Crystal structure of 2-enoyl-CoA hydratase 2 from human peroxisomal multifunctional enzyme type 2

2-Enoyl-CoA hydratase 2 is the middle part of the mammalian peroxisomal multifunctional enzyme type 2 (MFE-2), which is known to be important in the beta-oxidation of very-long-chain and alpha-methyl-branched fatty acids as well as in the synthesis of bile acids. Here, we present the crystal structure of the hydratase 2 from the human MFE-2 to 3A resolution. The three-dimensional structure resembles the recently solved crystal structure of hydratase 2 from the yeast, Candida tropicalis, MFE-2 having a two-domain subunit structure with a C-domain complete hot-dog fold housing the active site, and an N-domain incomplete hot-dog fold housing the cavity for the aliphatic acyl part of the substrate molecule. The ability of human hydratase 2 to utilize such bulky compounds which are not physiological substrates for the fungal ortholog, e.g. CoA esters of C26 fatty acids, pristanic acid and di/trihydroxycholestanoic acids, is explained by a large hydrophobic cavity formed upon the movements of the extremely mobile loops I-III in the N-domain. In the unliganded form of human hydratase 2, however, the loop I blocks the entrance of fatty enoyl-CoAs with chain-length >C8. Therefore, we expect that upon binding of substrates bulkier than C8, the loop I gives way, contemporaneously causing a secondary effect in the CoA-binding pocket and/or active site required for efficient hydration reaction. This structural feature would explain the inactivity of human hydratase 2 towards short-chain substrates. The solved structure is also used as a tool for analyzing the various inactivating mutations, identified among others in MFE-2-deficient patients. Since hydratase 2 is the last functional unit of mammalian MFE-2 whose structure has been solved, the organization of the functional units in the biologically active full-length enzyme is also discussed.     

Binary structure of the two-domain (3R)-hydroxyacyl-CoA dehydrogenase from rat peroxisomal multifunctional enzyme type 2 at 2.38 A resolution

The crystal structure of (3R)-hydroxyacyl-CoA dehydrogenase of rat peroxisomal multifunctional enzyme type 2 (MFE-2) was solved at 2.38 A resolution. The catalytic entity reveals an alpha/beta short chain alcohol dehydrogenase/reductase (SDR) fold and the conformation of the bound nicotinamide adenine dinucleotide (NAD(+)) found in other SDR enzymes. Of great interest is the separate COOH-terminal domain, which is not seen in other SDR structures. This domain completes the active site cavity of the neighboring monomer and extends dimeric interactions. Peroxisomal diseases that arise because of point mutations in the dehydrogenase-coding region of the MFE-2 gene can be mapped to changes in amino acids involved in NAD(+) binding and protein dimerization.     

Crystal structure of the flavohemoglobin from Alcaligenes eutrophus at 1.75 A resolution.

The molecular structure of the flavohemoglobin from Alcaligenes eutrophus has been determined to a resolution of 1.75 A and refined to an R-factor of 19.6%. The protein comprises two fused modules: a heme binding module, which belongs to the globin family, and an FAD binding oxidoreductase module, which adopts a fold like ferredoxin reductase. The most striking deviation of the bacterial globin structure from those of other species is the movement of helix E in a way to provide more space in the vicinity of the distal heme binding site. A comparison with other members of the ferredoxin reductase family shows similar tertiary structures for the individual FAD and NAD binding domains but largely different interdomain orientations. The heme and FAD molecules approach each other to a minimal distance of 6.3 A and adopt an interplanar angle of 80 degrees. The electron transfer from FAD to heme occurs in a predominantly polar environment and may occur directly or be mediated by a water molecule.     

Characterization of the promoter region of the human peroxisomal multifunctional enzyme type 2 gene

Peroxisomal multifunctional enzyme type 2 (perMFE-2) catalyzes conversion of (24E)-3alpha,7alpha, 12alpha-trihydroxy-5beta-cholest-24-enoyl-CoA to (24-keto)-3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoyl-CoA, which are physiological intermediates in cholic acid synthesis. In contrast to long chain fatty acid oxidizing enzymes clofibrate does not induce peroxisomal enzymes metabolizing bile acid intermediates. We proposed the existence of PPAR-independent regulation of cholesterol side chain oxidation in the process of bile acid synthesis. In the present study, we characterized the promoter region of the human perMFE-2 gene. The promoter contains the Sp1/AP2 binding site (-151/-142) within 197 base pairs upstream of the translation start site. Mutation of the Sp1/AP2 binding site decreases the promoter activity. Analysis by the luciferase assay revealed that the activity of the promoter region is strong in HepG2 and HeLa cell lines, although the activity in HepG2 cells was five- to sixfold higher than that in HeLa cells. Transient transfection assays have confirmed that AP2alpha and AP2gamma were able to transactivate the perMFE-2 promoter/luciferase chimeric gene. Cotransfections with Sp1 expression plasmid decreased the promoter activity. We suggest that perMFE-2 promoter activity is the result of both the abundance of AP2 and Sp1 family members and their relative ratios.     

