Structure of a microbial homologue of mammalian platelet-activating factor acetylhydrolases: Streptomyces exfoliatus lipase at 1.9 A resolution.

BACKGROUND: Neutral lipases are ubiquitous and diverse enzymes. The molecular architecture of the structurally characterized lipases is similar, often despite a lack of detectable homology at the sequence level. Some of the microbial lipases are evolutionarily related to physiologically important mammalian enzymes. For example, limited sequence similarities were recently noted for the Streptomyces exfoliatus lipase (SeL) and two mammalian platelet-activating factor acetylhydrolases (PAF-AHs). The determination of the crystal structure of SeL allowed us to explore the structure-function relationships in this novel family of homologous hydrolases. RESULTS: The crystal structure of SeL was determined by multiple isomorphous replacement and refined using data to 1.9 A resolution. The molecule exhibits the canonical tertiary fold of an alpha/beta hydrolase. The putative nucleophilic residue, Ser131, is located within a nucleophilic elbow and is hydrogen bonded to His209, which in turn interacts with Asp177. These three residues create a triad that closely resembles the catalytic triads found in the active sites of other neutral lipases. The mainchain amides of Met132 and Phe63 are perfectly positioned to create an oxyanion hole. Unexpectedly, there are no secondary structure elements that could render the active site inaccessible to solvent, like the lids that are commonly found in neutral lipases. CONCLUSIONS: The crystal structure of SeL reinforces the notion that it is a homologue of the mammalian PAF-AHs. We have used the catalytic triad in SeL to model the active site of the PAF-AHs. Our model is consistent with the site-directed mutagenesis studies of plasma PAF-AH, which implicate Ser273, His351 and Asp296 in the active site. Our study therefore provides direct support for the hypothesis that the plasma and isoform II PAF-AHs are triad-containing alpha/beta hydrolases.

The crystal and molecular structure of the Rhizomucor miehei triacylglyceride lipase at 1.9 A resolution.

The crystal and molecular structure of a triacylglyceride lipase (EC 3.1.1.3) from the fungus Rhizomucor miehei was analyzed using X-ray single crystal diffraction data to 1.9 A resolution. The structure was refined to an R-factor of 0.169 for all available data. The details of the molecular architecture and the crystal structure of the enzyme are described. A single polypeptide chain of 269 residues is folded into a rather unusual singly wound beta-sheet domain with predominantly parallel strands, connected by a variety of hairpins, loops and helical segments. All the loops are right-handed, creating an uncommon situation in which the central sheet is asymmetric in that all the connecting fragments are located on one side of the sheet. A single N-terminal alpha-helix provides the support for the other, distal, side of the sheet. Three disulfide bonds (residues 29-268, 40-43, 235-244) stabilize the molecule. There are four cis peptide bonds, all of which precede proline residues. In all, 230 ordered water molecules have been identified; 12 of them have a distinct internal character. The catalytic center of the enzyme is made up of a constellation of three residues (His257, Asp203 and Ser144) similar in structure and function to the analogous (but not homologous) triad found in both of the known families of serine proteinases. The fourth residue in this system equivalent to Thr/Ser in proteinases), hydrogen bonded to Asp, is Tyr260. The catalytic site is concealed under a short amphipatic helix (residues 85 to 91), which acts as "lid", opening the active site when the enzyme is adsorbed at the oil-water interface. In the native enzyme the "lid" is held in place by hydrophobic interactions.

(His)C epsilon-H...O=C < hydrogen bond in the active sites of serine hydrolases.

Close interactions of the C-H...O type are known to occur in a variety of organic crystals, although it had been often argued that they do not represent true hydrogen bonds. During an extensive comparative study of all structurally characterized serine hydrolases containing an Asp(Glu)-His-Ser catalytic triad at their active centers (i.e. serine proteinases, lipases, acetylcholinesterase and a thioesterase), we have discovered that the C epsilon 1 atom of the active site histidine is invariably in a close contact with a carbonyl oxygen. The stereochemistry of these contacts suggests a cohesive, predominantly electrostatic interaction, fully consistent with the requirements imposed by the generally accepted definition of a hydrogen bond. A study of a sample of protein structures refined at high resolution revealed that similar hydrogen bonds involving (His) C epsilon 1-H are found in approximately 15% of non-active site histidine residues. The ubiquitous occurrence of this hitherto underestimated contact in the active sites of serine hydrolases suggests functional significance. We propose that the (His)C epsilon 1-H...O=C bond affects the charge distribution within the imidazolium ion so as to weaken the N epsilon 2-H bond, thereby facilitating general acid catalysis by the active site histidine during both the acylation and deacylation steps of hydrolysis.

