Background SIR2 is an NAD+-dependent deacetylase [1]–[3] implicated in the regulation of lifespan in species as diverse as yeast [4], worms [5], and flies [6]. We previously reported that the level of SIRT1, the mammalian homologue of SIR2 [7], [8], is coupled to the level of mitotic activity in cells both in vitro and in vivo [9]. Cells from long-lived mice maintained SIRT1 levels of young mice in tissues that undergo continuous cell replacement by proliferating stem cells. Changes in SIRT1 protein level were not associated with changes in mRNA level, suggesting that SIRT1 could be regulated post-transcriptionally. However, other than a recent report on sumoylation [10] and identification of SIRT1 as a nuclear phospho-protein by mass spectrometry [11], post-translational modifications of this important protein have not been reported.Methodology/Principal FindingsWe identified 13 residues in SIRT1 that are phosphorylated in vivo using mass spectrometry. Dephosphorylation by phosphatases in vitro resulted in decreased NAD+-dependent deacetylase activity. We identified cyclinB/Cdk1 as a cell cycle-dependent kinase that forms a complex with and phosphorylates SIRT1. Mutation of two residues phosphorylated by Cyclin B/Cdk1 (threonine 530 and serine 540) disturbs normal cell cycle progression and fails to rescue proliferation defects in SIRT1-deficient cells [12], [13].Conclusions/SignificancePharmacological manipulation of SIRT1 activity is currently being tested as a means of extending lifespan in mammals. Treatment of obese mice with resveratrol, a pharmacological activator of SIRT1, modestly but significantly improved longevity and, perhaps more importantly, offered some protection against the development of type 2 diabetes mellitus and metabolic syndrome [14]–[16]. Understanding the endogenous mechanisms that regulate the level and activity of SIRT1, therefore, has obvious relevance to human health and disease. Our results identify phosphorylation by cell cycle dependent kinases as a major mechanism controlling the level and function of this sirtuin and complement recent reports of factors that inhibit [17], [18] and activate [19] SIRT1 by protein-protein interactions.

Crystal structures of two orthologs of the regulatory subunit of acetohydroxyacid synthase III (AHAS, EC 2.2.1.6) from Thermotoga maritima (TM0549) and Nitrosomonas europea (NE1324) were determined by single-wavelength anomalous diffraction methods with the use of selenomethionine derivatives at 2.3 Å and 2.5 Å , respectively. TM0549 and NE1324 share the same fold, and in both proteins the polypeptide chain contains two separate domains of a similar size. Each protein contains a C-terminal domain with ferredoxin-type fold and an N-terminal ACT domain, of which the latter is characteristic for several proteins involved in amino acid metabolism. The ferredoxin domain is stabilized by a calcium ion in the crystal structure of NE1324 and by a Mg(H 2 O) 6 2+ ion in TM0549. Both TM0549 and NE1324 form dimeric assemblies in the crystal lattice.Keywords: acetohydroxyacid synthase; actolactate synthase; regulatory subunit; ACT domain; AHAS; protein refolding Acetohydroxyacid synthase (AHAS, EC 2.2.1.6) catalyzes two physiologically significant reactions in the synthesis of isoleucine, valine, and leucine. In the first reaction, which is an initial step in the synthesis of isoleucine, 2-aceto-2-hydroxy butyrate is produced by condensation of 2-ketobutyrate with pyruvate. In the second reaction, 2-acetolactate is synthesized from two pyruvate molecules, and this provides substrates for synthesis of valine and leucine (Umbarger 1978(Umbarger , 1987Chipman et al. 1998). This pathway of branched-chain amino acid biosynthesis is characteristic for bacteria, fungi, algae, and higher plants, but not for animals. Accordingly, AHAS inhibitors have been developed as herbicides which have found broad application (Short and Colborn 1999). The inhibitors of branched-chain amino acids synthesis are also being tested as potential antituberculosis agents (Grandoni et al. 1998;Zohar et al. 2003;Choi et al. 2005).Three different FAD-dependent AHAS isozymes (I, II, and III) are found in enterobacteria (e.g., Escherichia ), but most other organisms from the bacteria encode only a single AHAS enzyme, similar to isozyme III from E. coli. The acetohydroxyacid synthases are multimeric proteins, and most frequently their biological unit is composed of two catalytic subunits (CSU) and two small regulatory subunits (SSU). In the absence of the SSU subunit, the CSU dimer is unstable (Vyazmensky et al. 1996), suggesting that in the holoenzyme the AHAS dimer is stabilized by a SSU dimer. The large catalytic subunits also show very weak activity without their associated regulatory subunits (Weinstock et al. 1992;Pang and Duggleby 1999). The E. coli AHAS enzyme can be reconstituted with SSU subunits from other organisms (Porat et al. 2004), and the reconstituted enzymes form stable heterotetramers. The properly associated holoenzymes show full catalytic activity, and are also susceptible to valine inhibition.Previous mutagenesis studies (Mendel et al. 2001;Kaplun et al. 2006) revealed that the valine binding sites are located at the dimerizatio...

