Structural Basis for Sirtuin Function: What We Know and What We Don’t

2.1 The Rossmann-fold domain

The large domain has a classical open α/β Rossmann-fold structure. Six parallel β strands form a central β sheet which is sandwiched between several α helices (the exact number of α helices depends upon the specific sirtuin protein) on either side of the β sheet [29] (Figure 2). The Rossmann-fold domain contains many of the hallmarks of a typical NAD-binding site such as a conserved Gly-X-Gly sequence important for phosphate binding, a pocket to accommodate an NAD⁺ molecule, and charged residues responsible for ribose group binding [30]. Interestingly, the Rossmann-fold domain of the sirtuin proteins is most similar to the inverted Rossmann-fold enzymes, where the adenine base of NAD⁺ binds to the C-terminal half and the nicotinamide group of NAD⁺ binds to the N-terminal half of the β sheet [31]. Structures of sirtuin enzymes bound to NAD⁺ (discussed below) confirm that NAD⁺ does bind to this protein family in an inverted conformation.

2.2 The Zn-binding domain

The small, structural Zn²⁺-binding domain results from two insertions in the Rossmann-fold domain and is the most diverse region between sirtuins for which structures have been determined in terms of primary sequence, three-dimensional structure and position relative to the large domain (Figure 2). The two structural modules of the small domain consist of a three-stranded antiparallel β sheet and a variable α helical region dependent on the sirtuin protein [30]. A Zn²⁺ molecule is bound by the sulphydryl groups of two pairs of strictly conserved cysteine residues in the β sheet module in a tetrahedral conformation [29], with the exception of CobB, which is liganded by only two visible cysteine residues and contains only three of the four expected zinc-coordinating cysteine residues according to sequence alignment (Figure 1). The three-stranded four-cysteine metal binding site resembles the Zn-ribbon structure of transcription factors TFIIS [32], TFIIB [33], and the RNA polymerase II subunit RPB9 [34]. The small domain contains the conserved sequence motif Cys-X_2–4-Cys-X_15–40-Cys-X_2–4-Cys, a characteristic Zn-binding motif. Although the location of the zinc ion is too far from the enzyme active site to participate in catalysis, its presence is required for deacetylase activity, as mutation of all four zinc-binding cysteines to alanine or addition of the zinc chelator o-phenanthroline inhibits the in vitro deacetylase activity of sirtuin enzymes [29]. The role of the zinc ion seems to be structural, as it is postulated to be required for holding together the β strands in the small domain. One of the most interesting characteristics of the small domain is a conserved salt bridge (between R/K178 and E186, Hst2 numbering, unless otherwise indicated) [29, 35], which seems to be important for positioning the small domain with respect to the large domain.

The small domains of the archaeal and bacterial Sir2 enzymes show a similar overall topology, while the small domains of the eukaryotic Sir2 enzymes show the greatest variability in secondary structure (Figure 2). One proposal to explain the divergence in primary sequence, secondary and tertiary structures among Sir2 homologues is that divergent small domains may be involved in substrate-specific binding. Since each homologue likely deacetylates different protein targets, the greater variability of the small domain in eukaryotic proteins might reflect the greater number of sirtuin proteins that exist in eukaryotes and therefore the greater number of substrates that need to be discriminated between [36]. The position of the small domain relative to the Rossmann-fold domain is also dependent on the conformation of bound NAD⁺ [37] or the presence of acetyl-lysine in the enzyme active site (discussed below). Human SIRT5, for example, contains an extra loop and helix (helix α9) inserted into the small domain (Figure 2H) that is sequentially conserved in other mitochondrial sirtuins and may prove to be structurally important for mitochondrial localization [35]. Taken together, the diversity in the small domain may have a potential role in regulating protein-protein interactions, which may be important for substrate specificity, enzyme localization, and potentially for modulation of enzyme activity. Indeed, structural differences between sirtuin proteins within the small domain might be exploited as binding sites for sirtuin-selective small molecule inhibitors.

