Crystal structure of the complex between 4-hydroxybutyrate CoA-transferase from Clostridium aminobutyricum and CoA.

Clostridium aminobutyricum ferments 4-aminobutyrate (γ-aminobutyrate, GABA) to ammonia, acetate and butyrate via 4-hydroxybutyrate that is activated to the CoA-thioester catalyzed by 4-hydroxybutyrate CoA-transferase. Then, 4-hydroxybutyryl-CoA is dehydrated to crotonyl-CoA, which disproportionates to butyryl-CoA and acetyl-CoA. Cocrystallization of the CoA-transferase with the alternate substrate butyryl-CoA yielded crystals with non-covalently bound CoA and two water molecules at the active site. Most likely, butyryl-CoA reacted with the active site Glu238 to CoA and the mixed anhydride, which slowly hydrolyzed during crystallization. The structure of the CoA is similar but less stretched than that of the CoA-moiety of the covalent enzyme-CoA-thioester in 4-hydroxybutyrate CoA-transferase from Shewanella oneidensis. In contrast to the structures of the apo-enzyme and enzyme-CoA-thioester, the structure described here has a closed conformation, probably caused by a flip of the active site loop (residues 215-219). During turnover, the closed conformation may protect the anhydride intermediate from hydrolysis and CoA from dissociation from the enzyme. Hence, one catalytic cycle changes conformation of the enzyme four times: free enzyme-open conformation, CoA+ anhydride 1-closed, enzyme-CoA-thioester-open, CoA + anhydride-2-closed, free enzyme-open.

Contribution of globular death domains and unstructured linkers to MyD88.IRAK-4 heterodimer formation: an explanation for the antagonistic activity of MyD88s.

Homotypic interactions of death domains (DD) mediate complex formation between MyD88 and IL-1 receptor-associated kinases (IRAKs). A truncated splice variant of MyD88, MyD88s, cannot recruit IRAK-4 and fails to elicit inflammatory responses. We have generated recombinant DD of MyD88 and IRAK-4, both alone and extended by the linkers to TIR or kinase domains. We show that both MyD88 DD variants bind to the linker-extended IRAK-4 DD and pull-down full-length IRAK-4 from monocyte extracts. By contrast, residues up to Glu(116) from the DD-kinase connector of IRAK-4 are needed for strong interactions with the adaptor. Our findings indicate that residues 110-120, which form a C-terminal extra helix in MyD88, but not the irregular linker between DD and TIR domains, are required for IRAK-4 recruitment, and provide a straightforward explanation for the negative regulation of innate immune responses mediated by MyD88s.

Crystal structure of 4-hydroxybutyrate CoA-transferase from Clostridium aminobutyricum.

4-Hydroxybutyrate CoA-transferases (4-HB-CoAT) takes part in the fermentation of 4-aminobutyrate to ammonia, acetate, and butyrate in anaerobic bacteria such as Clostridium aminobutyricum and Porphyromonas gingivalis or facultative anaerobic bacteria such as Shewanella oneidensis. Site-directed mutagenesis of the highly active enzyme has identified the catalytic glutamate residue as E238. Crystal structure of this enzyme has been determined at a resolution of 1.85 A. The 438-amino acid residue polypeptide chain folds into two topologically similar domains with an open alpha/beta-fold, which is also found in other CoAT family I and family II members. The data indicate that the members of CoAT families I and II are closely related; the latter only lacking the catalytic glutamate residue. A putative co-substrate binding site for the 4-HB-CoAT was identified, in which a 4-hydroxybutyrate molecule has been modeled. This site is also responsible for binding the acetyl group of acetyl-CoA or the succinyl group of succinyl-CoA in succinyl-CoA:3-oxoacid CoA-transferase from mammalian mitochondria. Mutations of relevant active site amino acid residues have been produced and their activities tested to corroborate the proposed structural model for substrate binding. 4-HB-CoAT from C. aminobutyricum represents the only functionally characterized 4-HB-CoAT present in the structural database.

Functional definition of the tobacco protoporphyrinogen IX oxidase substrate-binding site.

