Structure of a Sir2 Enzyme Bound to an Acetylated p53 Peptide

Molecular Cell, Vol. 10, 523–535, September, 2002, Copyright 2002 by Cell Press

Structure of a Sir2 Enzyme Bound to an Acetylated p53 Peptide Jose L. Avalos,1 Ivana Celic,2 Shabazz Muhammad,1,4 Michael S. Cosgrove,1 Jef D. Boeke,2,3 and Cynthia Wolberger1,4,5 1 Department of Biophysics and Biophysical Chemistry 2 Department of Molecular Biology and Genetics 3 Department of Oncology and the 4 Howard Hughes Medical Institute Johns Hopkins University School of Medicine 725 North Wolfe Street Baltimore, Maryland 21205

Summary Sir2 proteins are NADⴙ-dependent protein deacetylases that play key roles in transcriptional regulation, DNA repair, and life span regulation. The structure of an archaeal Sir2 enzyme, Sir2-Af2, bound to an acetylated p53 peptide reveals that the substrate binds in a cleft in the enzyme, forming an enzyme-substrate ␤ sheet with two flanking strands in Sir2-Af2. The acetyllysine inserts into a conserved hydrophobic tunnel that contains the active site histidine. Comparison with other structures of Sir2 enzymes suggests that the apoenzyme undergoes a conformational change upon substrate binding. Based on the Sir2-Af2 substrate complex structure, mutations were made in the other A. fulgidus sirtuin, Sir2-Af1, that increased its affinity for the p53 peptide. Introduction The Sir2-like proteins, also known as sirtuins, form a family of NAD⫹-dependent protein deacetylases found in all eukaryotes and in many archaea and prokaryotes (Brachmann et al., 1995; Frye, 1999, 2000). These highly conserved enzymes are involved in a broad variety of critical biological processes including transcriptional silencing, DNA repair, chromosome stability, and aging. In an enzymatic reaction unique in biology, Sir2 enzymes catalyze the removal of an acetyl group from the ⑀-amino group of lysine (Imai et al., 2000; Landry et al., 2000; Smith et al., 2000; Tanner et al., 2000; Tanny and Moazed, 2001), with an accompanying cleavage of one NAD⫹ molecule for each lysine deacetylated (Tanner et al., 2000; Tanny and Moazed, 2001). The products of the reaction are a mixture of 2⬘ and 3⬘-O-acetyl ADPribose, nicotinamide, and the deacetylated peptide (Sauve et al., 2001; Tanner et al., 2000; Tanny and Moazed, 2001). ADP-ribosyltransferase activity has also been reported for Sir2 enzymes (Bell et al., 2002; Frye, 1999; Tanny et al., 1999), although the significance of this observation and its connection with the more robust deacetylase activity remains to be elucidated. The enzymatic activity of Sir2 proteins is carried out by an ⵑ260 5

Correspondence: [email protected]

amino acid core domain that is conserved among Sir2 family members (Brachmann et al., 1995; Frye, 1999). The structure of the conserved Sir2 core domain has been determined for two crystal forms of archaeal Sir2Af1 bound to NAD⫹ (Min et al., 2001) and for a fragment of human apo-SIRT2 (Finnin et al., 2001). These studies reveal that the Sir2 core consists of a Rossmann fold domain that binds NAD⫹ and a smaller domain composed of two insertions in the Rossmann fold that form a zinc binding module and a helical module, respectively. The two Sir2-Af1 structures differ in the conformation of a flexible loop region located in the helical module and also in the conformation of the bound NAD⫹, suggesting considerable flexibility in the enzyme. In addition, the relative position of the Rossmann fold and zinc binding/helical module domains differs between the Sir2-Af1 and SIRT2 structures, although it was not possible to ascertain whether these were static variations or were due to the differently liganded states of the enzymes. A key question regarding the biological activity of Sir2 proteins centers on their substrate specificity. The beststudied of these enzymes, budding yeast Sir2p, deacetylates specific lysine residues in the N-terminal tails of histones critical for transcriptional silencing (Imai et al., 2000; Braunstein et al., 1993, 1996; Landry et al., 2000; Thompson et al., 1994). However, the yeast genome encodes five Sir2-like proteins that serve distinct cellular functions (Shore et al., 1984; Brachmann et al., 1995; Rine and Herskowitz, 1987; Xie et al., 1999) and presumably recognize different substrates. Most eukaryotic genomes encode four to seven Sir2 family members, and it is likely that these, too, will have distinct targets. It is clear that not all targets are chromatin-associated proteins, as recent studies have revealed that the p53 tumor suppressor protein is deacetylated by the human SIRT1 protein (Luo et al., 2001; Vaziri et al., 2001). For many eukaryotic sirtuins, target specificity may be mediated, at least in part, by N- and C-terminal domains outside the conserved enzymatic core. In support of this idea, mutations in yeast SIR2 that selectively affect distinct forms of silencing are located in the terminal extension domains (Cuperus et al., 2000). However, the archaeal and prokaryotic sirtuins, as well as some of the eukaryotic enzymes, consist of the core domain and little else. For these enzymes, the conserved Sir2 core may be sufficient for substrate recognition. The thermophile Archaeoglobus fulgidus encodes two Sir2 homologs, Sir2-Af1 and Sir2-Af2, that consist of the minimal conserved enzymatic core and share 46% amino acid sequence identity. Although the in vivo substrates of these two enzymes are unknown, a recent study of another archaeon, Sulfolobus solfataricus, showed that an archaeal nonhistone chromatin protein, Alba, is a Sir2 substrate (Bell et al., 2002). In vitro, Sir2Af2 has been shown to deacetylate peptides corresponding to the C terminus of p53 (Sauve et al., 2001). Here we present evidence that Sir2-Af1 can also deacetylate a p53 peptide, although Sir2-Af2 exhibits distinctly higher rates of enzymatic activity on this substrate.