Refined crystal structure of type III chloramphenicol acetyltransferase at 1.75 A resolution

High level bacterial resistance to chloramphenicol is generally due to O-acetylation of the antibiotic in a reaction catalysed by chloramphenicol acetyltransferase (CAT, EC 2.3.1.28) in which acetyl-coenzyme A is the acyl donor. The crystal structure of the type III enzyme from Escherichia coli with chloramphenicol bound has been determined and refined at 1.75 A resolution, using a restrained parameter reciprocal space least squares procedure. The refined model, which includes chloramphenicol, 204 solvent molecules and two cobalt ions has a crystallographic R-factor of 18.3% for 27,300 reflections between 6 and 1.75 A resolution. The root-mean-square deviation in bond lengths from ideal values is 0.02 A. The cobalt ions play a crucial role in stabilizing the packing of the molecule in the crystal lattice. CAT is a trimer of identical subunits (monomer Mr 25,000) and the trimeric structure is stabilized by a number of hydrogen bonds, some of which result in the extension of a beta-sheet across the subunit interface. Chloramphenicol binds in a deep pocket located at the boundary between adjacent subunits of the trimer, such that the majority of residues forming the binding pocket belong to one subunit while the catalytically essential histidine belongs to the adjacent subunit. His195 is appropriately positioned to act as a general base catalyst in the reaction, and the required tautomeric stabilization is provided by an unusual interaction with a main-chain carbonyl oxygen.     

Crystal structure of activin receptor type IIB kinase domain from human at 2.0 Angstrom resolution

Activin receptor type IIB (ActRIIB), a type II TGF-beta serine/threonine kinase receptor, is integral to the activin and myostatin signaling pathway. Ligands such as activin and myostatin bind to activin type II receptors (ActRIIA, ActRIIB), and the GS domains of type I receptors are phosphorylated by type II receptors. Myostatin, a negative regulator of skeletal muscle growth, is regarded as a potential therapeutic target and binds to ActRIIB effectively, and to a lesser extent, to ActRIIA. The high-resolution structure of human ActRIIB kinase domain in complex with adenine establishes the conserved bilobal architecture consistent with all other catalytic kinase domains. The crystal structure reveals that the adenine has a considerably different orientation from that of the adenine moiety of ATP observed in other kinase structures due to the lack of an interaction by ribose-phosphate moiety and the presence of tautomers with two different protonation states at the N9 nitrogen. Although the Lys217-Glu230 salt bridge is absent, the unphosphorylated activation loop of ActRIIB adopts a conformation similar to that of the fully active form. Unlike the type I TGF-beta receptor, where a partially conserved Ser280 is a gatekeeper residue, the AcRIIB structure possesses Thr265 with a back pocket supported by Phe247. Taken together, these structural features provide a molecular basis for understanding the coupled activity and recognition specificity for human ActRIIB kinase domain and for the rational design of selective inhibitors.     

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 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 muconate lactonizing enzyme at 3 A resolution

The crystal structure of muconate lactonizing enzyme has been solved at 3 A resolution, and an unambiguous alpha-carbon backbone chain trace made. The enzyme contains three domains; the central domain is a parallel-stranded alpha-beta barrel, which has previously been reported in six other enzymes, including triose phosphate isomerase and pyruvate kinase. One novel feature of this enzyme is that its alpha-beta barrel has only seven parallel alpha-helices around the central core of eight parallel beta-strands; all other known alpha-beta barrels contain eight such helices. The N-terminal (alpha + beta) and C-terminal domains cover the cleft where the eighth helix would be. The active site of muconate lactonizing enzyme has been found by locating the manganese ion that is essential for catalytic activity, and by binding and locating an inhibitor, alpha-ketoglutarate. The active site lies in a cleft between the N-terminal and barrel domains; when the active sites of muconate lactonizing enzyme and triose phosphate isomerase are superimposed, barrel-strand 1 of triose phosphate isomerase is aligned with barrel-strand 3 of muconate lactonizing enzyme. This implies that structurally homologous active-site residues in the two enzymes are carried on different parts of the primary sequence; the ancestral gene would had to have been transposed during its evolution to the modern proteins, which seems unlikely. Therefore, these two enzymes may be related by convergent, rather than divergent, evolution.     

