Alexei Savchenko

Structural proteomics of an archaeon.

A set of 424 nonmembrane proteins from Methanobacterium thermoautotrophicum were cloned, expressed and purified for structural studies. Of these, ∼20% were found to be suitable candidates for X-ray crystallographic or NMR spectroscopic analysis without further optimization of conditions, providing an estimate of the number of the most accessible structural targets in the proteome. A retrospective analysis of the experimental behavior of these proteins suggested some simple relations between sequence and solubility, implying that data bases of protein properties will be useful in optimizing high throughput strategies. Of the first 10 structures determined, several provided clues to biochemical functions that were not detectable from sequence analysis, and in many cases these putative functions could be readily confirmed by biochemical methods. This demonstrates that structural proteomics is feasible and can play a central role in functional genomics.

Data Mining Crystallization Databases: Knowledge-Based Approaches to Optimize Protein Crystal Screens

Protein crystallization is a major bottleneck in protein X‐ray crystallography, the workhorse of most structural proteomics projects. Because the principles that govern protein crystallization are too poorly understood to allow them to be used in a strongly predictive sense, the most common crystallization strategy entails screening a wide variety of solution conditions to identify the small subset that will support crystal nucleation and growth. We tested the hypothesis that more efficient crystallization strategies could be formulated by extracting useful patterns and correlations from the large data sets of crystallization trials created in structural proteomics projects. A database of crystallization conditions was constructed for 755 different proteins purified and crystallized under uniform conditions. Forty‐five percent of the proteins formed crystals. Data mining identified the conditions that crystallize the most proteins, revealed that many conditions are highly correlated in their behavior, and showed that the crystallization success rate is markedly dependent on the organism from which proteins derive. Of the proteins that crystallized in a 48‐condition experiment, 60% could be crystallized in as few as 6 conditions and 94% in 24 conditions. Consideration of the full range of information coming from crystal screening trials allows one to design screens that are maximally productive while consuming minimal resources, and also suggests further useful conditions for extending existing screens. Proteins 2003;51:562–568.

Structure of Escherichia coli ribose-5-phosphate isomerase: a ubiquitous enzyme of the pentose phosphate pathway and the Calvin cycle.

Ribose-5-phosphate isomerase A (RpiA; EC 5.3.1.6) interconverts ribose-5-phosphate and ribulose-5-phosphate. This enzyme plays essential roles in carbohydrate anabolism and catabolism; it is ubiquitous and highly conserved. The structure of RpiA from Escherichia coli was solved by multiwavelength anomalous diffraction (MAD) phasing, and refined to 1.5 A resolution (R factor 22.4%, R(free) 23.7%). RpiA exhibits an alpha/beta/(alpha/beta)/beta/alpha fold, some portions of which are similar to proteins of the alcohol dehydrogenase family. The two subunits of the dimer in the asymmetric unit have different conformations, representing the opening/closing of a cleft. Active site residues were identified in the cleft using sequence conservation, as well as the structure of a complex with the inhibitor arabinose-5-phosphate at 1.25 A resolution. A mechanism for acid-base catalysis is proposed.

Strategies for structural proteomics of prokaryotes: Quantifying the advantages of studying orthologous proteins and of using both NMR and X-ray crystallography approaches

Only about half of non‐membrane‐bound proteins encoded by either bacterial or archaeal genomes are soluble when expressed in Escherichia coli (Yee et al., Proc Natl Acad Sci USA 2002;99:1825–1830 ; Christendat et al., Prog Biophys Mol Biol 200;73:339–345) . This property limits genome‐scale functional and structural proteomics studies, which depend on having a recombinant, soluble version of each protein. An emerging strategy to increase the probability of deriving a soluble derivative of a protein is to study different sequence homologues of the same protein, including representatives from thermophilic organisms, based on the assumption that the stability of these proteins will facilitate structural analysis. To estimate the relative merits of this strategy, we compared the recombinant expression, solubility, and suitability for structural analysis by NMR and/or X‐ray crystallography for 68 pairs of homologous proteins from E. coli and Thermotoga maritima. A sample suitable for structural studies was obtained for 62 of the 68 pairs of homologs under standardized growth and purification procedures. Fourteen (eight E. coli and six T. maritima proteins) samples generated NMR spectra of a quality suitable for structure determination and 30 (14 E. coli and 16 T. maritima proteins) samples formed crystals. Only three (one E. coli and two T. maritima proteins) samples both crystallized and had excellent NMR properties. The conclusions from this work are: (1) The inclusion of even a single ortholog of a target protein increases the number of samples for structural studies almost twofold; (2) there was no clear advantage to the use of thermophilic proteins to generate samples for structural studies; and (3) for the small proteins analyzed here, the use of both NMR and crystallography approaches almost doubled the number of samples for structural studies. Proteins 2003;50:392–399.

