Steven Beasley

The crystal structure of spermidine synthase with a multisubstrate adduct inhibitor.

Polyamines are essential in all branches of life. Spermidine synthase (putrescine aminopropyltransferase, PAPT) catalyzes the biosynthesis of spermidine, a ubiquitous polyamine. The crystal structure of the PAPT from Thermotoga maritima (TmPAPT) has been solved to 1.5 Å resolution in the presence and absence of AdoDATO (S-adenosyl-1,8-diamino-3-thiooctane), a compound containing both substrate and product moieties. This, the first structure of an aminopropyltransferase, reveals deep cavities for binding substrate and cofactor, and a loop that envelops the active site. The AdoDATO binding site is lined with residues conserved in PAPT enzymes from bacteria to humans, suggesting a universal catalytic mechanism. Other conserved residues act sterically to provide a structural basis for polyamine specificity. The enzyme is tetrameric; each monomer consists of a C-terminal domain with a Rossmann-like fold and an N-terminal β-stranded domain. The tetramer is assembled using a novel barrel-type oligomerization motif.

Structure of Thermotoga maritima stationary phase survival protein SurE: a novel acid phosphatase.

BACKGROUND The rpoS, nlpD, pcm, and surE genes are among many whose expression is induced during the stationary phase of bacterial growth. rpoS codes for the stationary-phase RNA polymerase sigma subunit, and nlpD codes for a lipoprotein. The pcm gene product repairs damaged proteins by converting the atypical isoaspartyl residues back to L-aspartyls. The physiological and biochemical functions of surE are unknown, but its importance in stress is supported by the duplication of the surE gene in E. coli subjected to high-temperature growth. The pcm and surE genes are highly conserved in bacteria, archaea, and plants. RESULTS The structure of SurE from Thermotoga maritima was determined at 2.0 A. The SurE monomer is composed of two domains; a conserved N-terminal domain, a Rossman fold, and a C-terminal oligomerization domain, a new fold. Monomers form a dimer that assembles into a tetramer. Biochemical analysis suggests that SurE is an acid phosphatase, with an optimum pH of 5.5-6.2. The active site was identified in the N-terminal domain through analysis of conserved residues. Structure-based site-directed point mutations abolished phosphatase activity. T. maritima SurE intra- and intersubunit salt bridges were identified that may explain the SurE thermostability. CONCLUSIONS The structure of SurE provided information about the proteins fold, oligomeric state, and active site. The protein possessed magnesium-dependent acid phosphatase activity, but the physiologically relevant substrate(s) remains to be identified. The importance of three of the assigned active site residues in catalysis was confirmed by site-directed mutagenesis.

Identification of a Novel Zn2+-binding Domain in the Autosomal Recessive Juvenile Parkinson-related E3 Ligase Parkin

Missense mutations in park2, encoding the parkin protein, account for ∼50% of autosomal recessive juvenile Parkinson disease (ARJP) cases. Parkin belongs to the family of RBR (RING-between-RING) E3 ligases involved in the ubiquitin-mediated degradation and trafficking of proteins such as Pael-R and synphillin-1. The proposed architecture of parkin, based largely on sequence similarity studies, consists of N-terminal ubiquitin-like and C-terminal RBR domains. These domains are separated by a ∼160-residue unique parkin sequence having no recognizable domain structure. We used limited proteolysis experiments on bacterially expressed and purified parkin to identify a new domain (RING0) within the unique parkin domain sequence. RING0 comprises two distinct, conserved cysteine-rich clusters between Cys150–Cys169 and Cys196–His215 consisting of CX2-3CX11CX2C and CX4–6CX10–16-CX2(H/C) motifs. The positions of the cysteine/histidine residues in this region bear similarity to parkin RING1 and RING2 domains, as well as other E3 ligase RING domains. However, in parkin a 26-residue linker region separates the motifs, which is not typical of other RING domain structures. Further, the RING0 domain includes all but one of the known ARJP mutation sites between the ubiquitin-like and RBR regions of parkin. Using electrospray ionization mass spectrometry and inductively coupled plasma-atomic emission spectrometry analysis, we determined that the RING0, RING1, IBR, and RING2 domains each bind two Zn2+ ions, the first observation of an E3 ligase with the ability to bind eight metal ions. Removal of the zinc from parkin causes near complete unfolding of the protein, an observation that rationalizes cysteine-based ARJP mutations found throughout parkin, including RING0 (C212Y) that form cellular inclusions and/or are defective for ubiquitination likely because of poor zinc binding and misfolding. The identification of the RING0 domain in parkin provides a new overall domain structure for the protein that will be important in assessing the roles of ARJP mutations and designing experiments aimed at understanding the disease.

