Crystal structure of muconate lactonizing enzyme at 6.5 A resolution

We have obtained crystals of Pseudomonas putida muconate lactonizing enzyme. They diffract to better than 2.4 A resolution and have two monomers in the asymmetric unit, related by a non-crystallographic 2-fold axis. The cell dimensions are 139.3 A X 139.3 A X 84.1 A, and the space group is I4. The electron density map at 6.5 A resolution shows that the enzyme is an octamer with D4 symmetry.     

Crystal structure of a deubiquitinating enzyme (human UCH-L3) at 1.8 A resolution.

Ubiquitin C-terminal hydrolases catalyze the removal of adducts from the C-terminus of ubiquitin. We have determined the crystal structure of the recombinant human Ubiquitin C-terminal Hydrolase (UCH-L3) by X-ray crystallography at 1.8 A resolution. The structure is comprised of a central antiparallel beta-sheet flanked on both sides by alpha-helices. The beta-sheet and one of the helices resemble the well-known papain-like cysteine proteases, with the greatest similarity to cathepsin B. This similarity includes the UCH-L3 active site catalytic triad of Cys95, His169 and Asp184, and the oxyanion hole residue Gln89. Papain and UCH-L3 differ, however, in strand and helix connectivity, which in the UCH-L3 structure includes a disordered 20 residue loop (residues 147-166) that is positioned over the active site and may function in the definition of substrate specificity. Based upon analogy with inhibitor complexes of the papain-like enzymes, we propose a model describing the binding of ubiquitin to UCH-L3. The UCH-L3 active site cleft appears to be masked in the unliganded structure by two different segments of the enzyme (residues 9-12 and 90-94), thus implying a conformational change upon substrate binding and suggesting a mechanism to limit non-specific hydrolysis.     

QM/MM study of the reaction mechanism of Cl-cis,cis-muconate with muconate lactonizing enzyme

The lactonization process of Cl-cis,cis-muconate catalyzed by anti-muconate lactonizing enzyme (anti-MLE) was studied theoretically with the aid of a combined quantum mechanics/molecular mechanics (QM/MM) approach. Two elementary processes steps involved in the lactanization process were investigated. The calculated energy barriers agree well with the experimental values. The present work provided the explicit structures of the enolate anion intermediates. The electrostatic influence analysis highlighted residues Arg51, Gln294 and TIP383 for the MLE-Cl-2 system and the residue Asn193 for the MLE-Cl-4 system as the possible mutation targets for rational design of anti-MLE in future enzyme modification.     

Crystal structure of thermitase at 1.4 A resolution

The crystal structure of thermitase, a subtilisin-type serine proteinase from Thermoactinomyces vulgaris, was determined by X-ray diffraction at 1.4 A resolution. The structure was solved by a combination of molecular and isomorphous replacement. The starting model was that of subtilisin BPN' from the Protein Data Bank, determined at 2.5 A resolution. The high-resolution refinement was based on data collected using synchrotron radiation with a Fuji image plate as detector. The model of thermitase refined to a conventional R factor of 14.9% and contains 1997 protein atoms, 182 water molecules and two Ca ions. The tertiary structure of thermitase is similar to that of the other subtilisins although there are some significant differences in detail. Comparison with subtilisin BPN' revealed two major structural differences. The N-terminal region in thermitase, which is absent in subtilisin BPN', forms a number of contacts with the tight Ca2+ binding site and indeed provides the very tight binding of the Ca ion. In thermitase the loop of residues 60 to 65 forms an additional (10) beta-strand of the central beta-sheet and the second Ca2+ binding site that has no equivalent in the subtilisin BPN' structure. The observed differences in the Ca2+ binding and the increased number of ionic and aromatic interactions in thermitase are likely sources of the enhanced stability of thermitase.     