2.3 The cofactor binding loop region

The four loops that connect the small and large domains form the cleft that acts as the enzyme active site. Both the NAD⁺ and acetyl-lysine substrates bind in this cleft (Figure 3). This region has the most sequence homology among the protein family, and mutation of several residues in this region disrupt histone deacetylase activity, underscoring the importance of this region in sirtuin catalysis. Generally the largest of these four loops (the β1-α2 loop in Hst2) is one of the most conformationally dynamic regions of the sirtuin enzymes (Figure 4). Since this loop forms part of the cofactor NAD⁺ binding site and its conformation is largely dependent on the conformation and identity of the bound NAD⁺ molecule or intermediate, we will refer to it as the cofactor binding loop [29]. The cofactor binding loop appears to be disordered or highly flexible (as evidenced by high B-factors) when NAD⁺ is not bound (Figure 4A), and becomes ordered upon NAD⁺ binding into a relatively open conformation with residues of the loop (D43-Y52) about 7 Å away from the NAD⁺ binding site (Figure 4B) [38]. Residues (A33, G34, T37, F44 and D43) of the cofactor binding loop form part of the so-called “C pocket,” the region of the enzyme where the nicotinamide moiety of NAD⁺ binds, and they mediate almost half of the enzyme contacts to NAD⁺ [38, 39], which implies that this cofactor binding loop plays an important role in NAD⁺ binding. The relationship between the presence of bound NAD⁺ and the relative order and positioning of the cofactor binding loop is consistent among all homologues for which structures have been determined [29, 30, 40, 41], and can be concurrent with a rigid body rotation of the small domain relative to the large domain [42]. The cofactor binding loop region maintains the open, ordered conformation when a molecule designed to mimic the dissociative transition state of nicotinamide-dissociated NAD⁺, DADMe-NAD⁺ [43], or the oxocarbenium ion intermediate-like molecules, ADP-Ribose or ADP-HPD, are bound to the enzyme [38, 44], implying that nicotinamide cleavage does not affect the conformation of the preferred microstate of the dynamic cofactor binding loop. When the sirtuin reaction product 2’-O-acetyl-ADP-ribose is bound to the enzyme active site, the cofactor binding loop adopts a more closed conformation bringing residues (D43-Y52) of the loop ~5.5 Å closer to the nicotinamide binding pocket, partially occluding the site (Figure 4C) [38, 41, 45], suggesting that the loop may also play a role in nicotinamide release. The switch between the open and closed conformations of the cofactor binding loop seems to be dependent upon the the presence of the bound alkylamidate intermediate directly after nucleophilic attack of acetyl-lysine on an NAD⁺-derived intermediate and not upon nicotinamide cleavage or complete reaction product formation, as the closed conformation of the loop is also seen when an analog of the O-alkylamidate intermediate, S-alkylamidate, is bound to the enzyme [43]. When the reaction product nicotinamide is bound in the C-pocket either in complex with acetyl-lysine, NAD⁺ or ADP-ribose, the cofactor binding loop adopts slightly different conformations which facilitate slightly different interactions between nicotinamide and the C-pocket residues, although it is not clear whether the binding of different ligands of the ternary complex or the different nicotinamide conformations cause the differences in conformation of the cofactor binding loop [44, 46].