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.     

Crystal structure of human serum albumin at 2.5 A resolution.

A new triclinic crystal form of human serum albumin (HSA), derived either from pool plasma (pHSA) or from a Pichia pastoris expression system (rHSA), was obtained from polyethylene glycol 4000 solution. Three-dimensional structures of pHSA and rHSA were determined at 2.5 A resolution from the new triclinic crystal form by molecular replacement, using atomic coordinates derived from a multiple isomorphous replacement work with a known tetragonal crystal form. The structures of pHSA and rHSA are virtually identical, with an r.m. s. deviation of 0.24 A for all Calpha atoms. The two HSA molecules involved in the asymmetric unit are related by a strict local twofold symmetry such that the Calpha atoms of the two molecules can be superimposed with an r.m.s. deviation of 0.28 A in pHSA. Cys34 is the only cysteine with a free sulfhydryl group which does not participate in a disulfide linkage with any external ligand. Domains II and III both have a pocket formed mostly of hydrophobic and positively charged residues and in which a very wide range of compounds may be accommodated. Three tentative binding sites for long-chain fatty acids, each with different surroundings, are located at the surface of each domain.     

Crystal structure of human annexin I at 2.5 A resolution.

cDNA coding for N-terminally truncated human annexin I, a member of the family of Ca(2+)-dependent phospholipid binding proteins, has been cloned and expressed in Escherichia coli. The expressed protein is biologically active, and has been purified and crystallized in space group P2(1)2(1)2(1) with cell dimensions a = 139.36 A, b = 67.50 A, and c = 42.11 A. The crystal structure has been determined by molecular replacement at 3.0 A resolution using the annexin V core structure as the search model. The average backbone deviation between these two structures is 2.34 A. The structure has been refined to an R-factor of 17.7% at 2.5 A resolution. Six calcium sites have been identified in the annexin I structure. Each is located in the loop region of the helix-loop-helix motif. Two of the six calcium sites in annexin I are not occupied in the annexin V structure. The superpositions of the corresponding loop regions in the four domains show that the calcium binding loops in annexin I can be divided into two classes: type II and type III. Both classes are different from the well-known EF-hand motif (type I).     

Crystal structure of the RIM1alpha C2B domain at 1.7 A resolution

RIM proteins play critical roles in synaptic vesicle priming and diverse forms of presynaptic plasticity. The C-terminal C2B domain is the only module that is common to all RIMs but is only distantly related to well-studied C2 domains, and its three-dimensional structure and interactions have not been characterized in detail. Using NMR spectroscopy, we now show that N- and C-terminal extensions beyond the predicted C2B domain core sequence are necessary to form a folded, stable RIM1alpha C2B domain. We also find that the isolated RIM1alpha C2B domain is not sufficient for previously described protein-protein interactions involving the RIM1alpha C-terminus, suggesting that additional sequences adjacent to the C2B domain might be required for these interactions. However, analytical ultracentrifugation shows that the RIM1alpha C2B domain forms weak dimers in solution. The crystal structure of the RIM1alpha C2B domain dimer at 1.7 A resolution reveals that it forms a beta-sandwich characteristic of C2 domains and that the unique N- and C-terminal extensions form a small subdomain that packs against the beta-sandwich and mediates dimerization. Our results provide a structural basis to understand the function of RIM C2B domains and suggest that dimerization may be a crucial aspect of RIM function.     

Three-dimensional structure/function analysis of SCP-2-like2 reveals differences among SCP-2 family members

Mosquito sterol carrier protein-2 (AeSCP-2) and sterol carrier protein-2-like2 (AeSCP-2L2) are members of the SCP-2 protein family with similar expression profiles in the mosquito life cycle. In an effort to understand how lipids can be transported by different SCP-2 proteins, the three-dimensional crystal structure of AeSCP-2L2 was solved at 1.7 A resolution. AeSCP-2L2 forms a dimer and binds three fatty acids, one of which resides in a position within the internal cavity at a right angle to the others. This first report of ligand-bound dimerized protein in the SCP-2 protein family indicates that the family has a much more divergent mode of interaction with ligands than previously reported. The potential function of AeSCP-2L2 was investigated via in vivo incorporation of [(3)H]cholesterol and [3H]palmitic acid. Overexpression of AeSCP-2L2 in mosquito cells leads to an increased uptake of free fatty acid, whereas knockdown of AeSCP-2L2 in adult females decreases the accumulation of free fatty acid in the fat body from a blood meal. In contrast, overexpression or knockdown of AeSCP-2L2 has no effect on cholesterol uptake. Our results suggest that the main function of AeSCP-2L2 is as a general intracellular fatty acid carrier, as opposed to having a dedicated role in cholesterol transport.     