Crystal structure of lactoperoxidase at 2.4 A resolution

Lactoperoxidase (LPO) is a member of the mammalian peroxidase superfamily. It catalyzes the oxidation of thiocyanate and halides. Freshly isolated and purified samples of caprine LPO were saturated with ammonium iodide and crystallized using 20% polyethylene glycol 3350 in a hanging drop vapor diffusion setup. The structure has been determined using X-ray crystallographic method and refined to R_cryst and R_free factors of 0.196 and 0.203, respectively. The structure determination revealed an unexpected phosphorylation of Ser198 in LPO, which is also confirmed by anti-phosphoserine antibody binding studies. The structure is also notable for observing densities for glycan chains at all the four potential glycosylation sites. Caprine LPO consists of a single polypeptide chain of 595 amino acid residues and folds into an oval-shaped structure. The structure contains 20 well-defined α-helices of varying lengths including a helix, H_2a, unique to LPO, and two short antiparallel β-strands. The structure confirms that the heme group is covalently linked to the protein through two ester linkages involving carboxylic groups of Glu258 and Asp108 and modified methyl groups of pyrrole rings A and C, respectively. The heme moiety is slightly distorted from planarity, but pyrrole ring B is distorted considerably. However, an iron atom is displaced only by 0.1 Å from the plane of the heme group toward the proximal site. The substrate diffusing channel in LPO is cylindrical in shape with a diameter of approximately 6 Å. Two histidine residues and six buried water molecules are connected through a hydrogen-bonded chain from the distal heme cavity to the surface of protein molecule and seemingly form the basis of proton relay for catalytic action. Ten iodide ions have been observed in the structure. Out of these, only one iodide ion is located in the distal heme cavity and is hydrogen bonded to the water molecule W1. W1 is also hydrogen bonded to the heme iron as well as to distal His109. The structure contains a calcium ion that is coordinated to seven oxygen atoms and forms a typical pentagonal bipyramidal coordination geometry.     

Crystal structure of Escherichia coli UDPMurNAc-tripeptide d-alanyl-d-alanine-adding enzyme (MurF) at 2.3 A resolution

MurF is required to catalyze the final step in the synthesis of the cytoplasmic precursor of the bacterial cell wall peptidoglycan, rendering it an attractive target for antibacterial drug development. The crystal structure of the MurF apo-enzyme has been determined using the multiwavelength anomalous dispersion method and refined to 2.3 A resolution. It contains three consecutive open alpha/beta-sheet domains. In comparison with the complex crystal structures of MurD and its substrates, The topology of the N-terminal domain of MurF is unique, while its central and C-terminal domains exhibit similar mononucleotide and dinucleotide-binding folds, respectively. The apo-enzyme of MurF crystal structure reveals an open conformation with the three domains juxtaposed in a crescent-like arrangement creating a wide-open space where substrates are expected to bind. As such, catalysis is not feasible and significant domain closure is expected upon substrate binding.     

Crystal structure of human alpha-lactalbumin at 1.7 A resolution

The three-dimensional X-ray structure of human alpha-lactalbumin, an important component of milk, has been determined at 1.7 A (0.17 nm) resolution by the method of molecular replacement, using the refined structure of baboon alpha-lactalbumin as the model structure. The two proteins are known to have more than 90% amino acid sequence identity and crystallize in the same orthorhombic space group, P2(1)2(1)2. The crystallographic refinement of the structure using the simulated annealing method, resulted in a crystallographic R-factor of 0.209 for the 11,373 observed reflections (F greater than or equal to 2 sigma (F)) between 8 and 1.7 A resolution. The model comprises 983 protein atoms, 90 solvent atoms and a bound calcium ion. In the final model, the root-mean-square deviations from ideality are 0.013 A for covalent bond distances and 2.9 degrees for bond angles. Superposition of the human and baboon alpha-lactalbumin structures yields a root-mean-square difference of 0.67 A for the 123 structurally equivalent C alpha atoms. The C terminus is flexible in the human alpha-lactalbumin molecule. The striking structural resemblance between alpha-lactalbumins and C-type lysozymes emphasizes the homologous evolutionary relationship between these two classes of proteins.     

