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Structural basis for sirtuin function: what we know and what we don't - PubMed

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Structural basis for sirtuin function: what we know and what we don't

Brandi D Sanders et al. Biochim Biophys Acta. 2010 Aug.

Abstract

The sirtuin (silent information regulator 2) proteins are NAD(+)-dependent deacetylases that are implicated in diverse biological processes including DNA regulation, metabolism, and longevity. Homologues of the prototypic yeast Sir2p have been identified in all three kingdoms of life, and while bacteria and archaea typically contain one to two sirtuins, eukaryotic organisms contain multiple members. Sirtuins are regulated in part by the cellular concentrations of the noncompetitive inhibitor, nicotinamide, and several synthetic modulators of these enzymes have been identified. The x-ray crystal structures of several sirtuin proteins in various liganded forms have been determined. This wealth of structural information, together with related biochemical studies, have provided important insights into the catalytic mechanism, substrate specificity, and inhibitory mechanism of sirtuin proteins. Implications for future structural studies to address outstanding questions in the field are also discussed.

Copyright 2009 Elsevier B.V. All rights reserved.

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Figures

Figure 1
Figure 1

Sequence alignments of Sirtuins for which three-dimensional structures have been determined. An alignment of S. cerevisiae Hst2 and Sir2p, A. fulgidus Sir2-Af1 and Sir2-Af2, T. maritima Sir2Tm, E. Coli cobB, and H. sapiens SIRT2 and SIRT5. The secondary structural elements for Hst2 are shown above the sequence alignment; β sheets are indicated with orange arrows, α helices with green rectangles, loops with black lines and dashed lines indicate unstructured regions. Cyan circles indicate residues that participate in NAD+ binding, purple circles indicate residues that participate in acetyl-lysine peptide binding, a green star indicates the general base residue and blue triangles indicate the residues involved in zinc binding. Solid lines beneath the sequence alignment indicate which regions of the proteins compose the Rossmann-fold domain (magenta), cofactor binding loop (black), small domain (blue), and loop regions (green).

Figure 2
Figure 2

Three-dimensional structure of each sirtuin protein that has been determined. The enzymes are shown in cartoon representation without bound ligands. The Rossmann-fold domain is indicated in magenta, the small domain in blue, the loops in green, the cofactor binding loop in black, the Zn ion in red, the N-terminal region in orange, and the C-terminal region in yellow. Disordered regions are indicated with dashed lines. A) S. cerevisiae Hst2 (pdb accession number 1Q14) B) A. fulgidus Sir2-Af1 (1ICI) C) A. fulgidus Sir2-Af2 (1MA3) D) T. maritima Sir2Tm (2H4J) E) E. Coli cobB (1S5P) F) H. sapiens SIRT2 (1J8F) G) H. sapiens SIRT5 (2B4Y).

Figure 3
Figure 3

Sirtuin active site binding cleft. The Hst2 protein is shown in tan surface representation. The catalytic residues H135 and N116 are shown in red stick representation in the close-up view. The peptide substrate backbone is shown in green cartoon representation, the acetyl-lysine side chain is shown in green cpk stick representation, and the NAD+ substrate molecule is shown in cyan cpk stick representation.

Figure 4
Figure 4

Conformational changes of the cofactor binding loop upon substrate and product binding. The Hst2 protein is shown in tan cartoon representation, the C-terminus is shown in yellow, and the cofactor binding loop is shown in magenta. The peptide substrate is shown in green cartoon representation, while the acetyl-lysine residue is shown in green cpk stick representation. The substrate analog or product is shown in cyan cpk stick representation. A) The apo Hst2 enzyme, the cofactor binding loop is disordered (dashed lines). B) Hst2 with bound peptide substrate and carba-NAD+, the cofactor binding loop is in an open conformation. C) Hst2 with bound peptide substrate and 2’-O-acetyl-ADP-ribose, the cofactor binding loop is in a closed conformation.

Figure 5
Figure 5

Productive NAD+ binding interactions in several sirtuin proteins. Carba-NAD+ or NAD+ molecules are shown in cyan cpk stick representation and acetyl-lysine residues are shown in green cpk line representation. A) The Hst2 protein is shown in tan surface representation. The A, B, C and D sites are indicated by red circles. B) S. cerevisiae Hst2. Enzyme residues that make van der Waals interactions with NAD+ are shown in orange. Residues that make hydrogen bonds (red dashes) with NAD+ are shown in yellow. Residues that make both interactions are shown in magenta. C) T. maritima Sir2Tm. Residues are colored as in B.

Figure 6
Figure 6

Acetyl-lysine peptide binding to the sirtuin active site. A) A peptide containing an acetyl-lysine residue (green) is shown bound to the Sir2-Af2 sirtuin enzyme (tan, cartoon representation) active site forming the β staple with flanking β strands of the enzyme (cyan). B) The acetyl-lysine containing peptide (green) makes main chain β strand hydrogen bonds (red dashes) with main chain atoms of the enzyme (cyan). C) The acetyl-lysine residue (green) binds in a hydrophobic tunnel of the enzyme (tan, surface representation), making several van der Waals interactions with enzyme residues (orange) and one hydrogen bond (red dashes) with an enzyme residue.

Figure 7
Figure 7

The Sir2 catalytic mechanism. A) The steps of the catalytic mechanism for which there is structural support. The glycosidic bond is broken in a dissociative SN1-type manner, forming the oxocarbenium ion intermediate with an accompanying conformational change of the nicotinamide ribose ring. The acetyl-lysine is then positioned to nucleophilically attack C1’ of the oxocarbenium intermediate to form the alkylamidate intermediate, again with a conformational change in the ribose ring. F44 (Hst2 numbering) also flips to expel nicotinamide from the C pocket. The histidine general base can then deprotonate the 2’-OH directly to form the cyclic acyldioxilane intermediate. Through several steps and the addition of one water molecule the reaction products are formed. B) Nicotinamide cleavage via an SN2-type mechanism. Reaction steps after the formation of the akylamidate intermediate (E–I2) are identical as described in A). C) The reaction coordinate for the known steps of the Sir2 catalytic mechanism. S* indicates the destabilized substrate. E–I1, E–I2, and E–I3 indicate the oxocarbenium, alkylamidate and cyclic intermediates, respectively. E:T1–3 indicate the transition states required to form those intermediates, respectively. Since the remaining steps are unknown, their progress along the reaction coordinate is represented by a dashed line. D) The chemical structures of the transition state and intermediate-like molecules crystallized in complex with Sir2 enzymes.

Figure 8
Figure 8

The inhibitor binding sites. Sirtuin enzymes are shown in tan surface representation. Acetyl-lysine residues are shown in green cpk stick representation, the intermediate-like molecule, ADP-HPD, is shown in cyan cpk line representation, the nicotinamide molecules are shown in yellow cpk stick representation, and the suramin molecule is shown in orange cpk stick representation. Residues involved in van der Waals and hydrogen bonding (red dashes) interactions with nicotinamide are also shown in red stick representation. A) T. maritima Sir2Tm with nicotinamide bound in the C pocket. The cofactor binding loop is shown in cartoon instead of surface representation for clarity. B) S. cerevisiae Hst2 with nicotinamide bound in the D pocket. C) H. sapiens SIRT5 dimer with bound suramin. The red line indicates the 2-fold symmetry of the suramin molecule.

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