PPO (protoporphyrinogen IX oxidase) catalyses the flavin-dependent six-electron oxidation of protogen (protoporphyrinogen IX) to form proto (protoporphyrin IX), a crucial step in haem and chlorophyll biosynthesis. The apparent K(m) value for wild-type tobacco PPO2 (mitochondrial PPO) was 1.17 muM, with a V(max) of 4.27 muM.min(-1).mg(-1) and a catalytic activity k(cat) of 6.0 s(-1). Amino acid residues that appear important for substrate binding in a crystal structure-based model of the substrate docked in the active site were interrogated by site-directed mutagenesis. PPO2 variant F392H did not reveal detectable enzyme activity indicating an important role of Phe(392) in substrate ring A stacking. Mutations of Leu(356), Leu(372) and Arg(98) increased k(cat) values up to 100-fold, indicating that the native residues are not essential for establishing an orientation of the substrate conductive to catalysis. Increased K(m) values of these PPO2 variants from 2- to 100-fold suggest that these residues are involved in, but not essential to, substrate binding via rings B and C. Moreover, one prominent structural constellation of human PPO causing the disease variegate porphyria (N67W/S374D) was successfully transferred into the tobacco PPO2 background. Therefore tobacco PPO2 represents a useful model system for the understanding of the structure-function relationship underlying detrimental human enzyme defects.

Crystal structures of human tissue kallikrein 4: activity modulation by a specific zinc binding site.

Human tissue kallikrein 4 (hK4) belongs to a 15-member family of closely related serine proteinases. hK4 is predominantly expressed in prostate, activates hK3/PSA, and is up-regulated in prostate and ovarian cancer. We have identified active monomers of recombinant hK4 besides inactive oligomers in solution. hK4 crystallised in the presence of zinc, nickel, and cobalt ions in three crystal forms containing cyclic tetramers and octamers. These structures display a novel metal site between His25 and Glu77 that links the 70-80 loop with the N-terminal segment. Micromolar zinc as present in prostatic fluid inhibits the enzymatic activity of hK4 against fluorogenic substrates. In our measurements, wild-type hK4 exhibited a zinc inhibition constant (IC50) of 16 microM including a permanent residual activity, in contrast to the zinc-independent mutants H25A and E77A. Since the Ile16 N terminus of wild-type hK4 becomes more accessible for acetylating agents in the presence of zinc, we propose that zinc affects the hK4 active site via the salt-bridge formed between the N terminus and Asp194 required for a functional active site. hK4 possesses an unusual 99-loop that creates a groove-like acidic S2 subsite. These findings explain the observed specificity of hK4 for the P1 to P4 substrate residues. Moreover, hK4 shows a negatively charged surface patch, which may represent an exosite for prime-side substrate recognition.

Crystal structure of the catalytic domain of human atypical protein kinase C-iota reveals interaction mode of phosphorylation site in turn motif.

Atypical protein kinases C (aPKCs) play critical roles in signaling pathways that control cell growth, differentiation and survival. Therefore, they constitute attractive targets for the development of novel therapeutics against cancer. The crystal structure of the catalytic domain of atypical PKCiota in complex with the bis(indolyl)maleimide inhibitor BIM1 has been determined at 3.0A resolution within the frame of the European Structural Proteomics Project SPINE. The overall structure exhibits the classical bilobal kinase fold and is in its fully activated form. Both phosphorylation sites (Thr403 in the activation loop, and Thr555 in the turn motif) are well defined in the structure and form intramolecular ionic contacts that make an important contribution in stabilizing the active conformation of the catalytic subunit. The phosphorylation site in the hydrophobic motif of atypical PKCs is replaced by the phosphorylation mimic glutamate and this is also clearly seen in the structure of PKCiota (residue 574). This structure determination for the first time provides the architecture of the turn motif phosphorylation site, which is characteristic for PKCs and PKB/AKT, and is completely different from that in PKA. The bound BIM1 inhibitor blocks the ATP-binding site and puts the kinase domain into an intermediate open conformation. The PKCiota-BIM1 complex is the first kinase domain crystal structure of any atypical PKC and constitutes the basis for rational drug design for selective PKCiota inhibitors.

Structural basis for stereo-specific catalysis in NAD(+)-dependent (R)-2-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans.