Crystal structure of muconolactone isomerase at 3.3 A resolution

The crystal structure of muconolactone isomerase from Pseudomonas putida, a unique molecule with ten 96 amino acid subunits and 5-fold, and 2-fold symmetries, has been solved at 3.3 A resolution. The non-crystallographic symmetries were used to refine the initial single isomorphous replacement phases and produce an interpretable 10-fold averaged map. The backbone trace is complete and confirmed by the amino acid sequence fit. Each subunit is composed of a body with two alpha-helices and an antiparallel twisted beta-sheet of four strands, and an extended arm. The helices and the sheet fold to form a two-layered structure with an enclosed hydrophobic core and a partially formed putative active site pocket. The C-terminal arm of another subunit related by a local dyad symmetry extends over the core to complete this pocket. The decameric protein is almost spherical, with the helices forming the external coat. There is a large hydrophilic cavity in the center with open ends along the 5-fold axis. Molecular interactions between subunits are extensive. Each subunit contacts four neighbors and loses nearly 40% of its solvent contact area on oligomerization.     

Crystal structure of thioltransferase at 2.2 A resolution.

We report here the first three-dimensional structure of a mammalian thioltransferase as determined by single crystal X-ray crystallography at 2.2 A resolution. The protein is known for its thiol-redox properties and dehydroascorbate reductase activity. Recombinant pig liver thioltransferase expressed in Escherichia coli was crystallized in its oxidized form by vapor diffusion technique. The structure was determined by multiple isomorphous replacement method using four heavy-atom derivatives. The protein folds into an alpha/beta structure with a four-stranded mixed beta-sheet in the core, flanked on either side by helices. The fold is similar to that found in other thiol-redox proteins, viz. E. coli thioredoxin and bacteriophage T4 glutaredoxin, and thus seems to be conserved in these functionally related proteins. The active site disulfide (Cys 22-Cys 25) is located on a protrusion on the molecular surface. Cys 22, which is known to have an abnormally low pKa of 3.8, is accessible from the exterior of the molecule. Pro 70, which is in close proximity to the disulfide bridge, assumes a conserved cis-peptide configuration. Mutational data available on the protein are in agreement with the three-dimensional structure.     

Crystal structure of uncleaved ovalbumin at 1.95 A resolution

Ovalbumin, the major protein in avian egg-white, is a non-inhibitory member of the serine protease inhibitor (serpin) superfamily. The crystal structure of uncleaved, hen ovalbumin was solved by the molecular replacement method using the structure of plakalbumin, a proteolytically cleaved form of ovalbumin, as a starting model. The final refined model, including four ovalbumin molecules, 678 water molecules and a single metal ion, has a crystallographic R-factor of 17.4% for all reflections between 6.0 and 1.95 A resolution. The root-mean-square deviation from ideal values in bond lengths is 0.02 A and in bond angles is 2.9 degrees. This is the first crystal structure of a member of the serpin family in an uncleaved form. Surprisingly, the peptide that is homologous to the reactive centre of inhibitory serpins adopts an alpha-helical conformation. The implications for the mechanism of inhibition of the inhibitory members of the family is discussed.     

Crystal structure of Rab geranylgeranyltransferase at 2.0 A resolution

BACKGROUND: Rab geranylgeranyltransferase (RabGGT) catalyzes the addition of two geranylgeranyl groups to the C-terminal cysteine residues of Rab proteins, which is crucial for membrane association and function of these proteins in intracellular vesicular trafficking. Unlike protein farnesyltransferase (FT) and type I geranylgeranyltransferase, which both prenylate monomeric small G proteins or short peptides, RabGGT can prenylate Rab only when Rab is in a complex with Rab escort protein (REP). RESULTS: The crystal structure of rat RabGGT at 2.0 A resolution reveals an assembly of four distinct structural modules. The beta subunit forms an alpha-alpha barrel that contains most of the residues in the active site. The alpha subunit consists of a helical domain, an immunoglobulin (Ig)-like domain, and a leucine-rich repeat (LRR) domain. The N-terminal region of the alpha subunit binds to the active site in the beta subunit; residue His2alpha directly coordinates a zinc ion. The prenyl-binding pocket of RabGGT is deeper than that in FT. CONCLUSIONS: LRR and Ig domains are often involved in protein-protein interactions; in RabGGT they might participate in the recognition and binding of REP. The binding of the N-terminal peptide of the alpha subunit to the active site suggests an autoinhibition mechanism that might contribute to the inability of RabGGT to recognize short peptides or Rab alone as its substrate. Replacement of residues Trp102beta and Tyr154beta in FT by Ser48beta and Leu99beta, respectively, in RabGGT largely determine the different lipid-binding specificities of the two enzymes.     