3.1 NAD⁺ Binding Site

The Rossmann-fold domain forms the bottom of the NAD⁺ binding site while the loop regions, particularly the cofactor binding loop, forms the remainder of the pocket. When bound to a sirtuin enzyme, the NAD⁺ molecule adopts an extended conformation, which is commonly found when NAD⁺ is bound to a protein having a Rossmann-fold [29]. Unusually, the NAD⁺ molecule binds in an inverted direction relative to most NAD dehydrogenases [47]: the adenine base binds to the C-terminal half of the β sheet and the nicotinamide group binds to the N-terminal half. The NAD⁺ binding site can be divided into three regions: site A, the adenine-ribose binding site; site B, the nicotinamide-ribose binding site; and site C, the nicotinamide moiety binding site [29] (Figure 5A). The A site is shallow, mostly hydrophobic and exposed. The adenine base is surrounded by hydrophobic amino acids making van der Waals interactions, including several conserved glycines (G34 and G223) (Figure 5B). The amide nitrogen of the adenine base makes a hydrogen bond with a glutamate or serine residue in the A site. The N1 atom of the adenine base makes a hydrogen bond with a main chain amide in some sirtuin homologues. The 2’ and 3’ hydroxyls of the adenine ribose make several hydrogen bonds with asparagine residues within the A site. The oxygens of the phosphate groups make several hydrogen bonds with residues in the A site, although the exact hydrogen bonds depend upon the conformation of the cofactor binding loop [29, 41, 42]. The cofactor binding loop also makes up the ‘ceiling’ of the B site, so its conformation can also affect nicotinamide ribose binding. When the cofactor binding loop is in the closed conformation (Figure 4C), the nicotinamide ribose makes van der Waals interactions with conserved hydrophobic residues (F44, W58, and V228) (Figure 5B). Absolutely conserved residues (H135 and Q115) are located on the floor of the B site and make interactions with the 3’ oxygen and sometimes the 2’ oxygen of the nicotinamide ribose [29, 35, 41, 42] (Figure 5B). The conformation of the nicotinamide ribose is variable depending on the NAD⁺ analog bound (ADP-Ribose, non-productive NAD⁺, productive NAD⁺ or carba-NAD⁺), which other substrates are also bound to the enzyme, or even the particular sirtuin homologue [29, 35, 37, 38, 42, 45].

Different conformations of the nicotinamide ribose suggest different reaction mechanisms for the dissociation of nicotinamide from the rest of the NAD⁺ molecule. For example, in yeast Hst2, when carba-NAD⁺ (a non-hydrolysable NAD⁺ analog) and an acetyl-lysine containing peptide are bound to the enzyme, the nicotinamide ribose ring (in this case a cyclopentane) is positioned in such a way that both the 2’ and 3’-hydroxyls make hydrogen bonds with the acetyl-lysine carbonyl oxygen, which is on the β face of the ribose group, thereby sterically occluding the acetyl-lysine carbonyl oxygen from nucleophilic attack of C1’ of the nicotinamide ribose group, suggesting that the glycosidic bond between nicotinamide and the rest of the NAD⁺ molecule is broken in a dissociative fashion (Figure 5B) [38]. When an oxocarbenium intermediate-like molecule, either ADP-Ribose or ADP-HPD, is bound the nicotinamide ribose ring is rotated by about 45° [38, 44] relative to the substrate analog complex. Finally, when the reaction product, 2’-O-acetyl-ADP-ribose is bound, the nicotinamide ribose ring is rotated about 90° relative to the substrate bound complex and in the opposite direction of the intermediate-like molecule [42]. This variability of the nicotinamide-ribose ring along the reaction coordinate highlights the importance of the conformation of this ring during catalysis. However, in the complex of Sir2Tm bound to an acetyl-lysine containing peptide and NAD⁺ in a productive conformation, the nicotinamide ribose ring is rotated 30° around the glycosidic bond relative to its position in the Hst2 structure, orienting the acetyl-lysine carbonyl toward the α face of the ribose ring, placing it within 3.2 Å from the C1’ of the ribose and positioned for nucleophilic attack (Figure 5C) [45], thus supporting a direct nucleophilic role for acetyl-lysine in nicotinamide cleavage. These differences may be explained by the use of different catalytic mechanisms for archaeal and eukaryotic sirtuins, although it seems unlikely that enzymes with conserved catalytic residues would evolve different catalytic mechanisms. The difference in conformation of the nicotinamide ribose ring may be due to the presence of a cyclopentane instead of a ribose in this position for the Hst2 structure. Alternatively, a conformational change in the ribose ring of the Sir2Tm structure may be required for enzyme catalysis, which may explain why the cognate substrates have not turned over in the time course of crystallization and why the conformation of the nicotinamide ribose is different than in other structures of this homologue with productively bound NAD⁺ [37].