The occurrence of C-H...O hydrogen bonds in proteins.

Hydrogen bonds are a major feature of protein structure. By a generally accepted definition, they occur whenever a proton is shared by two electronegative atoms. Hence, only hydrogens bonded to nitrogen and oxygen atoms are usually considered in analyses of protein hydrogen bond networks. However, X-ray and neutron diffraction studies have shown that crystals of various organic compounds exhibit close C-H...X contacts (where X is an electronegative atom, in most cases oxygen) which show all the stereochemical hallmarks of hydrogen bonds. In this work, we describe an analysis of short C-H...O interactions in a sample of known protein structures representing different categories of tertiary folds and refined at a resolution of at least 2 A. Although our analysis is based on the calculated coordinates of hydrogen atoms, its results are statistically significant: we find strong evidence that a large percentage of short C...O contacts constitute cohesive interactions. Moreover, the stereochemical study of C-H...O = C contacts, in which the orientation of free electron orbitals on the acceptor oxygen atom can be predicted, reveals that these interactions exhibit stereochemical features typical of hydrogen bonds. Among the hydrogen atoms involved in these contacts, the most common are those bonded to alpha carbon. This is consistent with the fact that these hydrogens are more acidic than others. We describe four different categories of C-H...O = C bonds. Those found between C alpha-H groups and main chain oxygens in adjacent strands of beta sheets are the most ubiquitous. Our results call for a revision of crystallographic restrained refinement programs which treat close carbon-oxygen contacts as purely repulsive; they may also have implications for the understanding of some enzymatic reaction mechanisms.

X-ray analysis of the single chain B29-A1 peptide-linked insulin molecule. A completely inactive analogue.

A crystal structure of a totally inactive insulin molecule has been determined. For this insulin molecule, the first without detectable activity to be characterized, the A and B-chains are linked by a peptide bond between A1 Gly and B29 Lys. The molecule has retained all its normal self-association properties and it can also accommodate the two different conformations designated T and R, as seen in 4Zn native pig insulin crystals. The hexamers of the crosslinked insulin molecule were crystallized using the 4Zn insulin recipe of Schlichtkrull. The structure has been crystallographically refined with data extending to 2 A using restrained least-square methods. Comparison of the B29-A1 peptide crosslink insulin and the 4Zn native insulin reveals close structural similarities with the native dimer. The analysis of the structure confirms the earlier hypothesis that insulin structures in crystals are not in an active conformation and that a separation of N-terminal A-chain and C-terminal B-chain is required for interaction with the insulin receptor.

The crystal structure of a major dust mite allergen Der p 2, and its biological implications.

The crystal structure of the common house mite (Dermatophagoides sp.) Der p 2 allergen was solved at 2.15 A resolution using the MAD phasing technique, and refined to an R-factor of 0.209. The refined atomic model, which reveals an immunoglobulin-like tertiary fold, differs in important ways from the previously described NMR structure, because the two beta-sheets are significantly further apart and create an internal cavity, which is occupied by a hydrophobic ligand. This interaction is structurally reminiscent of the binding of a prenyl group by a regulatory protein, the Rho guanine nucleotide exchange inhibitor. The crystal structure suggests that binding of non-polar molecules may be essential to the physiological function of the Der p 2 protein.

Relationships among serine hydrolases: evidence for a common structural motif in triacylglyceride lipases and esterases.