Crystal structure of secretory protein Hcp3 from Pseudomonas aeruginosa

The Type VI secretion pathway transports proteins across the cell envelope of Gram-negative bacteria. Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen infecting humans, uses the type VI secretion pathway to export specific effector proteins crucial for its pathogenesis. The HSI-I virulence locus encodes for several proteins that has been proposed to participate in protein transport including the Hcp1 protein, which forms hexameric rings that assemble into nanotubes in vitro. Two Hcp1 paralogues have been identified in the P. aeruginosa genome, Hsp2 and Hcp3. Here, we present the structure of the Hcp3 protein from P. aeruginosa. The overall structure of the monomer resembles Hcp1 despite the lack of amino-acid sequence similarity between the two proteins. The monomers assemble into hexamers similar to Hcp1. However, instead of forming nanotubes in head-to-tail mode like Hcp1, Hcp3 stacks its rings in head-to-head mode forming double-ring structures.

X‐ray crystal structure of CutA from Thermotoga maritima at 1.4 Å resolution

The structure of the CutA protein from Thermotoga maritima (tmCutA) was determined at 1.4 A resolution using the Se-Met multiwavelength anomalous diffraction (MAD) technique. This protein (TIGR annotation - TM1056, DNA bases 1,069,580–1,069,885) is conserved in numerous bacteria, archaea and eucarya, including plants and mammals (COG1324, Fig. 1). The CutA Escherichia coli homolog—CutA1 (35% ID) is involved in divalent cation homeostasis,1 while the mammalian homolog—mCutA (40% ID) was found to be associated with cell surface acetylcholinesterase.2 However, the biological function of the CutA proteins is yet to be determined. n n n nFig. 1 n nMultiple sequence alignment of CutA from Thermotoga maritima (Thema) against other periplasmic divalent cation tolerance proteins from bacteria (Aquae = Aquifex aeolicus, Deira = Deinococcus radiodurans) and archaea (Pyrho = Pyrococcus horikoshii, Arcfu ... n n n nThe tmCutA monomer assumes a two-layer α/β α/β sandwich fold (β1β2α1β3β4α3β5β6α5) shared by a large number of proteins (CATH number 3.30.70.120). Two short (β2β5 and two long (β3β4) β-strands arranged in an antiparallel fashion β5↑β2↓β3↑β4↓ form a curved sheet. Three α-helices cover one side of the molecule (Fig. 2a). Each monomer is elongated with overall dimensions approximately 25 × 30× 60 A (Fig. 2a). The monomers are assembled into a trimer with dimensions 55 × 55 × 35 A through interaction of the edges of three β-strands (β3 on the top and β5β6 on the bottom) (Fig. 2b). In the trimer, the subunits are related by crystallographic three fold axes. The same oligomeric state was observed for tmCutA in solution by size exclusion experiments (see Methods) and appears to be functionally relevant. In the trimer, there is a cavity that is accessible from outside via three solvent accessible channels. The inner surface of the cavity is lined with several conserved residues including Cys35, which may serve as a potential metal binding site. n n n nFig. 2 n nRibbon representations of the tmCutA monomer (a), trimer (b) and superimposed structures of tmCutA and GlnK’ complex with ATP (c). a: The α-helixes and β-strains are colored in red and blue, respectively. b: Each monomer in the ... n n n nStructural homologues of tmCutA were identified using Dali search3 and SSAP structure comparison program 4. The closest structural matches, with a Dali score of 9.4 Z, were the E. coli signal transducer proteins GlnB (PII)5 and GlnK6 (Fig. 2c). These two proteins are 67% identical to each other but have insignificant sequence similarity (less than 15% ID) to tmCutA. GlnB and GlnK, which are also trimeric, are involved in maintaining nitrogen homeostasis7–9 and have been shown to cooperatively bind ATP and α-ketoglutarate.6,10 The ATP binding sites of GlnB and GlnK are located in clefts between the momoners,11 but most of the conserved amino acids that mediate ATP binding in PII proteins are not found in tmCutA. The cavity on the side of tmCutA is formed by a number of conserved aromatic (Tyr45, Trp47, Tyr81, and Trp101) and charged residues (Asp54, Glu56, Glu82) (Figs. 1 and u200band2c),2c), which makes this cleft a strong candidate for a conserved function. n nIn PII-type proteins, the two central β-strains β3 and β4 are joined by a flexible loop called the T-loop, which is composed of 17 amino acids. The T-loop plays a key role in protein-protein interactions with downstream effector proteins.12 The β3 and β4 strands in tmCutA, while longer than the corresponding strands in the PII proteins (12 residues in the β3 strand of tmCutA compared with 9 in the β2 strand of PII proteins), are joined by only a two-amino acid (Lys48Gly49) turn and form a hairpin. Although the residues in the C-terminal extension of β3-strand in tmCutA proteins (Tyr45, Trp46, and Trp47, Fig. 1) are conserved among tmCutA homologs, the structure of this region in tmCutA is different than in PII proteins and thus this region of tmCutA may not participate in protein-protein interactions. n nCutA is annotated as being involved in metal homeostasis. Several other structural homologs of tmCutA, including the PII proteins, metallochaperone Atx1 (5.9 Z score) and the metal-binding domain of the Menkes copper-transporting ATPase (4.5 Z score) have metal-binding properties.13,14 These proteins coordinate the metal through a CXXC sequence motif,15,16 but this motif is not found in CutA. Therefore, tmCutA may represent a new branch of this family that differs from PII and metal binding proteins. The biochemical experiments are under way to define possible ligands and functional partners of CutA proteins.