Density for the nicotinamide moiety of NAD⁺ is often not visible in crystal structure solutions of sirtuin proteins crystallized in the presence of NAD⁺, either because it is disordered or hydrolyzed in the crystallization solution. Occasionally, the nicotinamide moiety of NAD⁺ can been seen in a crystal structure, but it is bound in a so-called non-productive conformation. The nicotinamide moiety of non-productively bound NAD⁺ is not bound in the C site and instead adopts alternate conformations which fail to account for the invariance of key residues required for catalysis located in the C site, and this conformation of NAD⁺ is incompatible with acetyl-lysine binding [37, 46]. When an acetyl-lysine containing peptide, a PEG ion, or the N-terminus of a symmetry-related oligomer is simultaneously bound to the enzyme active site, the nicotinamide moiety of productively bound NAD⁺ can bind in the C site where it makes direct contact with many invariant residues [37, 38, 45, 46]. Interestingly, the hydrophobic tunnel which acts as the acetyl-lysine binding site and not simply the peptide binding site must be occupied to facilitate the productive binding of NAD⁺ [37]. In this productive conformation, the bound NAD⁺ is compatible with both acetyl-lysine binding and enzyme catalysis. In the productive conformation of NAD⁺, the coplanarity of the nicotinamide moiety with the glycosidic bond is strained and the positive charge is buried in a largely hydrophobic environment, thereby destabilizing the ground state of NAD⁺ (Figure 5B&C) [37]. NAD⁺ in the productive conformation makes hydrophobic and van der Waals contacts with the invariant Gly-Ala-Gly motif, which inserts between the nicotinamide moiety and the nicotinamide ribose, and other conserved residues (I41, F40 and N116). The carbonyl oxygen of the nicotinamide moiety of NAD⁺ forms a hydrogen bond with the backbone nitrogen of a conserved isoleucine (117); the carbonyl amino group forms a hydrogen bond with the side chain of a conserved Asp (118) [37]. A highly conserved serine (36) is required for catalytic activity of the protein [29] and it is clear from the conformation of the nicotinamide moiety of NAD⁺ that the serine residue, through its network of direct and water-mediated hydrogen bonds with invariant residues involved in nicotinamide group binding, serves to maintain the protein fold or the enzyme-nicotinamide interactions it mediates [38]. The pi electron cloud of a conserved phenylalanine (44) is positioned directly above the ribose oxygen, possibly to stabilize the carbocation transition state through the formation of a cation-pi interaction, and also may act to shield the glycosidic bond and oxocarbenium intermediate from water hydrolysis [45]. The C site adopts a more closed conformation after acetyl-lysine transfer, not directly after nicotinamide cleavage [38]. The structures of sirtuin homologues with bound product (2’ or 3’-O-acetyl-ADP-ribose) reveal that residues of the cofactor binding loop (D43-R45) shift by about 5.5 Å toward the nicotinamide binding pocket and these residues along with a conserved phenylalanine (44) serve to occlude the C site and probably act to expel the product nicotinamide from this site after cleavage. Finally, when nicotinamide is absent from the C site, a series of water molecules form a network which is conserved in all sirtuin structures, mediates interactions between several residues in the nicotinamide-binding site and seems to be required to maintain the integrity of the core domain [38].