Crystal structure of the FMN-binding domain of human cytochrome P450 reductase at 1.93 A resolution

The crystal structure of the FMN-binding domain of human NADPH-cytochrome P450 reductase (P450R-FMN), a key component in the cytochrome P450 monooxygenase system, has been determined to 1.93 A resolution and shown to be very similar both to the global fold in solution (Barsukov I et al., 1997, J Biomol NMR 10:63-75) and to the corresponding domain in the 2.6 A crystal structure of intact rat P450R (Wang M et al., 1997, Proc Nat Acad Sci USA 94:8411-8416). The crystal structure of P450R-FMN reported here confirms the overall similarity of its alpha-beta-alpha architecture to that of the bacterial flavodoxins, but reveals differences in the position, number, and length of the helices relative to the central beta-sheet. The marked similarity between P450R-FMN and flavodoxins in the interactions between the FMN and the protein, indicate a striking evolutionary conservation of the FMN binding site. The P450R-FMN molecule has an unusual surface charge distribution, leading to a very strong dipole, which may be involved in docking cytochrome P450 into place for electron transfer near the FMN. Several acidic residues near the FMN are identified by mutagenesis experiments to be important for electron transfer to P4502D6 and to cytochrome c, a clear indication of the part of the molecular surface that is likely to be involved in substrate binding. Somewhat different parts are found to be involved in binding cytochrome P450 and cytochrome c.     

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 a bacterial albumin-binding domain at 1.4 A resolution

The albumin-binding domain, or GA module, of the peptostreptococcal albumin-binding protein expressed in pathogenic strains of Finegoldia magna is believed to be responsible for the virulence and increased growth rate of these strains. Here we present the 1.4A crystal structure of this domain, and compare it with the crystal structure of the GA-albumin complex. An analysis of protein-protein interactions in the two crystals, and the presence of multimeric GA species in solution, indicate the GA module is "sticky", and is capable of forming contacts with a range of protein surfaces. This might lead to interactions with different host proteins.     

Crystal structure of human bone morphogenetic protein-2 at 2.7 A resolution

Homodimeric bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor beta (TGF-beta) superfamily that induces bone formation and regeneration, and determines important steps during early stages of embryonic development in vertebrates and non-vertebrates. BMP-2 can interact with two types of receptor chains, as well as with proteins of the extracellular matrix and several regulatory proteins. We report here the crystal structure of human BMP-2 determined by molecular replacement and refined to an R-value of 24.2 % at 2.7 A resolution. A common scaffold of BMP-2, BMP-7 and the TGF-betas, i.e. the cystine-knot motif and two finger-like double-stranded beta-sheets, can be superimposed with r. m.s. deviations of around 1 A. In contrast to the TGF-betas, the structure of BMP-2 shows differences in the flexibility of the N terminus and the orientation of the central alpha-helix as well as two external loops at the fingertips with respect to the scaffold. This is also known from the BMP-7 model. Small secondary structure elements in the loop regions of BMP-2 and BMP-7 seem to be specific for the respective BMP-subgroup. Two identical helix-finger clefts and two distinct cavities located around the central 2-fold axis of the dimer show characteristic shapes, polarity and surface charges. The possible function of these specific features in the interaction of BMP-2 with its binding partners is discussed.     

Crystal structure of a ribonuclease from the seeds of bitter gourd (Momordica charantia) at 1.75 A resolution.

The ribonuclease MC1 (RNase MC1) from seeds of bitter gourd (Momordica charantia) consists of 190 amino acid residues with four disulfide bridges and belongs to the RNase T(2) family, including fungal RNases typified by RNase Rh from Rhizopus niveus and RNase T(2) from Aspergillus oryzae. The crystal structure of RNase MC1 has been determined at 1.75 A resolution with an R-factor of 19.7% using the single isomorphous replacement method. RNase MC1 structurally belongs to the (alpha+beta) class of proteins, having ten helices (six alpha-helices and four 3(10)-helices) and eight beta-strands. When the structures of RNase MC1 and RNase Rh are superposed, the close agreement between the alpha-carbon positions for the total structure is obvious: the root mean square deviations calculated only for structurally related 151 alpha-carbon atoms of RNase MC1 and RNase Rh molecules was 1.76 A. Furthermore, the conformation of the catalytic residues His-46, Glu-105, and His-109 in RNase Rh can be easily superposed with that of the possible catalytic residues His-34, Glu-84, and His-88 in RNase MC1. This observation strongly indicates that RNase MC1 from a plant origin catalyzes RNA degradation in a similar manner as fungal RNases.