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In order to shed light on the molecular basis of substrate binding and specificity, we have determined the 2.0 A˚ resolution structure of the A. fulgidus enzyme, Sir2-Af2, bound to an acetylated 18 amino acid p53 peptide that is a substrate for Sir2-Af2 in vitro (Sauve et al., 2001). The structure shows that the acetylated peptide binds in the cleft between the two domains of Sir2-Af2, forming a “␤ staple” interaction that links the Rossmann fold and zinc binding module through a staggered enzyme-substrate ␤ sheet, and inserting the acetyl-lysine side chain into a conserved, largely hydrophobic tunnel. Many of the key contacts with the substrate are with peptide backbone atoms and are mediated by residues that are either invariant or highly conserved among Sir2 homologs. This is consistent with our findings that many Sir2 enzymes can deacetylate the p53 peptide that was cocrystallized with Sir2-Af2. However, there are variable Sir2 residues that also interact with the substrate peptide, and we show that mutating three of these residues in Sir2-Af1 can increase its affinity for the p53 peptide. A comparison of the Sir2-Af2-p53 peptide structure with the previously determined structures of Sir2 homologs reveals a significant difference in protein conformation that appears to be triggered by substrate binding and that may play a role in the mechanism of the deacetylation reaction. Results Overall Structure of Sir2-Af2 Bound to an Acetylated p53 Peptide The 2.0 A˚ resolution crystal structure contains the fulllength Sir2-Af2 protein in complex with an 18 residue p53 peptide acetylated at Lys382 (Figure 1A). Sir2-Af2 adopts the canonical Sir2 catalytic core fold seen in the two previously determined structures of Sir2 family members (Finnin et al., 2001; Min et al., 2001). The fold consists of two characteristic domains (Figures 1B and 2): a larger, inverted Rossmann fold domain (residues 1–27, 77–118, and 170–253) that binds NAD⫹ and a smaller domain composed of residues from two insertions in the Rossmann fold that form a helical module (residues 28–76) and a zinc binding module (residues 119–161), respectively (Bellamacina, 1996; Finnin et al., 2001; Min et al., 2001). The p53 peptide lies in the large groove between the Rossmann fold and the small domain. Eight of the eighteen peptide residues (380–387) are well ordered in the electron density map (Figure 1C). The peptide binds to Sir2-Af2 by forming ␤ sheet-like interactions with two flanking strands in the enzyme: one located in the Rossmann fold and the second in a loop between the Rossmann fold and the zinc binding module (residues 162–169), which we call the “FGE loop” for the highly conserved FGExL motif that it contains (Figures 1B and 2). The peptide is oriented with its N terminus closer to the zinc binding module and its C terminus closer to the flexible loop region (Figure 1B). The acetyl-lysine side chain of KAc382 inserts into a tunnel, located in the cleft between the Rossmann fold and the zinc binding module, that leads to the NAD⫹ binding site. The bound peptide residues are well ordered, as reflected in an overall temperature factor of 25.4 A˚2 for peptide residues as compared with an average B factor

of 21.2 A˚2 for the entire structure. The acetyl-lysine residue, KAc382, is particularly well ordered (side chain B factor of 15.8 A˚2), as are the immediately flanking peptide residues 381–385 (average B factor of 19.1 A˚2). Binding of the Acetylated Peptide by Formation of an Enzyme-Substrate ␤ Sheet Sir2-Af2 binds the acetylated p53 peptide primarily through ␤ sheet interactions with the main chain atoms of the peptide, forming an enzyme-substrate ␤ sheet that we refer to as a ␤ staple for the way in which the substrate links together two distinct domains of Sir2Af2 (Figures 3A and 3B). The eight peptide residues visible in the electron density map correspond to the p53 sequence, 380-HKKAcLMFKT-387 (Figure 1C). Residues 380–385 of the peptide comprise the middle strand of a staggered antiparallel three-stranded ␤ sheet. The bottom strand is contributed by strand ␤9 (residues 195– 197) of the Sir2-Af2 Rossmann fold domain, and the top strand is contributed by ␤7 (residues 166–169), which is part of the FGE loop (Figures 1B and 3A). The FGE loop itself contains a Type II ␤ turn formed by hydrogen bonding between the carbonyl oxygen of L164 and the amide nitrogen of E167 (Figure 3C). Peptide main chain atoms from residues H380 and KAc382 form hydrogen bonds with main chain atoms of residues G166, E167, and L169 in the FGE loop, while peptide backbone atoms of residues L383 and F385 form hydrogen bonds with residues V195 and Y197 in ␤9 (Figures 3A and 3B). The residues that make H bonds with the main chain atoms of the acetyl-lysine, G166 and E167, are both in the ␤7 segment of the conserved FGE loop and are engaged in atypical antiparallel ␤ sheet H bonds with the acetyllysine. The remaining contacts between Sir2-Af2 and peptide residues flanking the acetyl-lysine consist of van der Waals interactions and two water-mediated hydrogen bonds (data not shown). The acetyl-lysine side chain, KAc382, inserts into a hydrophobic tunnel located at the interface between the Rossmann fold and the FGE loop (Figures 1B and 3D) in an orientation similar to that proposed by Min et al. (2001). The acetyl-lysine side chain is in its extended conformation and lies atop H118, an invariant histidine residue required for enzymatic activity (Min et al., 2001; Tanny et al., 1999). The side chains of residues H118, V163, F165, L169, and V196 form van der Waals interactions along the length of the aliphatic portion of the side chain (Figures 3A and 3D). A single hydrogen bond between the N⑀ of the acetyl-lysine and the carbonyl oxygen of V163 in the FGE loop helps position the acetyllysine side chain within the tunnel. The acetyl methyl group is in van der Waals contact with I102 and the C␤ of H118. The terminal carbonyl of the acetyl group is unliganded and points directly into the NAD⫹ binding pocket (Figure 3A and 3D). Most of the key Sir2-Af2 peptide binding residues are conserved in other Sir2 proteins (Figure 2). As seen in Figure 3E, the residues surrounding the opening of the acetyl-lysine binding tunnel are highly conserved, while those within the tunnel are either invariant or chemically conserved. Some of the residues in the Rossmann fold domain that interact with the peptide are also highly conserved, although the identities of residues 195 and

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Figure 1. Overall Structure of the Sir2-Af2-p53-KAc382 Complex (A) Schematic representation of the domains of p53 with the amino acid sequence of the regulatory C-terminal tail and its derived peptides: p53-KAc382, which was cocrystalized with Sir2-Af2, and p53-KAc373. The asterisk marks sites of acetylation in the C-terminal tail. (B) Cartoon representation of the overall structure of the Sir2-Af2-p53-KAc382 complex. The reverse Rossmann fold is shown in green and orange. The helical module is shown in yellow, with the disordered region represented as small circles. The zinc binding module is shown in dark blue, the FGE loop in cyan, and the p53-KAc382 peptide in red. (C) A 2Fo-Fc electron density map of the p53-KAc382 peptide calculated at 2.0 A˚ resolution and contoured at 1␴, superimposed on the structure of the peptide. A ribbons representation of Sir2-Af2 is shown in green. The visible residues of the peptide span H380 to T387 and are labeled in black.