3.2 Acetyl-lysine binding site

Structures of several sirtuin homologues bound to peptides containing acetyl-lysine bearing substrates have been determined [40]. The peptide itself binds to the cleft between the small and large domains with the acetyl-lysine side chain inserting into a hydrophobic tunnel located within the cleft. The peptide backbone containing the acetyl-lysine side chain forms β sheet-like interactions with two flanking strands in the enzyme (Figure 6A) [40]. This staggered, antiparallel three-stranded β sheet is referred to as the β staple. Main chain atoms of the peptide make several hydrogen bonds with main chain atoms of catalytic residues from the flanking strands of the β staple (Figure 6B). A comparison of sirtuin enzymes with and without bound acetyl-lysine containing peptide suggest that peptide binding induces a significant shift in the position of the small domain relative to the large domain, bringing the two domains together to form the β staple motif and resulting in the correct positioning of conserved residues for formation of the acetyl-lysine binding tunnel [48]. It has been hypothesized that NAD⁺ and peptide binding may be cooperative, as the region surrounding the peptide binding site adopts the more closed conformation seen in the peptide bound structure when NAD⁺ is bound but not in the absence of NAD⁺ [40]. The aliphatic region of the acetyl-lysine side chain makes several van der Waals interactions with conserved hydrophobic residues (H135, V228, F184, and L188). The acetyl group appears to be positioned by a single hydrogen bond between the Nε atom of the lysine side chain and the main chain of a Val residue (182) and van der Waals interactions between the acetyl methyl group and other highly conserved residues (I117 and H135), which position the unliganded carbonyl oxygen toward the NAD⁺ binding B site (Figure 6C) [40]. When the peptide is bound in a ternary complex with the intermediate-like molecule ADP-ribose, or the reaction product 2’-O-acetyl-ADP-ribose, the acetyl-lysine carbonyl oxygen hydrogen bonds to the 1’-OH of the nicotinamide ribose ring [38, 42]. The β staple binding motif and the residues involved in peptide binding seem to be conserved features of acetyl-lysine peptide binding, as structures of several sirtuin proteins with bound peptides make similar interactions [36, 39, 42, 48].

Since only main chain atoms from both the peptide and the enzyme participate in hydrogen bonding, substrate specificity does not appear to be conferred by the primary sequence of the acetyl-lysine containing peptide, particularly in the immediate vicinity of the acetyl-lysine residue, although the enzyme residues involved in peptide binding are some of the most highly conserved in the enzyme (Figure 1). The relatively small number of protein-peptide interactions, especially outside of the acetyl-lysine residue, may reflect the fact that the peptide substrates bound to the enzymes in the crystal structures are not true in vivo targets or that substrate specificity determinants may involve other regions of the Sir2 proteins and/or regions of the substrate that are distal to the acetyl-lysine [42]. Consistent with this, comparisons of the same peptide (histone H4) bound to different sirtuin homologues or different peptides bound to different homologues show that peptide residues outside of those immediately flanking the acetyl-lysine residue diverge in direction and make variable contacts to their respective proteins. However, a study of several peptides that vary in primary sequence bound to the same sirtuin homologue, Sir2Tm, show that primary sequence in the residue immediately N-terminal (−1 position) and the residue 2 positions C-terminal (+2 position) to acetyl-lysine may govern sequence specific interactions in this homologue as these residues may hydrogen bond to the side chain of residue N165 and the main chain of G163, and make van der Waals interactions with the side chains of residues F162 and V193, respectively (Sir2Tm numbering) [48]. These van der Waals interactions have been proposed to stabilize β staple interactions when the cleft closes upon peptide binding [48]. In the same study, several peptides failed to crystallize bound to Sir2Tm, and these residues all lacked hydrophobic side chains at the +2 position and had residues other than lysine or arginine at position −1. Since residue N165 of Sir2Tm makes a hydrogen bond to the side chain of the residue in the −1 position of the peptide and residue 165 varies among sirtuin homologues, this interaction may point to one mechanism by which different sirtuins discriminate amongst substrates [48]. Interestingly, comparison of the same acetylated and deacetylated peptide bound to Sir2Tm revealed that the same lysine residue inserts into the hydrophobic tunnel in both structures despite the presence of three other lysine residues in the deacetylated peptide. This suggests that the sequence of the residues immediately N- and C- terminal to the target lysine residue do play some role in governing which lysine residue enters the hydrophobic tunnel [48].