Crystal structures of TM0549 and NE1324--two orthologs of E. coli AHAS isozyme III small regulatory subunit.

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(H2O)62+ ion in TM0549. Both TM0549 and NE1324 form dimeric assemblies in the crystal lattice.

The crystal structure of a novel SAM-dependent methyltransferase PH1915 from Pyrococcus horikoshii

The S‐adenosyl‐L‐methionine (SAM)‐dependent methyltransferases represent a diverse and biologically important class of enzymes. These enzymes utilize the ubiquitous methyl donor SAM as a cofactor to methylate proteins, small molecules, lipids, and nucleic acids. Here we present the crystal structure of PH1915 from Pyrococcus horikoshii OT3, a predicted SAM‐dependent methyltransferase. This protein belongs to the Cluster of Orthologous Group 1092, and the presented crystal structure is the first representative structure of this protein family. Based on sequence and 3D structure analysis, we have made valuable functional insights that will facilitate further studies for characterizing this group of proteins. Specifically, we propose that PH1915 and its orthologs are rRNA‐ or tRNA‐specific methyltransferases.

Crystal structure of glutamine amidotransferase from Thermotoga maritima

Glutamine amidotransferases (GATases) are ubiquitous enzymes that transfer the amide nitrogen of glutamine to a variety of substrates.1 GATases catalyze two separate reactions at two active sites, which are located either on a single polypeptide chain or on different subunits. In the glutaminase reaction, glutamine is hydrolyzed to glutamate and ammonia, which is added to an acceptor substrate in the synthase reaction. Imidazole glycerol phosphate synthase (IGPS) is a GATase that catalyzes the bifurcation step of the histidine and de novo purine biosynthetic pathways. In yeast, IGPS is a single polypeptide,2 whereas in bacteria IGPS activity is effected by a complex of a glutaminase subunit, HisH, and a synthase subunit, HisF.3,4 IGPS is a class I GATase, which is characterized by a Cys–His–Glu catalytic triad. IGPS is a potential target for antibiotic and herbicide development because the histidine pathway does not occur in mammals. n nHere, we present the crystal structure of the HisH protein from Thermotoga maritima (TmHisH), which complements the known structure of HisF from the same organism (TmHisF)5 [Protein Data Bank (PDB) ID 1THF] and HisF from Pyrobaculum aerophilum6 (PDB ID 1H5Y) and shows high structural similarity with the glutaminase domain of the recently determined structure of IGSP from yeast7 (HIS7) (PDB ID 1JVN).

The structure of a putative S-formylglutathione hydrolase from Agrobacterium tumefaciens

The structure of the Atu1476 protein from Agrobacterium tumefaciens was determined at 2 Å resolution. The crystal structure and biochemical characterization of this enzyme support the conclusion that this protein is an S‐formylglutathione hydrolase (AtuSFGH). The three‐dimensional structure of AtuSFGH contains the α/β hydrolase fold topology and exists as a homo‐dimer. Contacts between the two monomers in the dimer are formed both by hydrogen bonds and salt bridges. Biochemical characterization reveals that AtuSFGH hydrolyzes Cuf8ffO bonds with high affinity toward short to medium chain esters, unlike the other known SFGHs which have greater affinity toward shorter chained esters. A potential role for Cys54 in regulation of enzyme activity through S‐glutathionylation is also proposed.