197 that form all three H bonds in the ␤ sheet interaction between ␤9 and the peptide are highly variable in sirtuins (Figures 2, 3A, and 3B). In contrast, the variable residue in ␤7 (P168), which corresponds to residue x of the FGExL motif, interacts with the p53 peptide only through van der Waals interactions (Figures 3B and 3E). Conformational Differences between Sir2 Structures A conformational difference relating the larger Rossmann fold domain and the smaller zinc binding/helical domain was previously observed between the Sir2-Af1/ NAD⫹ binary complex and the SIRT2 apoprotein (Finnin et al., 2001). Here we observe a third significantly different conformation in the structure of Sir2-Af2 with bound

substrate in the absence of NAD⫹ (Figure 4A). Compared to the structure of NAD⫹-bound Sir2-Af1, the zinc binding/helical module in Sir2-Af2 is rotated clockwise in the view shown in Figure 4A, shifting backbone atoms by up to 10.8 A˚. By contrast, the zinc binding/helical module domain of apo-SIRT2 is shifted in roughly the opposite direction relative to the structure of Sir2-Af1 and exhibits a protein backbone displacement from Sir2-Af2 of up to 14.3 A˚. The helical module also differs significantly from the conformations observed in previous structures. This module contains a loop region followed by three ␣ helices, ␣3, ␣4, and ␣5 (Figure 1B). In the structure of Sir2Af2 bound to peptide, ten residues of the loop region

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Figure 2. Sequence Alignment of Ten Eukaryotic and Prokaryotic Sirtuins Red indicates positions in which the amino acid is identical in nine or more sequences; yellow indicates residues that are identical in seven or eight sequences, or that are chemically similar in seven or more sequences. The secondary structure is shown aligned above the sequences and is color coded as in the tertiary structure in Figure 1B: Rossmann fold in green, helical module in yellow, zinc binding module in dark blue, and FGE loop in cyan. The disordered region in the helical module is shown in yellow and gray. The flag code is in the lower right-hand corner of the alignment.

(aa 30–39) are disordered (Figure 4B), some of which correspond to side chains that in Sir2-Af1 contact NAD⫹ (Figure 4C). These include Sir2-Af1 residues E26, F32, and R33, which correspond to Sir2-Af2 NAD⫹ binding residues, E29, F35, and R36 (Figures 4B and 4C). In addition, Sir2-Af2 residues A28 and W42 adopt different conformations from their NAD⫹ binding Sir2-Af1 counterparts, A25 and W39 (Figures 4B and 4C). In the human apo-SIRT2 structure, residues F96, R97, and Y104 in the helical module (corresponding to residues F35, R36, and

W42 in Sir2-Af2) are also shifted relative to the NAD⫹ binding pocket, although all residues of the helical module are ordered in that structure (Figure 4D). The different flexible loop conformations observed are all the more striking in light of the fact that this region has the most highly conserved sequence in the helical module (Figure 2). Taken together, these observations suggest a reordering of the helical module’s flexible loop upon NAD⫹ binding. Consistent with this idea is the fact that the closed and open forms of the Sir2-Af1/NAD⫹ binary

Structure of a Sir2-p53 Peptide Complex 527

Figure 3. The p53 Peptide Binding to Sir2-Af2 (A) Schematic drawing of p53-KAc382 binding by Sir2-Af2. The substrate peptide is shown in black over a pink background, and the acetyllysine is shown in red. ␤7 and ␤9 of Sir2-Af2 are shown in cyan and green, respectively. H bonds are shown as dashed lines, and the residues that form the hydrophobic tunnel are represented with yellow semicircles. Single boxes indicate peptide residues in p53-KAc382, and double boxes indicate residues in Sir2-Af2. The general region of NAD⫹ binding is indicated with a turquoise shadow. (B) Stereo view of the enzyme-substrate ␤ sheet that constitutes the ␤ staple. (C) Stereo view of the FGE loop (cyan), which has a Type II ␤ turn and makes four H bonds with the substrate peptide (white), three of which are with the acetyl-lysine (*). The FGE loop also interacts with the helical module (yellow), the zinc binding module (blue), and the Rossmann fold (green). H bonds are shown in gray and the salt bridge in magenta. (D) The transparent van der Waals surface of the hydrophobic acetyl-lysine binding tunnel. The asterisk labels the carbonyl oxygen of V163 that H bonds with the N⑀ of the acetyl-lysine. The peptide binding surface is located toward the foreground of the figure, while the NAD⫹ binding pocket is located in the distal end of the tunnel. (E) The peptide binding surface of Sir2-Af2 colored according to sequence conservation (same orientation as Figure 1B). Conserved enzyme residues are colored red, similar residues yellow, and variable residues green (as defined in Figure 2). P168, Y197, and M222 are the residues whose counterparts in Sir2-Af1 were mutated as described in the text. The full p53-KAc382 peptide is shown with all side chains labeled.

complex contain differences in the flexible loop region coupled to a change in the position of the ribose ring of the nicotinamide moiety of NAD⫹ (Min et al., 2001). The FGE Loop: A Central and Dynamic Structural Component The FGE loop, which plays a central role in binding the acetyl-lysine and the peptide, adopts a markedly

different conformation in the structure of apo-SIRT2 (inset of Figure 4A). As compared with its position in the Sir2-Af2 peptide complex, the FGE loop of apo-SIRT2 is shifted by as much as 8.7 A˚, yielding a more open substrate binding cleft in which FGE loop residues are farther from the invariant H187 in SIRT2 and other Rossmann fold residues (Figures 4A and 4D). As a result, the acetyl-lysine binding tunnel is disrupted in apo-SIRT2

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Figure 4. Structural Comparison of Sir2-Af2 with Other Sirtuin Structures (A) Superposition of Sir2-Af2 (green) with the Sir2-Af1 open (yellow) and closed (red) conformations and the structure of the human apo-SIRT2 (blue). The p53 peptide bound to Sir2-Af2 is shown in silver. The superposition minimizes the distances between the Rossmann folds of the four structures (lower half of the figure). The inset shows a close-up view of the significant shift in the FGE loop of the apo-SIRT2 protein with respect to its position in the Sir2-Af1-NAD⫹ and Sir2-Af2 peptide structures. (B) Structure of Sir2-Af2, with the acetyl-lysine molecule shown in red and the disordered region of the helical module shown with small circles. Residues that form the hydrophobic tunnel (blue) bind the acetyl-lysine and orient it toward the NAD⫹ binding pocket. In the absence of cofactor, the NAD⫹ binding residues from the flexible loop (yellow) are shifted or disordered. The carbonyl oxygen of V163 is labeled with an asterisk, and the H bond it makes with the N⑀ of the acetyl-lysine is shown in gray. (C) Structure of Sir2-Af1-NAD⫹ complex in the closed conformation, with the NAD⫹ molecule shown in green. NAD⫹ binding residues from the flexible loop (yellow) pack against the NAD⫹ molecule, while the residues that form the hydrophobic tunnel (blue) are in the proper orientation to bind acetyl-lysine. The asterisk labels the carbonyl oxygen of V157 that H bonds the N⑀ of acetyl-lysine. (D) Structure of apo-SIRT2. NAD⫹ binding residues from the flexible loop (yellow) are ordered but shifted from their relative position in Sir2Af1. In the absence of peptide, the hydrophobic tunnel (blue) is distorted. The asterisk labels the carbonyl oxygen of V233 that H bonds the N⑀ of acetyl-lysine.