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.

Structure of the Parkin in-between-ring domain provides insights for E3-ligase dysfunction in autosomal recessive Parkinson's disease

Mutations in Parkin are one of the predominant hereditary factors found in patients suffering from autosomal recessive juvenile Parkinsonism. Parkin is a member of the E3 ubiquitin ligase family that is defined by a tripartite RING1-in-between-ring (IBR)-RING2 motif. In Parkin, the IBR domain has been shown to augment binding of the E2 proteins UbcH7 and UbcH8, and the subsequent ubiquitination of the proteins synphilin-1, Sept5, and SIM2. To facilitate our understanding of Parkin function, the solution structure of the Parkin IBR domain was solved by using NMR spectroscopy. Folding of the IBR domain (residues M327–S378) was found to be zinc dependent, and the structure reveals the domain forms a unique pair scissor-like and GAG knuckle-like zinc-binding sites, different from other zinc-binding motifs such as the RING, LIM, PHD, or B-box motifs. The N terminus of the IBR domain, residues E307–E322, is unstructured. The disease causing mutation T351P causes global unfolding, whereas the mutation R334C causes some structural rearrangement of the domain. In contrast, the protein containing the mutation G328E appears to be properly folded. The structure of the Parkin IBR domain, in combination with mutational data, allows a model to be proposed where the IBR domain facilitates a close arrangement of the adjacent RING1 and RING2 domains to facilitate protein interactions and subsequent ubiquitination.

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 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. Here, 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).

Solution structure of the E3 ligase HOIL-1 Ubl domain

The E3 ligases HOIL‐1 and parkin are each comprised of an N‐terminal ubiquitin‐like (Ubl) domain followed by a zinc‐binding region and C‐terminal RING–In‐between‐RING–RING domains. These two proteins, involved in the ubiquitin‐mediated degradation pathway, are the only two known E3 ligases to share this type of multidomain architecture. Further, the Ubl domain of both HOIL‐1 and parkin has been shown to interact with the S5a subunit of the 26S proteasome. The solution structure of the HOIL‐1 Ubl domain was solved using NMR spectroscopy to compare it with that of parkin to determine the structural elements responsible for S5a intermolecular interactions. The final ensemble of 20 structures had a β‐grasp Ubl‐fold with an overall backbone RMSD of 0.59 ± 0.10 Å in the structured regions between I55 and L131. HOIL‐1 had a unique extension of both β1 and β2 sheets compared to parkin and other Ubl domains, a result of a four‐residue insertion in this region. A similar 15‐residue hydrophobic core in the HOIL‐1 Ubl domain resulted in a comparable stability to the parkin Ubl, but significantly lower than that observed for ubiquitin. A comparison with parkin and other Ubl domains indicates that HOIL‐1 likely uses a conserved hydrophobic patch (W58, V102, Y127, Y129) found on the β1 face, the β3–β4 loop and β5, as well as a C‐terminal basic residue (R134) to recruit the S5a subunit as part of the ubiquitin‐mediated proteolysis pathway.