NAD(+)-dependent (R)-2-hydroxyglutarate dehydrogenase (HGDH) catalyses the reduction of 2-oxoglutarate to (R)-2-hydroxyglutarate and belongs to the d-2-hydroxyacid NAD(+)-dependent dehydrogenase (d-2-hydroxyacid dehydrogenase) protein family. Its crystal structure was determined by phase combination to 1.98 A resolution. Structure-function relationships obtained by the comparison of HGDH with other members of the d-2-hydroxyacid dehydrogenase family give a chemically satisfying view of the substrate stereoselectivity and catalytic requirements for the hydride transfer reaction. A model for substrate recognition and turnover is discussed. The HGDH active site architecture is structurally optimized to recognize and bind the negatively charged substrate 2-oxoglutarate. The structural position of the side chain of Arg52, and its counterparts in other family members, strongly correlates with substrate specificity towards substitutions at the C3 atom (linear or branched substrates). Arg235 interacts with the substrate's alpha-carboxylate and carbonyl groups, having a dual role in both substrate binding and activation, and the gamma-carboxylate group can dock at an arginine cluster. The proton-relay system built up by Glu264 and His297 permits His297 to act as acid-base catalyst and the 4Re-hydrogen from NADH is transferred as hydride to the carbonyl group Si-face leading to the formation of the correct enantiomer (R)-2-hydroxyglutarate.

Crystallization and preliminary X-ray analysis of the tungsten-dependent acetylene hydratase from Pelobacter acetylenicus.

Acetylene hydratase is a tungsten-containing hydroxylase that converts acetylene to acetaldehyde in a unique reaction that requires a strong reductant. The subsequent disproportionation of acetaldehyde yields acetate and ethanol. Crystals of the tungsten/iron-sulfur protein acetylene hydratase from Pelobacter acetylenicus strain WoAcy 1 (DSM 3246) were grown by the vapour-diffusion method in an N2/H2 atmosphere using polyethylene glycol as precipitant. Growth of crystals suitable for X-ray analysis strictly depended on the presence of Ti(III) citrate or dithionite as reducing agents.

Crystal structure and putative mechanism of 3-methylitaconate-delta-isomerase from Eubacterium barkeri.

3-Methylitaconate-Delta-isomerase (Mii) participates in the nicotinate fermentation pathway of the anaerobic soil bacterium Eubacterium barkeri (order Clostridiales) by catalyzing the reversible conversion of (R)-3-methylitaconate (2-methylene-3-methylsuccinate) to 2,3-dimethylmaleate. The enzyme is also able to catalyze the isomerization of itaconate (methylenesuccinate) to citraconate (methylmaleate) with ca 10-fold higher K(m) but > 1000-fold lower k(cat). The gene mii from E. barkeri was cloned and expressed in Escherichia coli. The protein produced with a C-terminal Strep-tag exhibited the same specific activity as the wild-type enzyme. The crystal structure of Mii from E. barkeri has been solved at a resolution of 2.70 A. The asymmetric unit of the P2(1)2(1)2(1) unit cell with parameters a = 53.1 A, b = 142.3 A, and c = 228.4 A contains four molecules of Mii. The enzyme belongs to a group of isomerases with a common structural feature, the so-called diaminopimelate epimerase fold. The monomer of 380 amino acid residues has two topologically similar domains exhibiting an alpha/beta-fold. The active site is situated in a cleft between these domains. The four Mii molecules are arranged as a tetramer with 222 symmetry for the N-terminal domains. The C-terminal domains have different relative positions with respect to the N-terminal domains resulting in a closed conformation for molecule A and two distinct open conformations for molecules B and D. The C-terminal domain of molecule C is disordered. The Mii active site contains the putative catalytic residues Lys62 and Cys96, for which mechanistic roles are proposed based on a docking experiment of the Mii substrate complex. The active sites of Mii and the closely related PrpF, most likely a methylaconitate Delta-isomerase, have been compared. The overall architecture including the active-site Lys62, Cys96, His300, and Ser17 (Mii numbering) is similar. This positioning of (R)-3-methylitaconate allows Cys96 (as thiolate) to deprotonate C-3 and (as thiol) to donate a proton to the methylene carbon atom of the resulting allylic carbanion. Interestingly, the active site of isopentenyl diphosphate isomerase type I also contains a cysteine that cooperates with glutamate rather than lysine. It has been proposed that the initial step in this enzyme is a protonation generating a tertiary carbocation intermediate.