(Figure 4D). Since the residues that comprise the tunnel and interact with acetyl-lysine are identical in the two proteins, it is likely that SIRT2 would need to adopt a conformation similar to that in the Sir2-Af2 peptide complex in order to bind an acetylated peptide. The position of the FGE loop in the Sir2-Af2 peptide complex is closely coupled to the first helix in the helical module, ␣3. The backbone atoms of F165 in the FGE loop make H bonds with backbone atoms of A51 and I53 of the helical module, in addition to van der Waals contacts between the FGE loop and residues at the N terminus of ␣3 (Figure 3C). The FGE loop is also bonded

to the zinc binding module by a conserved salt bridge between the E167 of the FGE loop and K159 of the zinc binding module (Figure 3C). These interactions are largely conserved in the structures of Sir2-Af1 and SIRT2. Mutagenesis experiments done on the yeast homolog, HST2, show that the residue corresponding to E167, which makes the FGE loop salt bridge interaction with the zinc binding module, is essential for enzymatic activity (Min et al., 2001). Finally, the FGE loop also makes an H bond with the backbone of the invariant H118, located in the Rossmann fold domain (Figure 3C). In the Sir2-Af2 peptide complex, the conformation of

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the FGE loop is closer to that observed in the Sir2-Af1NAD⫹ complex (C␣ rms of 1.4 A˚) than to that observed in the apo-SIRT2 structure (C␣ rms of 6.4 A˚) (Figure 4A). The difference in the FGE loop position between the Sir2-Af1-NAD⫹ complex and the SIRT2 apoenzyme is most closely coupled to a shift of residues in and N-terminal to ␣3 of the helical module of Sir2-Af1. These residues from ␣3 that interact with the FGE loop are connected to the flexible loop region that contacts NAD⫹. The link between the FGE loop and the helical module raises the possibility that NAD⫹ binding favors a closing of the cofactor binding cleft, which in turn causes the FGE loop to shift to its peptide binding conformation, as seen in the Sir2-Af2 peptide complex (Figures 4B and 4C). This suggests that NAD⫹ and substrate may bind sirtuins in a cooperative fashion.

viously observed that Sir2-Af2 and Sir2-Af1 differ in the efficiency with which they deacetylate selected substrates (I.C. and J.D.B., unpublished data). To better quantitate the differences between the two enzymes, we determined the apparent kinetic parameters for deacetylation of the monoacetylated p53 peptide used in the crystallographic study, using glucose-6-phosphate dehydrogenase to measure NAD⫹ consumption during the reaction (see Experimental Procedures). The results (Figure 5C) show that the apparent Kcat of Sir2Af2 is nearly double that of Sir2-Af1, while the apparent KM of Sir2-Af2 is about five times lower than that of Sir2Af1. Sir2-Af2 therefore has an apparent Kcat/KM seven and a half times greater than Sir2-Af1, consistent with earlier observations that this peptide is a better substrate for Sir2-Af2 than for Sir2-Af1.

Sir2 Enzymes Can Deacetylate a Variety of Peptide Substrates The structure of the Sir2-Af2 peptide complex reveals how this Sir2 family member specifically recognizes an acetylated p53 peptide. We had previously shown that this peptide is an in vitro substrate of Sir2-Af2 (Sauve et al., 2001), for which the physiological target is unknown. Acetylated histone N-terminal tails have been proposed to be a major substrate for Sir2 in yeast and other organisms. However, the histones of archaea such as A. fulgidus, from which the Sir2-Af1 and Sir2-Af2 enzymes are derived, lack eukaryote-like N-terminal tails. The recent demonstration that the p53 tumor suppressor protein is deacetylated by Sir2 enzymes from various species (Luo et al., 2001; Vaziri et al., 2001; Sauve et al., 2001) and that the archaeal S. solfataricus Sir2 deacetylates a major chromatin protein, Alba, in the host species (Bell et al., 2002) demonstrates that Sir2 proteins are likely to have a broad array of cellular targets. An important question that remains is how substrate specificity is achieved in Sir2 enzymes. To examine the issue of target specificity, we compared the ability of Sir2-Af1, Sir2-Af2, human SIRT2, and yeast Sir2p to deacetylate a variety of proteins. The substrates assayed were acetylated histones and two 18 residue monoacetylated peptides derived from the regulatory C-terminal tail of p53. One of the peptides, p53-KAc382, is acetylated at K382 and is identical to the one used in the structural study; the other peptide, p53-KAc373, is acetylated at K373 (Figure 1A). The deacetylation reaction was monitored by following the separation of acetylated and deacetylated peptides by HPLC (see Experimental Procedures). The results (Figure 5A) show that all four enzymes were active on all substrates tested. As previously shown (Imai et al., 2000; Landry et al., 2000; Smith et al., 2000), all deacetylation activity was dependent on the presence of NAD⫹. These results indicate that, at least for the proteins tested, Sir2 enzymes can deacetylate a broad range of peptide substrates. Sir2-Af2, yeast Sir2p, and human SIRT2 are also capable of deacetylating full-length p53 in vitro (Figure 5B), indicating that these enzymes can bind and deacetylate the target acetyl-lysine within the context of the native p53 protein. Biological specificity of individual Sir2 enzymes can be achieved if the relative deacetylation rate for a given substrate varies among different Sir2 enzymes. We pre-

Altering the Affinity of Sir2-Af1 for p53-KAc382 Peptide The enzyme-substrate interactions seen in the structure of the Sir2-Af2-p53 peptide complex suggest which residues in the substrate binding cleft might determine the relative affinity of the enzyme for particular peptides. Most of the substrate binding residues are identical between Sir2-Af1 and Sir2-Af2, with three exceptions: Sir2Af2 residues P168 in ␤7 of the FGE loop, Y197 in ␤9 of the Rossmann fold, and M222 that is outside the enzymesubstrate ␤ sheet, which correspond to residues M162, Q191, and P216 in Sir2-Af1, respectively (Figures 2 and 3E). In order to test whether these residues influence substrate specificity, we mutated the corresponding residues in Sir2-Af1 to their Sir2-Af2 counterparts and assayed the Sir2-Af1 (M162P, Q191Y, P216M)-triple mutant to calculate its apparent kinetic parameters. The reaction rates for Sir2-Af2, Sir2-Af1-wild-type (Sir2-Af1 WT), and Sir2-Af1-triple mutant (Sir2-Af1 TM) are compared in Figure 5D. The results show that the apparent KM for the Sir2-Af1 triple mutant is reduced 3-fold from that of the Sir2-Af1 wild-type enzyme, while the apparent Kcat/KM is 4-fold higher than that of the wild-type enzyme and only half that of Sir2-Af2 (Figure 5C). Most of this gain of activity can be attributed to the mutations at positions 162 and 191, based on analysis of the single mutants (data not shown), both of which fall on the enzyme-substrate ␤ sheet (Figures 2 and 3E). These substitutions therefore increase the relative affinity of the Sir2-Af1 enzyme for the p53 peptide, primarily by affecting the KM. Discussion Recognition of the Acetylated Peptide by Sir2 Enzymes The binding of an acetylated p53 peptide to Sir2-Af2 is dominated by two main enzyme-substrate interactions: the burying of the acetyl-lysine of the peptide in a highly conserved hydrophobic tunnel and the formation of an enzyme-substrate ␤ sheet. The burial of the acetyl-lysine side chain within the enzyme active site positions the acetyl group for reaction with the ribose ring of the nicotinamide moiety of NAD⫹ and puts it in contact with the catalytic histidine, H118. The acetyl-lysine is fixed at each end of the hydrophobic tunnel by a total of three H bonds. Deep within the tunnel, the N⑀ of the acetyllysine and the carbonyl oxygen of V163 make the first