7.1 Inhibitory nicotinamide binding

Nicotinamide is a reaction product and a non-competitive inhibitor of sirtuin proteins [2, 16]. The catalytic mechanism by which Sirtuins couple NAD⁺ cleavage to deacetylation and the mechanism of nicotinamide inhibition have important implications for Sir2 regulation by the physiological regulators NAD⁺ and nicotinamide, and for development of synthetic regulators of Sirtuins. Nicotinamide exerts its inhibitory effect on deacetylation by reacting with a reaction intermediate to reform β-NAD⁺ at the expense of deacetylation [18, 19]. The inhibitory nicotinamide molecule may react with either the oxocarbenium or the α-1’-O-alkylamidate intermediate to reform substrate. The identity of the binding site of the inhibitory nicotinamide molecule has implications for the development of rational activators of Sirtuins that exert their effect through relief of nicotinamide inhibition. Several potential binding sites for the inhibitory nicotinamide molecule have been proposed, including the C site (the binding site of the nicotinamide moiety of NAD⁺) [37] or another conserved pocket in the enzyme active site, termed the D site (Figure 5A) [38].

Structures of Sir2-Af2 bound to NAD⁺ and Sir2Tm bound to acetyl-lysine in the presence of high concentrations of nicotinamide reveal nicotinamide bound in the highly conserved C pocket [46] (Figure 5A). These structures show that nicotinamide can bind in several conformations within the C pocket, anchored by the carboxyamide group (Figure 8A). The carboxyamide amino of nicotinamide hydrogen bonds with the side chain of a conserved aspartic acid (D101) residue in the C pocket, while the carboxyamide oxygen hydrogen bonds with the backbone amino group of a conserved isoleucine (I100) and makes van der Waals interactions with several residues (N99 and I100, Sir2-Af2 numbering) [46]. The pyridine ring of nicotinamide makes more variable interactions with conserved residues (A22, I30, F33 and I100, Sir2-Af2 numbering) within the pocket, as it adopts different conformations in the structures and has correspondingly weaker electron density than the carboxyamide moiety (Figure 8A). The conformation of the cofactor binding loop also affects the interactions between the pyridine ring and residues within the C pocket. There are differences in binding between the nicotinamide moiety of NAD⁺ and the free nicotinamide molecule in the C site. As part of an NAD⁺ molecule, the carboxyamide moiety rotates approximately 150° from its most favored conformation (planar with the nicotinamide ring), which is seen in the free nicotinamide structure. Also, in the free nicotinamide structure, the pyridine ring may be flipped about the carboxyamide relative to its position in the NAD⁺ structure, allowing N1 of the pyridine ring to form favorable interactions with the backbones of hydrophobic residues (F44 and P42, Hst2 numbering) [46]. Although noncompetitive inhibitors like nicotinamide are expected to bind to the enzyme/substrate complex, the C site is known to bind the nicotinamide moiety of the substrate NAD⁺. Therefore, occupation of this binding pocket in the binary Sir2-Af2/(non-productive)NAD⁺ and Sir2Tm/acetyl-lysine complexes may be due to the high concentration of nicotinamide in the crystal condition. It is therefore unclear whether nicotinamide binding in this pocket is the only physiologically relevant binding site for an inhibitory nicotinamide molecule.