Crystal structure of a trapped phosphate intermediate in vanadium apochloroperoxidase catalyzing a dephosphorylation reaction.

The crystal structure of the apo form of vanadium chloroperoxidase from Curvularia inaequalis reacted with para-nitrophenylphosphate was determined at a resolution of 1.5 A. The aim of this study was to solve structural details of the dephosphorylation reaction catalyzed by this enzyme. Since the chloroperoxidase is functionally and evolutionary related to several acid phosphatases including human glucose-6-phosphatase and a group of membrane-bound lipid phosphatases, the structure sheds light on the details of the dephosphorylation catalyzed by these enzymes as well. The trapped intermediate found is bound to the active site as a metaphosphate anion PO3-, with its phosphorus atom covalently attached to the Nepsilon2 atom of His496. An apical water molecule is within hydrogen-bonding distance to the phosphorus atom of the metaphosphate, and it is in a perfect position for a nucleophilic attack on the metaphosphate-histidine intermediate to form the inorganic phosphate. This is, to our knowledge, the first structural characterization of a real reaction intermediate of the inorganic phosphate group release in a dephosphorylation reaction.

Binding and reduction of sulfite by cytochrome c nitrite reductase.

Pentaheme cytochrome c nitrite reductase (ccNiR) catalyzes the six-electron reduction of nitrite to ammonia as the final step in the dissimilatory pathway of nitrate ammonification. It has also been shown to reduce sulfite to sulfide, thus forming the only known link between the biogeochemical cycles of nitrogen and of sulfur. We have found the sulfite reductase activity of ccNiR from Wolinella succinogenes to be significantly smaller than its nitrite reductase activity but still several times higher than the one described for dissimilatory, siroheme-containing sulfite reductases. To compare the sulfite reductase activity of ccNiR with our previous data on nitrite reduction, we determined the binding mode of sulfite to the catalytic heme center of ccNiR from W. succinogenes at a resolution of 1.7 A. Sulfite and nitrite both provide a pair of electrons to form the coordinative bond to the Fe(III) active site of the enzyme, and the oxygen atoms of sulfite are found to interact with the three active site protein residues conserved within the enzyme family. Furthermore, we have characterized the active site variant Y218F of ccNiR that exhibited an almost complete loss of nitrite reductase activity, while sulfite reduction remained unaffected. These data provide a first direct insight into the role of the first sphere of protein ligands at the active site in ccNiR catalysis.

Phosphorylation of the human full-length protein kinase Ciota.

Atypical protein kinases C, including protein kinase Ciota (PKCiota), play critical roles in signaling pathways that control cell growth, differentiation and survival. This qualifies them as attractive targets for development of novel therapeutics for the treatment of various human diseases. In this study, the full-length PKCiota was expressed in Sf9 insect cells, purified, and digested with trypsin and endoproteinase Asp-N, and its phosphorylation analyzed by liquid chromatography-high accuracy mass spectrometry. This strategy mapped 97% of the PKCiota protein sequence and revealed seven new Ser/Thr phosphorylation sites, in addition to the two previously known, pThr403 in the activation loop and pThr555 in the turn motif of the kinase domain. Most of the newly identified phosphorylation sites had low estimated occupancies (below 2%). Two phosphorylation sites were located in domain connecting amino acid sequence stretches (pSer217 and pSer237/pSer238) and may contribute to an improved stability and solubility of the protein. The most interesting new phosphorylation site was detected in a well-accessible loop of the PB1 domain (pSer35/pSer37) and may be involved in the interactions of the PB1 domain with different partners in the relevant signaling pathways.

Influence of loop shortening on the metal binding site of cupredoxin pseudoazurin.

Atomic resolution structures of the pseudoazurin (PAZ) variant into which the shorter ligand-containing loop of amicyanin (AMI) is introduced have been determined. The mutated loop adopts a different conformation in PAZAMI than in AMI. The copper site structure is affected, with the major influence being an increased axial interaction resulting in the shortest Cu(II)-S(Met) bond observed for the cupredoxin family of electron-transfer proteins. This is accompanied by a lengthening of the important Cu-S(Cys) bond and enhanced tetragonal distortion, consistent with the influence of the PAZAMI loop contraction on the UV/vis spectrum. The change in active site geometry is the major cause of the 50 mV decrease in reduction potential. The copper site structure changes very little upon reduction, consistent with the distorted site still possessing the properties required to facilitate rapid electron transfer. The exposed His ligand on the loop protonates in the reduced protein and reasons for the increased pKa compared to that of PAZ are discussed. The area surrounding the His ligand is more hydrophobic in PAZAMI than in PAZ, while electron self-exchange, which involves homodimer formation via this surface patch, is decreased. The nature of the side chains in this region, as dictated by the sequence of the ligand-containing loop, is a more significant factor than hydrophobicity for facilitating transient protein interactions in PAZ. The structure of PAZAMI demonstrates the importance of loop-scaffold interactions for metal sites in proteins.