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Figure 5. Deacetylation Activity of Various Sirtuins (A) Activity of four Sir2 enzymes on histone and p53 substrates. (B) In vitro sirtuin activity on full-length p53 expressed in yeast, immunoprecipitated with anti-p53 DO-1, and acetylated in vitro with GSTCBP. The upper panel is immunoblotted with anti-acetyl p53 antibody specific to lysines 373 and 382. The lower panel is immunoblotted with antibody FL-393 reactive against acetylated and deacetylated forms of p53. (C) Apparent kinetic parameters of archaeal sirtuins Sir2-Af2, Sir2-Af1 wild-type (WT), and Sir2-Af1 triple mutant (TM) in the enzymatic deacetylation of acetylated p53KAc382 peptide. (D) Rates of reaction, as measured by NAD⫹ hydrolysis, of Sir2-Af2 (black), Sir2-Af1 wild-type (green), and Sir2-Af1 triple mutant (red), as a function of p53-KAc382 peptide concentration.

H bond (Figures 3A and 3D). The other two H bonds occur in ␤ sheet-like interactions between the FGE loop and the main chain atoms of the acetyl-lysine residue that determine the N- to C-terminal orientation of the peptide in the binding surface (Figures 3A and 3B). The hydrophobic character of the tunnel and the residues that line its entrance are likely to favor binding of acetyllysine over that of an unacetylated, charged lysine side chain. A large proportion of the peptide-contacting residues lie in the conserved FGE loop, making it highly likely that the interactions with the acetylated peptide observed here will be conserved in all Sir2 enzymesubstrate complexes. The dominant role of peptide backbone hydrogen bonds in substrate binding and the limited extent of substrate side chain burial results in only weak selectiv-

ity by Sir2-Af2 for particular peptide side chains flanking the acetyl-lysine. This is consistent with our observation that a variety of Sir2 enzymes are capable of deacetylating a broad array of substrates (Figure 5A). Nevertheless, the relative rates at which the two A. fulgidus sirtuins deacetylate a substrate peptide do differ to some extent, as shown by the larger apparent KM and lower apparent Kcat exhibited by Sir2-Af1 for the same acetylated p53 peptide used in the present structural study (Figure 5C). As we demonstrate by mutagenesis studies, several side chains in the peptide binding site that differ between Sir2-Af1 and Sir2-Af2 help determine the relative affinity of the Sir2 enzyme for a particular substrate. Since both of these A. fulgidus Sir2 enzymes consist solely of the minimal conserved Sir2 catalytic core, the differences we observe likely influence relative preferences of these

Structure of a Sir2-p53 Peptide Complex 531

enzymes for particular cellular substrates. However, the kinetic differences between Sir2-Af2 and wild-type Sir2Af1 are still relatively modest, which raises the possibility that most sirtuins depend upon domains outside the conserved catalytic core for precise targeting in the cell, either directly or through their involvement in the formation of multiprotein complexes that mediate substrate recognition. Yeast Sir2p, for example, contains domains flanking the conserved catalytic core involved in Sir2 binding to Sir4, Net1, and other proteins (Cockell et al., 2000; Cuperus et al., 2000). Regions of the Sir2 core domain outside the peptide binding surface might also interact with domains in the target protein that are separate from the portion of the substrate that inserts into the peptide binding cleft and forms part of the enzymesubstrate ␤ sheet. The helical module, in particular, exhibits the highest degree of variation in the catalytic core of sirtuins, and its position adjacent to the peptide binding groove would make possible additional interactions with a substrate protein. The nature of the interaction between Sir2-Af2 and an acetylated p53 peptide indicates that the substrates of Sir2 proteins are likely to be either unstructured peptides or segments of proteins that can unfold upon binding to Sir2. The C terminus of p53, corresponding to the peptide used in the present study, is deacetylated by human SIRT1 in vivo (Vaziri et al., 2001) and has been shown by NMR studies to be unstructured in solution (Rustandi et al., 2000). Acetylated histone N-terminal tails, which are substrates for yeast Sir2p, are similarly unstructured in the context of the nucleosome (Luger et al., 1997; Luger and Richmond, 1998). Human SIRT2, whose structure was determined in the absence of either substrate or NAD⫹, has a significantly wider cleft separating the FGE loop and the Rossmann fold that could in principle accommodate a larger substrate made up of two ␤ strands. However, we have shown that SIRT2 is able to deacetylate a p53 peptide of eighteen residues (Figure 5A), making it more likely that binding of either substrate or NAD⫹ to SIRT2 triggers a conformational change that narrows the substrate binding cleft (discussed in further detail below). The insertion of the substrate peptide between ␤ strands belonging to two different domains forms an interaction we refer to as a ␤ staple for the way in which it joins the two domains of the Sir2 protein through the formation of a staggered enzyme-substrate ␤ sheet. There are several examples of peptides that bind to other proteins by adding a strand to the edge of an existing ␤ sheet. This has been found for PTB and PDZ domains (Doyle et al., 1996; Zhou et al., 1995), which bind phosphorylated and nonphosphorylated peptides, respectively, and for the interaction of the flexible N-terminal arm of Mat␣2 with Mcm1 (Tan and Richmond, 1998). Most recently, the crystal structure of the chromodomain of HP1 bound to a K9 methylated peptide of the N-terminal tail of histone H3 revealed that the peptide binds by completing a ␤ sandwich construction with the chromodomain (Jacobs and Khorasanizadeh, 2002). Conformational Differences among Sir2 Enzymes There are significant differences in the conformation of the Sir2-Af2 enzyme bound to an acetylated p53 peptide