GGA (Golgi-localizing, -adaptin ear domain homology, ARFbinding) proteins, which constitute a family of clathrin coat adaptor proteins, have recently been shown to be involved in the ubiquitindependent sorting of receptors, through the interaction between the Cterminal three-helix-bundle of the GAT (GGA and Tom1) domain (C-GAT) and ubiquitin. We report the crystal structure of human GGA3 C-GAT in complex with ubiquitin. At the center of the interface, three pockets on the hydrophobic Ile44 surface of ubiquitin accommodate three hydrophobic residues from helices 1 and 2 of C-GAT. Two distinct orientations of ubiquitin Arg42 determine the shape and the charge distribution of the third pocket of the ubiquitin Ile44 surface, leading to tight and loose binding modes of C-GAT. The flexibility of the third pocket explains why ubiquitin Ile44 surface can interact with structurally divergent ubiquitin binding modules. In addition, biochemical and NMR data suggest another hydrophobic binding site on C-GAT helices 2 and 3, opposite to the first binding site, also binds ubiquitin although weakly. The double-sided ubiquitin binding provides the GAT domain with higher efficiency in recognizing ubiquitinated receptors for lysosomal receptor degradation.

The crystal structure of a putative transcriptional regulator protein TM1030 from Thermotoga maritima, a hyperthermophilic bacterium, was determined by an unusual multi-wavelength anomalous dispersion method at 2.0 Å resolution., in which data from two different crystals and two different beamlines were used. The protein belongs to the tetracycline repressor TetR superfamily. The three-dimensional structure of TM1030 is similar to the structures of proteins that function as multidrug-binding transcriptional repressors, and contains a large solvent-exposed pocket similar to the drug-binding pockets present in those repressors. The asymmetric unit in the crystal structure contains a single protein chain and the two-fold symmetry of the dimer is adopted by the crystal symmetry. The structure described in this paper is an apo-form of TM1030. Although it is known that the protein is significantly overexpressed during heat shock, its detailed function cannot be yet explained.

Transcriptional regulator protein TM1030 from the hyperthermophile Thermotoga maritima, as well as its complex with DNA, was crystallized at a wide range of temperatures. Crystallization plates were incubated at 4, 20, 37 and 50° C over 3 weeks. The best crystals of TM1030 in complex with DNA were obtained at 4, 20 and 37° C, while TM1030 alone crystallized almost equally well in all temperatures. The crystals grown at different temperatures were used for X-ray diffraction experiments and their structures were compared. Surprisingly, the models of TM1030 obtained from crystals grown at different temperatures are similar in quality. While there are some examples of structures of proteins grown at elevated temperatures in the PDB, these temperatures appear to be underrepresented. Our studies show that crystals of some proteins may be grown and are stable at broad range of temperatures. We suggest that crystallization experiments at elevated temperatures could be used as a standard part of the crystallization protocol.

The crystal structure of PA5185, a putative thioesterase from Pseudomonas aeruginosa strain PAO1, was solved using multi-wavelength anomalous diffraction to 2.4 Å. Analysis of the structure and information about the putative function of the protein were used to optimize crystallization conditions. The crystal growth was optimized by applying additives with chemical similarity to a fragment of a putative PA5185 substrate (CoA or its derivative). Using new crystallization conditions containing this function-biased set of additives, several new crystal forms were produced and structures of three of them (in three different space groups) were determined. One of the new crystal forms had an improved resolution limit of 1.9 Å, and another displayed an alternative conformation of the highly-conserved loop containing Asn26, which could play a physiological role. Surprisingly, none of the additives were ordered in the crystal structures. Application of function-biased additives could be used as a standard optimization protocol for producing improved diffraction, or new crystal forms, which may lead to better understanding of the biological functions of proteins.

3‐(1‐Pyridinio)propane­sulfonate, C8H11NO3S, and 3‐(benzyl­dimethyl­ammonio)propane­sulfonate monohydrate, C12H19NO3S·H2O, used as additives during protein refolding and crystallization, both crystallize in the monoclinic system in the P21/c space group, with one mol­ecule (or one set of mol­ecules) per asymmetric unit. The solvent water mol­ecule present in the second crystal structure results in the formation of a dimer through hydrogen bonds. The conformation of the propane­sulfonate moiety is similar in both structures.

Key indicatorsSingle-crystal X-ray study T = 106 K Mean (C-C) = 0.002 Å R factor = 0.034 wR factor = 0.092 Data-to-parameter ratio = 16.7For details of how these key indicators were automatically derived from the article, see