Structure of the non-redox-active tungsten/[4Fe:4S] enzyme acetylene hydratase.

The tungsten-iron-sulfur enzyme acetylene hydratase stands out from its class because it catalyzes a nonredox reaction, the hydration of acetylene to acetaldehyde. Sequence comparisons group the protein into the dimethyl sulfoxide reductase family, and it contains a bis-molybdopterin guanine dinucleotide-ligated tungsten atom and a cubane-type [4Fe:4S] cluster. The crystal structure of acetylene hydratase at 1.26 A now shows that the tungsten center binds a water molecule that is activated by an adjacent aspartate residue, enabling it to attack acetylene bound in a distinct, hydrophobic pocket. This mechanism requires a strong shift of pK(a) of the aspartate, caused by a nearby low-potential [4Fe:4S] cluster. To access this previously unrecognized W-Asp active site, the protein evolved a new substrate channel distant from where it is found in other molybdenum and tungsten enzymes.

Basic requirements for a metal-binding site in a protein: the influence of loop shortening on the cupredoxin azurin.

The main active-site loop of the copper-binding protein azurin (a cupredoxin) has been shortened from C(112)TFPGH(117)SALM(121) to C(112)TPH(115)PFM(118) (the native loop from the cupredoxin amicyanin) and also to C(112)TPH(115)PM(117). The Cu(II) site structure is almost unaffected by shortening, as is that of the Cu(I) center at alkaline pH in the variant with the C(112)TPH(115)PM(117) loop sequence. Subtle spectroscopic differences due to alterations in the spin density distribution at the Cu(II) site can be attributed mainly to changes in the hydrogen-bonding pattern. Electron transfer is almost unaffected by the introduction of the C(112)TPH(115)PFM(118) loop, but removal of the Phe residue has a sizable effect on reactivity, probably because of diminished homodimer formation. At mildly acidic pH values, the His-115 ligand protonates and dissociates from the cuprous ion, an effect that has a dramatic influence on the reactivity of cupredoxins. These studies demonstrate that the amicyanin loop adopts a conformation identical to that found in the native protein when introduced into azurin, that a shorter than naturally occurring C-terminal active-site loop can support a functional T1 copper site, that CTPHPM is the minimal loop length required for binding this ubiquitous electron transfer center, and that the length and sequence of a metal-binding loop regulates a range of structural and functional features of the active site of a metalloprotein.

SPINE high-throughput crystallization, crystal imaging and recognition techniques: current state, performance analysis, new technologies and future aspects.

This paper reviews the developments in high-throughput and nanolitre-scale protein crystallography technologies within the remit of workpackage 4 of the Structural Proteomics In Europe (SPINE) project since the project's inception in October 2002. By surveying the uptake, use and experience of new technologies by SPINE partners across Europe, a picture emerges of highly successful adoption of novel working methods revolutionizing this area of structural biology. Finally, a forward view is taken of how crystallization methodologies may develop in the future.

Radical-mediated dehydration reactions in anaerobic bacteria.