compared to the two structures reported of Sir2-Af1 bound to NAD⫹ (the “open” and “closed” forms; Min et al., 2001) and to the structure of the core domain of human apo-SIRT2 (Finnin et al., 2001; Figure 4A). All Sir2 proteins studied differ markedly in the position of the smaller zinc binding/helical module domain relative to the larger, Rossmann fold domain, as well as in the conformation of the flexible loop region. Each of the three Sir2 family members has been studied in a differently liganded state, raising the possibility that the observed differences are induced by ligand binding. Since there is natural sequence variation among the enzymes, particularly in the helical subdomain, one cannot rule out the possibility that these enzymes adopt inherently different conformations irrespective of substrate or cofactor binding. Nevertheless, there are striking differences that appear to be correlated with binding of ligands that are likely to be of significant mechanistic importance. The acetyl-lysine binding tunnel, which includes residues from the FGE loop and side chains in the Rossmann fold domain, is most strikingly different in the structure of apo-SIRT2 (Figures 4A and 4D). The difference is due, in great part, to a shift of up to 8.7 A˚ of the FGE loop relative to its position in the Sir2-Af2 peptide structure. This shift includes an ⵑ2 A˚ increase in the distance between the catalytically important H187 and the backbone carbonyl of V233 in SIRT2 (corresponding to H118 and V163, respectively, of Sir2-Af2), both of which must contact the acetyl-lysine side chain in the enzyme-substrate complex. In addition, the more open conformation of the apo-SIRT2 structure places the acetyl-lysine binding tunnel farther from the NAD⫹ binding site, making it unlikely that the acetyl group could bind close enough to the NAD⫹ to participate in the chemical reaction without a rearrangement of the hydrophobic tunnel (Figures 4B and 4D). Given that virtually all the key substrate binding residues are conserved in SIRT2, it is highly likely that this enzyme undergoes a conformational change upon substrate binding, shifting the FGE loop to the position observed in the Sir2-Af2 peptide complex where it can bind to acetyl-lysine. The observation that SIRT2 can deacetylate p53 KAc373 (Figure 5A) supports the argument that a conformational change takes place in SIRT2 that allows it to bind a single peptide-strand in an enzyme-substrate ␤ sheet. The FGE loop plays a pivotal role in linking the binding of either substrate or cofactor with the position of the zinc binding/helical module. This highly conserved loop interacts with the acetyl-lysine, forms the upper strand of the enzyme-substrate ␤ sheet, packs directly against the ␣3 helix of the helical module, forms a conserved salt bridge (E167–K159) with the zinc binding module, and hydrogen bonds with the backbone of the catalytic histidine (Figure 3C). Therefore, any conformational change induced by binding of either substrate or NAD⫹ could induce more global shifts of the zinc binding/ helical modules relative to the Rossmann fold domain. The relative position of the FGE loop in the structure of Sir2-Af1 bound to NAD⫹ is closer to the position observed in the Sir2-Af2 peptide complex than to that in apo-SIRT2 (Figure 4A). Since the position of the FGE loop is linked to that of the helical module, which participates in NAD⫹ binding (Min et al., 2001), it is possible

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Table 1. Crystallographic Statistics Diffraction Data Space group Unit cell Resolution (A˚) Measured reflections Unique reflections Completeness (%) Average I/␴ Multiplicity Mosaicity Rmeasa (%)

P212121 34.9, 35.6, 184.7 2.0 79,201 (4350) 15,490 (1414) 97.6 (92.4) 11.8 (4.2) 5.1 (3.1) 0.8 11.3 (34.2)

Refinement Statistics

Figure 6. Model Showing the Relative Orientation of Acetyl-Lysine and NAD⫹ in the Active Site The position of NAD⫹ in the ternary complex was modeled by superimposing the structure of Sir2-Af1 bound to NAD⫹ (Min et al., 2001) with the structure of Sirf-Af2 bound to an acetylated peptide. The close-up view shows the active site region of Sir2-Af2 (green) with acetyl-lysine KAc382 (colored by atom: C, white; N, blue; O, red; van der Waals radii indicated by stippled spheres) in the binding tunnel. The relative position of the NAD⫹ molecules in the enzyme active site is shown for the open (yellow) and closed (red) structures of Sir2-Af1-NAD⫹.

that a shift in the FGE loop position and consequent closing of the peptide binding cleft is also induced by NAD⫹ binding (Figure 4C). The coupling of a conformation change with either substrate or cofactor binding would provide favorable cooperative energy to the formation of the ternary enzyme-acetylated substrateNAD⫹ cofactor complex. Implications for Enzyme Mechanism Comparison of the Sir2-Af2-p53 peptide complex with the two crystal forms of the Sir2-Af1-NAD⫹ complex suggests that the NAD⫹ is likely to shift its position in the ternary enzyme-substrate-cofactor complex. In the structure of the Sir2-Af2 peptide complex, the acetyl group on the N⑀ of lysine is inserted into the nicotinamide ribose ring binding region (Figure 6). In the structural superpositions of the Sir2-Af2 substrate complex with the NAD⫹-containing structures of Sir2-Af1, the acetyl group of the acetyl-lysine actually occupies the same space as either the 2⬘OH or the 3⬘OH of the nicotinamide ribose ring in the open or closed Sir2-Af1 conformations, respectively (Figure 6). This implies that something in the active site must move in order to accommodate the ternary enzyme-substrate-cofactor complex. Several reasons make it much more likely that it is the nicotinamide-ribose moiety, rather than the acetyl-lysine, that moves upon ternary complex formation. First, the two conformations of Sir2-Af1 show the nicotinamide ribose ring in different conformations, suggesting that this part of the NAD⫹ is quite loosely bound. Second, the electron density of the nicotinamide ring is absent from both structures, indicating further flexibility. Finally, as all cur-

Resolution range (A˚) Reflections (I/␴ ⬎ 0) Working set Test set (5.17%) Total atoms Protein/peptide atoms Metal atoms (Zn) MES buffer atoms Water atoms Rfactorb (%) Rfreec (%) B factor (A˚2) Rms deviations Bond lengths (A˚) Bond angles (⬚)

2.0 15,497 14,696 801 2135 1982 1 12 140 20.9 25.4 21.2 0.006 1.17

Values in brackets correspond to the highest resolution shell. a Rmeas as described in Diederichs and Karplus (1997). b Rfactor ⫽ ⌺||Fo| ⫺ |Fc||/⌺|Fo|, where Fo is the amplitude of the observed structure factor and Fc is the structure factor calculated from the model. c Rfree is the R factor calculated with 5% of the reflection data randomly omitted from the refinement.