Crystal structure of glucose dehydrogenase from Bacillus megaterium IWG3 at 1.7 A resolution

The crystal structure of glucose dehydrogenase (GlcDH) from Bacillus megaterium IWG3 has been determined to an R-factor of 17.9% at 1.7 A resolution. The enzyme consists of four identical subunits, which are similar to those of other short-chain reductases/dehydrogenases (SDRs) in their overall folding and subunit architecture, although cofactor binding sites and subunit interactions differ. Whereas a pair of basic residues is well conserved among NADP(+)-preferring SDRs, only Arg39 was found around the adenine ribose moiety of GlcDH. This suggests that one basic amino acid is enough to determine the coenzyme specificity. The four subunits are interrelated by three mutually perpendicular diad axes (P, Q, and R). While subunit interactions through the P-axis for GlcDH are not so different from those of the other SDRs, those through the Q-axis differ significantly. GlcDH was found to have weaker hydrophobic interactions in the Q-interface. Moreover, GlcDH lacks the salt bridge that stabilizes the subunit interaction in the Q-interface in the other SDRs. Hydrogen bonds between Q-axis related subunits are also less common than in the other SDRs. The GlcDH tetramer dissociates into inactive monomers at pH 9.0, which can be attributed mainly to the weakness of the Q-axis interface.     

Crystal structure of haemoglobin from donkey (Equus asinus) at 3A resolution

Haemoglobin from donkey was purified and crystallized in space group C2. The present donkey haemoglobin model comprises of two subunits alpha and beta. These alpha and beta subunits comprise of 141 and 146 amino acid residues, respectively, and the haem groups. The donkey haemoglobin differs from horse only in two amino acids of alpha-chain (His20 to Asn and Tyr24 to Phe) and these substitutions do not significantly change the secondary structural features of donkey haemoglobin. The haem group region and subunit contacts are closely resemble with that of horse methaemoglobin.     

Crystal structure of brain-type creatine kinase at 1.41 A resolution

Excitable cells and tissues like muscle or brain show a highly fluctuating consumption of ATP, which is efficiently regenerated from a large pool of phosphocreatine by the enzyme creatine kinase (CK). The enzyme exists in tissue--as well as compartment-specific isoforms. Numerous pathologies are related to the CK system: CK is found to be overexpressed in a wide range of solid tumors, whereas functional impairment of CK leads to a deterioration in energy metabolism, which is phenotypic for many neurodegenerative and age-related diseases. The crystal structure of chicken cytosolic brain-type creatine kinase (BB-CK) has been solved to 1.41 A resolution by molecular replacement. It represents the most accurately determined structure in the family of guanidino kinases. Except for the N-terminal region (2-12), the structures of both monomers in the biological dimer are very similar and closely resemble those of the other known structures in the family. Specific Ca2+-mediated interactions, found between two dimers in the asymmetric unit, result in structurally independent heterodimers differing in their N-terminal conformation and secondary structure. The high-resolution structure of BB-CK presented in this work will assist in designing new experiments to reveal the molecular basis of the multiple isoform-specific properties of CK, especially regarding different subcellular locations and functional interactions with other proteins. The rather similar fold shared by all known guanidino kinase structures suggests a model for the transition state complex of BB-CK analogous to the one of arginine kinase (AK). Accordingly, we have modeled a putative conformation of CK in the transition state that requires a rigid body movement of the entire N-terminal domain by rms 4 A from the structure without substrates.     

Crystal structure of native chicken fibrinogen at 2.7 A resolution

The crystal structure of native chicken fibrinogen (320 kDa) complexed with two synthetic peptides has been determined at a resolution of 2.7 A. The structure provides the first atomic-resolution view of the polypeptide chain arrangement in the central domain where the two halves of the molecule are joined, as well as of a putative thrombin-binding site. The amino-terminal segments of the alpha and beta chains, including fibrinopeptides A and B, are not visible in electron density maps, however, and must be highly disordered. The alphaC domain is also very disordered. A residue by residue analysis of the coiled coils with regard to temperature factor shows a strong correlation between mobility and plasmin attack sites. It is concluded that structural flexibility is an inherent feature of fibrinogen that plays a key role in both its conversion to fibrin and its subsequent destruction by plasmin.     