Most dehydratases catalyse the elimination of water from beta-hydroxy ketones, beta-hydroxy carboxylic acids or beta-hydroxyacyl-CoA. The electron-withdrawing carbonyl functionalities acidify the alpha-hydrogens to enable their removal by basic amino acid side chains. Anaerobic bacteria, however, ferment amino acids via alpha- or gamma-hydroxyacyl-CoA, dehydrations of which involve the abstraction of a beta-hydrogen, which is ostensibly non-acidic (pK ca. 40). Evidence is accumulating that beta-hydrogens are acidified via transient conversion of the CoA derivatives to enoxy radicals by one-electron transfers, which decrease the pK to 14. The dehydrations of (R)-2-hydroxyacyl-CoA to (E)-2-enoyl-CoA are catalysed by heterodimeric [4Fe-4S]-containing dehydratases, which require reductive activation by an ATP-dependent one-electron transfer mediated by a homodimeric protein with a [4Fe-4S] cluster between the two subunits. The electron is further transferred to the substrate, yielding a ketyl radical anion, which expels the hydroxyl group and forms an enoxy radical. The dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA involves a similar mechanism, in which the ketyl radical anion is generated by one-electron oxidation. The structure of the FAD- and [4Fe-4S]-containing homotetrameric dehydratase is related to that of acyl-CoA dehydrogenases, suggesting a radical-based mechanism for both flavoproteins.

Novel bacterial molybdenum and tungsten enzymes: three-dimensional structure, spectroscopy, and reaction mechanism.

The molybdenum enzymes 4-hydroxybenzoyl-CoA reductase and pyrogallol-phloroglucinol transhydroxylase and the tungsten enzyme acetylene hydratase catalyze reductive dehydroxylation reactions, i.e., transhydroxylation between phenolic residues and the addition of water to a triple bond. Such activities are unusual for this class of enzymes, which carry either a mononuclear Mo or W center. Crystallization and subsequent structural analysis by high-resolution X-ray crystallography has helped to resolve the reaction centers of these enzymes to a degree that allows us to understand the interaction of the enzyme and the respective substrate(s) in detail, and to develop a concept for the respective reaction mechanism, at least in two cases.

Crystal structures of oxidized and reduced stellacyanin from horseradish roots.

Umecyanin (UMC) is a type 1 copper-containing protein which originates from horseradish roots and belongs to the stellacyanin subclass of the phytocyanins, a ubiquitous family of plant cupredoxins. The crystal structures of Cu(II) and Cu(I) UMC have been determined at 1.9 and 1.8 A, respectively. The protein has an overall fold similar to those of other phytocyanins. At the active site the cupric ion is coordinated by the N(delta1) atoms of His44 and His90, the S(gamma) of Cys85, and the O(epsilon)(1) of Gln95 in a distorted tetrahedral geometry. Both His ligands are solvent exposed and are surrounded by nonpolar and polar side chains on the protein surface. Thus, UMC does not possess a distinct hydrophobic patch close to the active site in contrast to almost all other cupredoxins. UMC has a large surface acidic patch situated approximately 10-30 A from the active site. The structure of Cu(I) UMC is the first determined for a reduced phytocyanin and demonstrates that the coordination environment of the cuprous ion is more trigonal pyramidal. This subtle change in geometry is primarily due to the Cu-N(delta1)(His44) and Cu-O(epsilon1)(Gln95) bond lengths increasing from 2.0 and 2.3 A in Cu(II) UMC to 2.2 and 2.5 A, respectively, in the reduced form, as a consequence of slight rotations of the His44 and Gln95 side chains. The limited structural changes upon redox interconversion at the active site of this stellacyanin are analogous to those observed in a typical type 1 copper site with an axial Met ligand and along with its surface features suggest a role for UMC in interprotein electron transfer.

Crystal structure of 4-hydroxybutyryl-CoA dehydratase: radical catalysis involving a [4Fe-4S] cluster and flavin.

Dehydratases catalyze the breakage of a carbon-oxygen bond leading to unsaturated products via the elimination of water. The 1.6-A resolution crystal structure of 4-hydroxybutyryl-CoA dehydratase from the gamma-aminobutyrate-fermenting Clostridium aminobutyricum represents a new class of dehydratases with an unprecedented active site architecture. A [4Fe-4S](2+) cluster, coordinated by three cysteine and one histidine residues, is located 7 A from the Re-side of a flavin adenine dinucleotide (FAD) moiety. The structure provides insight into the function of these ubiquitous prosthetic groups in the chemically nonfacile, radical-mediated dehydration of 4-hydroxybutyryl-CoA. The substrate can be bound between the [4Fe-4S](2+) cluster and the FAD with both cofactors contributing to its radical activation and catalytic conversion. Our results raise interesting questions regarding the mechanism of acyl-CoA dehydrogenases, which are involved in fatty acid oxidation, and address the divergent evolution of the ancestral common gene.