The structure of Hst2 bound to carba-NAD⁺ and an acetyl-lysine containing peptide reveals that the acetyl group of the acetyl-lysine substrate hydrogen bonds to the 2’ and 3’ hydroxyl groups of the cyclopentane ring, presumably to help position the nicotinamide group in the highly conserved C pocket for hydrolysis, leaving the acetyl group inappropriately positioned for nucleophilic attack of the 1’ carbon. A comparison of this structure with a ternary complex in which ADP-ribose replaces carba-NAD⁺ reveals that the ribose ring is rotated by about 90° relative to its corresponding position in the carba-NAD⁺ structure with the 1’-hydroxyl group of the ADP-ribose ring pointing into another highly conserved, hydrophobic D pocket that could accommodate an incoming nicotinamide group for a base exchange reaction to reform β-NAD⁺ [38]. The Hst2/acetyl-lysine/ADP-HPD (Figure 7D) complex crystals were soaked with nicotinamide in order to trap a free inhibitory nicotinamide molecule bound to a sirtuin enzyme in complex with an oxocarbenium ion intermediate-like molecule (ADP-HPD). The structure revealed the presence of one nicotinamide molecule bound to the D pocket, adjacent to the β face of the ADP-HPD molecule (Figure 8B) [44]. The D pocket is formed by several residues around the pyridine ring (E64, F67, N116 and F184) and proximal to the carboxyamide moiety (F44 and F117). The density for the carboxyamide is better defined than for the pyridine ring, suggesting greater flexibility of the pyridine ring. Both the carboxyamide nitrogen and oxygen make water mediated hydrogen bonds with the backbone of a highly conserved isoleucine residue (117), while the carboxyamide nitrogen also makes water mediated hydrogen bonds with the side chain of a conserved asparagine (116) and a phosphate oxygen. The pyridine ring makes van der Waals interactions with two phenylalanine residues (F44 and F67) (Figure 8B). Nicotinamide binding in this conformation is consistent with exclusive reformation of β-NAD⁺ upon base exchange and the noncompetitive nature of nicotinamide inhibition. Also, a nicotinamide molecule in the D pocket can be accommodated in the S-alkylamidate structure, while a nicotinamide molecule in the C pocket cannot because of the occupancy of the cofactor binding loop, specifically a conserved phenylalanine residue (44), in this pocket. Since this intermediate may be the one involved in base exchange [50], it is a requirement that nicotinamide would bind simultaneously to this complex. Taken together, these results seem to support the D pocket as a secondary nicotinamide binding site involved in nicotinamide inhibition.

7.2 Synthetic inhibitor complex

There has also been a reported structure of a human sirtuin homologue, SIRT5, in complex with a synthetic sirtuin inhibitor, suramin [35] (Figure 8C). This structure reveals the binding mode of the inhibitor and helps explain the inhibition mechanism. Monomeric SIRT5 dimerizes in solution upon addition of suramin, and in the crystal structure one suramin molecule links two monomers of SIRT5. The buried surface area of the dimer is very limited and the interface between the two monomers is mediated by only non-polar interactions (Figure 8C). The conformation of the cofactor binding loop of SIRT5 changes and becomes more ordered in the suramin structure relative to the SIRT5/ADP-ribose complex structure due to the presence of part of the suramin molecule in the C site. Further, in the suramin bound structure of SIRT5, structural elements in the A site become disordered because this pocket is not occupied. One sulphonyl and the naphthyl groups of suramin bind in the C and B NAD⁺ binding sites, respectively. Several of the benzene rings of suramin bind in the peptide, but not the acetyl-lysine, binding site. This is consistent with suramin acting as a competitive inhibitor of both substrates. The sulphonyl groups make several hydrogen bonds within the enzyme active site, while many residues in the catalytic cleft make van der Waals interactions with nonpolar groups of the suramin molecule [35]. Since suramin is a large, flexible, chemically multifunctional molecule and is not specific for sirtuin enzymes, suramin itself may not be an attractive sirtuin inhibitor candidate for further development. Nonetheless, the structure of suramin bound in the SIRT5 NAD⁺ and acetyl-lysine binding site does suggest that the trisulfonyl naphthyl group and adjacent moieties of the suramin molecule may serve as minimal inhibitor moieties that may be used as lead scaffolds for developing new sirtuin inhibitors with increased specificity for this class of enzymes.