Crystal structure of human coactosin-like protein at 1.9 A resolution

Human coactosin-like protein (CLP) shares high homology with coactosin, a filamentous (F)-actin binding protein, and interacts with 5LO and F-actin. As a tumor antigen, CLP is overexpressed in tumor tissue cells or cell lines, and the encoded epitopes can be recognized by cellular and humoral immune systems. To gain a better understanding of its various functions and interactions with related proteins, the crystal structure of CLP expressed in Escherichia coli has been determined to 1.9 A resolution. The structure features a central beta-sheet surrounded by helices, with two very tight hydrophobic cores on each side of the sheet. CLP belongs to the actin depolymerizing protein superfamily, and is similar to yeast cofilin and actophilin. Based on our structural analysis, we observed that CLP forms a polymer along the crystallographic b axis with the exact same repeat distance as F-actin. A model for the CLP polymer and F-actin binding has therefore been proposed.     

Crystal structure of spinach plastocyanin at 1.7 A resolution.

The crystal structure of plastocyanin from spinach has been determined using molecular replacement, with the structure of plastocyanin from poplar as a search model. Successful crystallization was facilitated by site-directed mutagenesis in which residue Gly8 was substituted with Asp. The region around residue 8 was believed to be too mobile for the wild-type protein to form crystals despite extensive screening. The current structure represents the oxidized plastocyanin, copper (II), at low pH (approximately 4.4). In contrast to the similarity in the core region as compared to its poplar counterpart, the structure shows some significant differences in loop regions. The most notable is the large shift of the 59-61 loop where the largest shift is 3.0 A for the C(alpha) atom of Glu59. This results in different patterns of electrostatic potential around the acidic patches for the two proteins.     

Crystal structure of transhydrogenase domain III at 1.2 A resolution

The nicotinamide nucleotide transhydrogenases (TH) of mitochondria and bacteria are membrane-intercalated proton pumps that transduce substrate binding energy and protonmotive force via protein conformational changes. In mitochondria, TH utilizes protonmotive force to promote direct hydride ion transfer from NADH to NADP, which are bound at the distinct extramembranous domains I and III, respectively. Domain II is the membrane-intercalated domain and contains the enzyme's proton channel. This paper describes the crystal structure of the NADP(H) binding domain III of bovine TH at 1.2 A resolution. The structure reveals that NADP is bound in a manner inverted from that previously observed for nucleotide binding folds. The non-classical binding mode exposes the NADP(H) nicotinamide ring for direct contact with NAD(H) in domain I, in accord with biochemical data. The surface of domain III surrounding the exposed nicotinamide is comprised of conserved residues presumed to form the interface with domain I during hydride ion transfer. Further, an adjacent region contains a number of acidic residues, forming a surface with negative electrostatic potential which may interact with extramembranous loops of domain II. Together, the distinctive surface features allow mechanistic considerations regarding the NADP(H)-promoted conformation changes that are involved in the interactions of domain III with domains I and II for hydride ion transfer and proton translocation.     

Crystal structure of eosinophil cationic protein at 2.4 A resolution

Eosinophil cationic protein (ECP) is located in the matrix of the eosinophil's large specific granule and has marked toxicity for a variety of helminth parasites, hemoflagellates, bacteria, single-stranded RNA virus, and mammalian cells and tissues. It belongs to the bovine pancreatic ribonuclease A (RNase A) family and exhibits ribonucleolytic activity which is about 100-fold lower than that of a related eosinophil ribonuclease, the eosinophil-derived neurotoxin (EDN). The crystal structure of human ECP, determined at 2.4 A, is similar to that of RNase A and EDN. It reveals that residues Gln-14, His-15, Lys-38, Thr-42, and His-128 at the active site are conserved as in all other RNase A homologues. Nevertheless, evidence for considerable divergence of ECP is also implicit in the structure. Amino acid residues Arg-7, Trp-10, Asn-39, His-64, and His-82 appear to play a key part in the substrate specificity and low catalytic activity of ECP. The structure also shows how the cationic residues are distributed on the surface of the ECP molecule that may have implications for an understanding of the cytotoxicity of this enzyme.