a) Detailed view of the a12 helix in the apoSirt2 structure (petrol) and its unwound form in the binary complex with ADPribose (yellow) with the positions of L297 side-chain. (b) Multiple sequence alignment of the seven human sirtuin isoforms (Sirt1 to Sirt7; uniprot code Q96EB6, Q8IXJ6, Q9NTG7, Q9Y6E7, Q9NXA8, Q8N6T7, and Q9NRC8, respectively) and of several non-human Sirt2 isoforms (Rattus norvegicus (Q5RJQ4), Bos Taurus (A6QP26), Gallus gallus (Q68BG1), Xenopus leavis (Q5U250), Danio rerio (Q7ZVK3), and Anolis carolinensis (H9GFI4)) as obtained from the web server TCOFFEE Espresso. The alignment of the region surrounding the a12 helix is shown with the secondary structure element identified in the apo-Sirt2 structure (Finnin et al. 2001). (c) The interaction between the a12 loop of the Sirt2/ADPr complex (chain B, purple) and the peptide substrate binding site (chain A, yellow) mimics a b-sheet association.

channel is, however, very different in both structures. In Sirt2, the channel is part of a large and elongated hydrophobic pocket that is the largest pocket so far described for human Sirtuin isoforms. Its dimensions (12  10  9 Å) are comparable to the pocket ob-

The structure of human Sirt2 in complex with ADPr presented here and its comparison to previously reported structures of apoform and various complexes of other Sirtuins reveal conformational changes induced upon substrate binding and catalytic turnover. The apo-structures of Sirt2 and Sirt3 represent relatively open conformations (Finnin et al., 2001; Jin et al., 2009). In both cases, binding of a substrate induces a closure of the two domains around the substrates. The movement of Sirt2 upon ADPr binding is similar to the one reported for Sirt3 with, respectively, 25° and 23° (3gls to 3glt – Dyndom server) rotation of the domain relative to each other. In case of human Sirt3, however, binding of the substrate peptide and NAD+ were proposed to be sequential events (Jin et al., 2009). Binding of the AceCS2 peptide induces a first rearrangement that brings the two Sirt3 domains closer to each others (3gls (apo) to 3glr (AceCS2) 18° – Dyndom server), which was suggested to be essential for NAD+ binding. The binding of NAD+ to peptide-bound Sirt3 induces only limited rearrangements, such as the folding down of the co-substrate binding loop around NAD+ (Jin et al., 2009). Efficient binding of NAD+ in the absence of substrate peptide was reported for other Sirtuins such as Sir2Tm or Sir2Af2 (Chang et al., 2002; Avalos et al., 2004; Hoff et al., 2006), and the different response to NAD+ indicates that Sirtuin subfamilies with different preferences concerning the substrate binding sequence might exist. In the present study, ADPr could be cocrystallized with human Sirt2 in the absence of substrate peptide. Nevertheless, the presence in the crystal packing of a neighboring molecule which mimics the binding of a substrate peptide makes it unclear whether Sirt2 behaves as suggested for Sirt3, or as Sir2Tm/ Sir2Af2. However, only threefold different Kd values were recently reported for NAD+ binding to Sirt3 in the absence of substrate peptide or under peptide saturation (0.71 ± 0.23 mM and 0.26 ± 0.15 mM, respectively) (Fischer et al., 2012) indicating that there might be no fixed order for this isoform as well. As shown by a serie of crystal structures of Sir2Tm (DADMeNAD+ analog, S-alkylamidate reaction intermediates as well as complexes with products or product analogs) the most dynamic part of NAD+ during catalysis is the N-ribose moiety (Hoff et al., 2006; Hawse et al., 2008) (Fig. 2f). ADPr is actually chemically more closely related to the product 20 -O-acetyl-ADP-ribose than to the co-substrate NAD+. The conformation of the N-ribose in our Sirt2 complex is indeed equivalent to those described for other ADPr and product complexes, with the a-face of the ribose pointing to the acetyllysine-binding site. This conformation of the N-ribose is not compatible with that of NAD+, which assumes a constrained conformation with its C1-nicotinamide pointing into the C-pocket. The N-ribose appears to rotate gradually along the reaction axis with three of its atoms (O4, C1 and C2) nearly staying in the same location (Fig. 2f). After cleavage of the N-glycosidic bond, a rotation of the sugar ring brings the C1 atom to the acetyl group for the formation of the O-alkylamidate. Then, the N-ribose

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Fig. 4. The potential nicotinamide exit/entry, Sirt2 hydrophobic pocket and substrate specificity. (a) Surface representation of the Sirt2/ADPr complex. Residues 140–141 (dark grey patch), not defined by the electron density, were modeled according to the re-refined apo-structure. The surfaces of the gate-keeper, F96, and of the acetyllysine mimic, L297 (chain B), are shown in red and purple, respectively. The ADPr and acetate molecule bound to Sirt2 active site are shown as sticks. (b) SIRT2 typical specific deacetylase activities against a-tubulin and CPS1 peptides are compared to the depalmitoylase and debutyrylase activities. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

ring rotates further and presents the 20 O-acetyl group on the C2 atom after the resolution of the 10 ,20 bicyclic intermediate. Consistent with this scheme, our ADPr complex present its 20 -hydroxyl function to the acetyl binding site which is characterized through the presence of an acetate from the crystallization condition. For Sirt2, in contrast to other crystal structures of Sirtuins, the conformation of the co-substrate binding loop is perfectly defined in both the apo- and the ligand-bound state. Similar to Sirt3, binding of the co-substrate analog induces partial unwinding and reorientation of the a3 helix and folding down of the flexible loop that contributes to ligand binding. Furthermore, Phe96 of the flexible loop is stacked on top of the N-ribose and partially occupies the C-site, consistent with its suggested ‘‘gate-keeper’’ function, expelling nicotinamide and preventing base exchange (Hoff et al., 2006). Interestingly, the side-chain of Phe96 and, more generally, the folding of the co-substrate-binding loop is closing a large hydrophobic pocket at the back of the active site which appears to be specific for Sirt2 and might be exploited for further development of isoformspecific drugs. The new Sirt2 conformation and the novel crystallization procedure described here now allow to study further Sirt2 complexes, e.g. ligands binding to this site, through docking and co-crystallization. The substrate peptide mimic observed in the Sirt2/ADPr complex is supporting the classical b-sheet-like binding mode for substrate already reported for other members of the Sirtuin family and refutes the original binding mode proposed for Sirt2 based on the apo-structure that involved the hydrophobic pocket. The new substrate mimicking conformation of the Sirt2 specific insertion (residues 294–307), however, is unique. This region appears to be conformationally flexible and the two stable states observed likely result from stabilization of these conformations through crystal packing interactions. It will be interesting to see what physiological function this Sirt2-specific region mediates and it is tempting to speculate that it involves interactions with Sirt2 binding partners, such as substrates, regulators, or partners mediating specific Sirt2localization.

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