Crystal structure of Escherichia coli EC1530, a glyoxylate induced protein YgbM

The crystal structure of YgbM (EC1530) (Fig. 1), a glyoxylate induced protein from Escherichia coli, has been determined and refined to 1.63 A by multiple-wavelength anomalous dispersion (MAD) method. YgbM is encoded by DNA bases 2862259–2863035 and belongs to a protein family of Pfam-B_7694.1 The gene is clustered with MutS (DNA mismatch repair protein), serine/threonine protein phosphatase, glycerol-3-phosphate regulon repressor, 3-hydroxyisobutyrate dehydrogenase, l-fuculose phosphate aldolase, gluconate permease, Rpos (RNA polymerase sigma factor), Nlpd (lipoprotein Nlpd), Pcm (protein-l-isoaspartate o-methyltransferase), SurE (stationary phase survival protein). Fig. 1 A: Protein sequences are compared between different species. B: The ribbon drawing shows the TIM structure of EC1530. α-helices, outside of the barrel, are shown in red, β-strands, inside of the barrel in cyan, and one of two Mg2+ (the ... The Se-Met derivative of YgbM crystallized in the C2 space group with unit cell dimensions of a = 104.907 A, b = 74.368 A, c = 39.376 A, β = 98.81°. There is one 258-residue protein per asymmetric unit. This structure adopts the common TIM (triosephosphate isomerase) barrel (β/α)8, in which an eight-membered cylindrical β-sheet is surrounded by eight helices.2 Similar to other TIM barrel structures, all of the turns between the α-helices and the subsequent β-strands at the N-terminal end of the barrel are composed of only three or four residues, whereas the corresponding loops at the C-terminal end are longer and form a part of the potential active site. Inside of the TIM barrel, several hydrophilic side-chains from the C-terminal loops as well as two well-ordered water molecules coordinate to a Mg2+, presumably forming an active site (Fig. 2). As expected, a Dali search3 found several structures with relatively high similarity, which include 4XIS, 1A0C-A, 1QUM-A, 1DE5, and 1BYB with Z scores higher than 10. Further biochemical and structural analyses are in progress. Fig. 2 Putative catalytic site including Mg2+ (major site) is shown. The Mg2+ is coordinated to an ordered water molecule, a formate, two glutamate, a glutamine, aspartate residues forming a square-bi-pyramid conformation. Materials and Methods Protein Cloning Expression and Purification. The ORF of ygbM was amplified by PCR from E. coli genomic DNA (ATCC). The gene was cloned into the NdeI and BamHI sites of a modified pET15b cloning vector (Novagen) in which the TEV protease cleavage site replaced the thrombin cleavage site and a double-stop codon was introduced downstream from the BamHI site. This construct provides for an N-terminal hexa-histidine tag separated from the gene by a TEV protease recognition site (ENLYFQ↓G). The fusion protein was overexpressed in E. coli BL21-Gold (DE3) (Stratagene) harboring an extra plasmid encoding three rare tRNAs (AGG and AGA for Arg, ATA for Ile). The cells were grown in LB at 37°C to an OD600 of approximately 0.6 and protein expression induced with 0.4 mM IPTG. After induction, the cells were incubated overnight with shaking at 15°C. The harvested cells were resuspended in binding buffer (500 mM NaCl, 5% Glycerol, 50 mM HEPES pH 7.5, 5 mM imidazole), flash-frozen in liquid N2, and stored at –70°C. The thawed cells were lysed by sonication after the addition of 0.5% NP-40 and 1 mM each of PMSF and benzamidine. The lysate was clarified by centrifugation (27000g for 30 min) and passed through a DE52 column preequilibrated in binding buffer. The flow-through fraction was then applied to a metal chelate affinity column charged with Ni2+. The hexa-histidine tag was eluted from the column in elution buffer (500 mM NaCl, 5% Glycerol, 50 mM HEPES pH 7.5, 500 mM imidazole), and the tag then cleaved from the protein by treatment with recombinant His-tagged TEV protease. The cleaved protein was then resolved from the cleaved His-tag and the His-tagged protease by flowing the mixture through a second Ni2+-column. The YgbM protein was dialyzed in 10 mM HEPES, pH 7.5, 500 mM NaCl, and concentrated by using a BioMax concentrator (Millipore). Before crystallization, any particulate matter was removed from the sample by passing through a 0.2-μm Ultrafree-MC centrifugal filter (Millipore). For the preparation of selenomethionine (SeMet) enriched protein, the E. coli YgbM was expressed in the methionine auxotroph strain B834(DE3) of E. coli (Novagen) and purified under the same conditions as the native protein in supplemented M9 media. The reducing reagent β-mercaptoethanol (5 mM) was added to all purification buffers.