rent crystal structures of Sir2 proteins reveal, there is much more space within the active site for the nicotinamide-ribose moiety to move than there is for the acetyllysine, which is tightly constrained in a hydrophobic tunnel and positioned by a hydrogen bond between the N⑀ of the lysine and the carbonyl oxygen of V163 (Figure 3D). Combining the structural results described here with our recent studies of the chemical mechanism of Sir2Af2 (Sauve et al., 2001) allows us to propose a likely conformation for the bound NAD⫹ in the enzyme-substrate-cofactor complex. The final products of the deacetylation reaction have been shown to be the deacetylated peptide, nicotinamide, and an ⵑ40/60 mixture of 2⬘-OH- and 3⬘-OH-acetyl-ADP-ribose (AADR); no ␤-1⬘-acetyl-ADP-ribose could be detected (Sauve et al., 2001). Kinetic measurements showed that 2⬘-OH-AADR spontaneously converts to the 3⬘ form and back again, leading ultimately to a 40/60 mixture of the two forms in vitro. The earliest detectable reaction product is 2⬘OH-AADR. However, O18 labeling experiments suggest that 2⬘-OH-AADR is not formed by direct attack of the 2⬘-OH on the acetyl carbon. Rather, the reaction mechanism appears to involve a nucleophilic attack at the 1⬘ position from the ␣ side by the acetyl group of acetyllysine, leading to the formation of a transient O-alkylamidate intermediate (Sauve et al., 2001). This mechanism is consistent with the Sir2-Af2 peptide structure

Structure of a Sir2-p53 Peptide Complex 533

that shows the acetyl-lysine in close proximity to the putative catalytic H118, which could make the acetyl group of the acetyl-lysine a better nucleophile by favoring a developing negative charge on the carbonyl oxygen during deacetylation. The mechanism is also consistent with the structure of the Sir2-Af1-NAD⫹ binary complex, which shows that there is sufficient space for the rearrangement of the NAD⫹ required to accommodate the acetyl-lysine for catalysis (explained above). Comparison of the Sir2-Af1 open and closed structures (Min et al., 2001) shows that the conformations of NAD⫹ in these two forms differ in large part by rotation around the N-phosphate-diester oxygen (␨N) bond (NAD⫹ bond nomenclature is per Bell et al. [1997]). Studies of NAD⫹ bound to various proteins show that many conformations of this angle, as well as the adjacent ␣N, ␤N, and ␥N angles, are compatible with protein-bound NAD⫹ (Bell et al., 1997). Model building shows that the ␣ face of the C1⬘ of the nicotinamide ribose can be readily oriented to allow attack by the acetyl-lysine oxygen (data not shown). We propose that the NAD⫹ binds the enzymepeptide complex in such a conformation to facilitate the deacetylation chemistry. Release of the final reaction products may be promoted by the positive charge on the deacetylated lysine, whose binding in the hydrophobic tunnel would be disfavored, and by dissociation of the nicotinamide. Either of these events could favor an opening of the substrate-cofactor binding cleft, further favoring product release.

Structure Determination and Refinement The structure of the Sir2-Af2-JB12 complex was solved by molecular replacement using the program MOLREP (CCP4, 1994). The search model was designed by substituting the amino acid sequence of Sir2-Af2 onto the backbone of the structure of Sir2-Af1 (Protein Data Bank ID code 1ICI) (Min et al., 2001) and deleting the flexible loop region (S30–V50). This model was then divided in two search models: the first one consisted of residues 51–76 and 119–168, approximating the small domain, and the second section consisted of residues 1–29, 77–118, and 169–251 approximating the Rossmann fold. The molecular replacement solution was verified with EPMR (Kissinger et al., 1999). An initial rigid body refinement was done on the model divided into the same two rigid sections described above. Subsequently, simulated annealing, energy minimization, and individual B-factor refinement was done using CNS (Brunger et al., 1998) with the maximum likelihood target. The electron density maps were converted to FSFOUR format to be visualized in XFIT (McRee, 1999) for model building. Details of the refinement are given in Table 1. The refinement converged after fifteen cycles of model building and simulated annealing/energy minimization refinements to an R/Rfree of 20.9%/25.4%. The final model contains residues 1–29 and 40–251 of Sir2-Af2 and residues 379–387 of the p53 peptide. The structure quality was assessed with PROCHECK (CCP4, 1994), and the figures of models and surfaces were drawn with PYMOL (DeLano, 2002).

Experimental Procedures

Sir2 Deacetylation Reaction Using p53 Protein as Substrate The histone acetyl transferase GST-CBP was purified from pGEX expression vector. Cells containing expression vector were induced with 300 ␮M IPTG at OD600 ⫽ 0.6 and grown at 30⬚C for 6 hr. Cells were harvested and protein purified using glutathione-Sepharose 4B beads according to the manufacturer’s instruction. The GSTCBP was used to acetylate recombinant p53 made in yeast. Yeast strain YPH500 was transfected with a CEN vector containing the p53 gene, pRB16 (Brachmann et al., 1996), and protein extracts were prepared from transformed cells using glass bead disruption. One milliliter of total yeast protein containing 4.6 mg of protein was incubated with 12 ␮g of anti-p53 antibody DO-1 conjugated to agarose (Santa Cruz) for 1 hr at 4⬚C. Beads were washed three times with 1 ml lysis buffer. After washing, the beads were incubated with 10 ␮l of GST-CBP and 37.5 ␮l of [3H]acetyl-CoA (3.7 Ci/mmol, 67 ␮M, Amersham-Pharmacia) in 5⫻ HAT buffer (250 mM Tris-Cl [pH 8.0], 50% glycerol, 0.5 mM EDTA, 5 mM DTT, 250 mM NaCl, and 20 mM sodium-butyrate) for 1 hr at 30⬚C. After the acetylation reaction, beads were pelleted by quick centrifugation and washed three times with lysis buffer. The HAT reaction was divided into four tubes, each containing 10 ␮l, and incubated with 5 ␮g of GST-SIRT2 (37⬚C), GST-Sir2p (30⬚C), and Sir2-Af2 (55⬚C), 200 ␮M NAD⫹, 50 mM TrisCl (pH 8.0) and 50 mM NaCl in a final volume of 30 ␮l. A control reaction was performed without the addition of any of the enzymes at 37⬚C; incubations were done for 3 hr. A volume of 15 ␮l of deacetylation reaction was washed three times with lysis buffer before the addition of SDS sample buffer and boiling. The samples were separated on 10% SDS-PAGE. After electrophoresis, the samples were transferred to a PVDF membrane, and an immunoblot was performed using FL-393 antibody according to the manufacturer’s instructions.

Protein Expression and Purification Sir2-Af2 and Sir2-Af1 wild-type and mutant enzymes were expressed and purified as previously described (Smith et al., 2002) with the following differences in the purification of Sir2-Af1 proteins: after heat denaturation, the solutions of Sir2-Af1 proteins were dialyzed in 20 mM MES (pH 6.0), 25 mM NaCl, 1 mM DTT, and 25 ␮M ZnCl2, and run through a Q sepharose fast flow column, where most of the impurities bound and the Sir2-Af1 eluted with the flowthrough at greater than 95% purity. The p53 peptides were synthesized by the Johns Hopkins Medical Institutions Sequencing and Synthesis Facility and purified by HPLC on a reverse phase C18 column developed with a 0%–100% acetonitrile gradient in 600 min at 1.5 ml/min. Crystallization and Data Collection Purified Sir2-Af2 at 20 mg/ml was dialyzed into 10 mM HEPES (pH 7.4) and 1 mM TCEP. Separately, 3 mM of p53-KAc382 peptide, which is identical in sequence to the previously used JB12 (Sauve et al., 2001), was dissolved in 3 mM HEPES (pH 7.5). Equal volumes of the protein solution and the peptide solution were mixed to make the complex solution, which contained 10 mg/ml Sir2-Af2 and 1.5 mM JB12. Crystals were grown by the hanging drop vapor diffusion method at 20⬚C by mixing 2 ␮l of the complex solution with 1 ␮l of the well solution containing 40 mM MES (pH 5.5), 10% PEG8000, 14% PEG1000, and 5mM NaCl. Prior to data collection, the crystals were soaked in a solution of 40 mM MES (pH5.5), 18% PEG8000, 18% PEG1000, 5mM NaCl, and 3 mM JB12, and then flash frozen in a N2 stream at ⫺180⬚C. The complex crystallized in the orthorhombic space group P212121, with unit cell dimensions, a ⫽ 34.9 A˚, b ⫽ 35.6 A˚, and c ⫽ 184.7 A˚. Diffraction data were collected at the National Synchrotron Light Source of the Brookhaven National Laboratory at beam line X25, using an X-ray wavelength of 1.1 A˚ and a Brandeis charged-coupled device (CCD Brandeis-b4) detector. The data were processed with the HKL package, DENZO, Xdisp, and Scalepack (Otwinowski and Minor, 1997) and are summarized in Table 1. The intensities from SCALEPACK were converted to structure factors using TRUNCATE from CCP4 v 4.0 (CCP4, 1994).

Sir2 Deacetylation Reaction Using p53-Derived Peptides as Substrate For p53-KAc373 and p53-KAc382 peptides (Figure 1A), 100 ␮g of each was incubated overnight with 10 ␮g of GST-Sir2p and 200 ␮M NAD⫹ at 30⬚C. Reaction products were separated on a C18 analytical column with a 0%–100% gradient of acetonitrile at 0.4 ml/min. The absorption was monitored at 210 nm. The same reaction conditions were used for Sir2-Af2 and GST-SIRT2 except that the incubation was performed at 55⬚C for Sir2-Af2 and at 37⬚C for GST-SIRT2.

Kinetics Measurements of Sir2 Protein Reactions Kinetic measurements were performed in a 50 ␮l reaction volume containing 50 mM Tris (pH 8.0), 1 mM NAD⫹ (previously neutralized with NaOH), 50 mM NaCl, 1mM EDTA, 0.5 mM DTT, and varying concentrations of JB12 to a maximum of 2.5 mM. Substrate activation was observed at higher concentrations of JB12 (from 4.2 mM

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to 9.6 mM), probably due to change in pH produced by the peptide. For the kinetic characterization of Sir2-Af2 and Sir2-Af1 wild-type and mutant enzymes, we used 20 ␮g/ml of protein in each reaction. All deacetylation reactions were carried out in a heat block at 50⬚C and covered with Chill-out 14 liquid wax, or in a heated cover thermal cycler to prevent evaporation. Under these conditions, we determined that the reaction behaved in a linear manner during the first 10–15 min. The reactions were quenched by adding 15 ␮l of 1 M HCl and incubating on ice. The extent of the reaction was measured by reacting to completion of the unconsumed NAD⫹ with glucose6-phosphate dehydrogenase (G6PD) to produce NADH, which can then be measured spectrophotometrically at 340 nm. To do this, the 65 ␮l of the quenched reaction was diluted to a final volume of 500 ␮l in G6PD buffer with a final concentration of 200 mM Tris (pH 8.0), 1.38 mM glucose-6-phosphate (G6P), and 0.1 mg/ml G6PD. Each sample was blanked in the spectrophotometer before adding the G6PD and then allowed to react with G6PD until the absorbance at 340 nm reached a constant value (normally within 3 min). JB12 deacetylation was directly demonstrated by separating the acetylated and deacetylated fractions by HPLC on a C18 column in H2O ⫹ 0.1% TFA, and running a 5%–35% acetonitrile gradient in 0.1% TFA for 60 min. This control was performed on Sir2-Af2 and Sir2-Af1 wild-type and triple mutant enzymes and displayed an average apparent ratio of NAD⫹ hydrolyzed to JB12 deacetylated of 0.91 ⫾ 0.19, similar to the previously reported stoichiometry (Tanny and Moazed, 2001). Acknowledgments We thank R.M. Xu for providing the coordinates of the Sir2-Af1 complex in its closed conformation and S. Berger for the CBP expression plasmid. We thank L. Berman and M. Becker for their help at beamline X25 of the National Synchrotron Light Source at Brookhaven National Laboratory, R. Campbell for his advice and help in the structure determination, and P. Cole, M. Amzel, M. Bianchet, M. Faig, A. Mildvan, A. VanDemark, and C. Garvie for helpful discussions. This work was supported by grant GM62385 (J.D.B. and C.W.) from the National Institutes of Health and by National Research Service Award CA84760-02 (M.S.C.) from the National Cancer Institute. Received: May 20, 2002 Revised: July 24, 2002 References Bell, C.E., Yeates, T.O., and Eisenberg, D. (1997). Unusual conformation of nicotinamide adenine dinucleotide (NAD) bound to diphtheria toxin: a comparison with NAD bound to the oxidoreductase enzymes. Protein Sci. 6, 2084–2096. Bell, S.D., Botting, C.H., Wardleworth, B.N., Jackson, S.P., and White, M.F. (2002). The interaction of Alba, a conserved archaeal chromatin protein, with Sir2 and its regulation by acetylation. Science 296, 148–151. Bellamacina, C.R. (1996). The nicotinamide dinucleotide binding motif: a comparison of nucleotide binding proteins. FASEB J. 10, 1257– 1269. Brachmann, C.B., Sherman, J.M., Devine, S.E., Cameron, E.E., Pillus, L., and Boeke, J.D. (1995). The SIR2 gene family, conserved from bacteria to humans, functions in silencing, cell cycle progression, and chromosome stability. Genes Dev. 9, 2888–2902. Brachmann, R.K., Vidal, M., and Boeke, J.D. (1996). Dominant-negative p53 mutations selected in yeast hit cancer hot spots. Proc. Natl. Acad. Sci. USA 93, 4091–4095.

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