A detailed analysis of the highly refined (1.9 A resolution) molecular model of the fungal (Rhizomucor miehei) triglyceride lipase reveals a unique conformation of the oligopeptide containing the active serine (Ser 144) residue. It consists of a six-residue beta-strand (strand 4 of the central sheet), a four-residue turn of type II' with serine in the epsilon conformation, and a buried alpha-helix packed in a parallel way against strands 4 and 5 of the central beta-pleated sheet. It is shown that the invariant glycines in positions (1) and (5) of the so-called lipase consensus sequence (G-X-S-X-G) are in extended and helical conformations, respectively, and that they are conserved owing to the steric restrictions imposed on these residues by the packing stereochemistry of this beta-epsilon Ser-alpha motif, and not by secondary structure requirements, as is the case in serine proteinases. Sequence homologies indicate that this unique motif is likely to be found in serine esterases and other lipases, indicating a possible evolutionary link of these families of hydrolytic enzymes.

The structure and function of platelet-activating factor acetylhydrolases.

Platelet-activating factor acetylhydrolases (PAF-AHs, EC 3.1.1.47) constitute a unique and biologically important family of phospholipase A2s. They are related to neither the well-characterized secretory nor cytosolic PLA2s, and unlike them do not require Ca2+ for catalytic activity. The distinguishing property of PAF-AHs is their unique substrate specificity: they act on the phospholipid platelet-activating factor (PAF), and in some cases on proinflammatory polar phospholipids, from which they remove a short acyl moiety--acetyl in the case of PAF--located at the sn-2 position. Because PAF is found both in the plasma and in the cytosol of many tissues, PAF-acetylhydrolases are equally widely distributed in an animal organism. Recent crystallographic studies shed new light on the complex structure-function relationships in PAF-AHs.

A novel variant of the catalytic triad in the Streptomyces scabies esterase.

The crystal structure of a novel esterase from Streptomyces scabies, a causal agent of the potato scab disease, was solved at 2.1 A resolution. The tertiary fold of the enzyme is substantially different from that of the alpha/beta hydrolase family and unique among all known hydrolases. The active site contains a dyad of Ser 14 and His 283, closely resembling two of the three components of typical Ser-His-Asp(Glu) triads from other serine hydrolases. Proper orientation of the active site imidazol is maintained by a hydrogen bond between the N delta-H group and a main chain oxygen. Thus, the enzyme constitutes the first known natural variation of the chymotrypsin-like triad in which a carboxylic acid is replaced by a neutral hydrogen-bond acceptor.

Molecular structure of insulin: the insulin monomer and its assembly.

The insulin molecule contains 51 amino acids; it is made up of two peptide chains linked by disulphide bonds. Although it is active as a monomer, during its biosynthesis and storage it assembles to dimers and, in the presence of zinc, to hexamers. X-ray analysis has revealed the 3-dimensional structure of the insulin molecule in its hexameric, dimeric and monomeric states. Two main conformations of insulin which differ in the extent of helix in the B chain (B9-B20 and B1-B20, respectively) have been identified. Other variations are seen in insulin when dimeric or monomeric. Reagents such as chloride and phenol govern the conformations present in the insulin hexamers and this can influence the behaviour and properties of insulin preparations employing them.

Crystal structure of the Escherichia coli thioesterase II, a homolog of the human Nef binding enzyme.

Here we report the solution and refinement at 1.9 A resolution of the crystal structure of the Escherichia coli medium chain length acyl-CoA thioesterase II. This enzyme is a close homolog of the human protein that interacts with the product of the HIV-1 Nef gene, sharing 45% amino acid sequence identity with it. The structure of the E. coli thioesterase II reveals a new tertiary fold, a 'double hot dog', showing an internal repeat with a basic unit that is structurally similar to the recently described beta-hydroxydecanoyl thiol ester dehydrase. The catalytic site, inferred from the crystal structure and verified by site directed mutagenesis, involves novel chemistry and includes Asp 204, Gln 278 and Thr 228, which synergistically activate a nucleophilic water molecule.

Catalysis at the interface: the anatomy of a conformational change in a triglyceride lipase.

The crystal structure of an extracellular triglyceride lipase (from a fungus Rhizomucor miehei) inhibited irreversibly by diethyl p-nitrophenyl phosphate (E600) was solved by X-ray crystallographic methods and refined to a resolution of 2.65 A. The crystals are isomorphous with those of n-hexylphosphonate ethyl ester/lipase complex [Brzozowski, A. M., Derewenda, U., Derewenda, Z. S., Dodson, G. G., Lawson, D. M., Turkenburg, J. P., Bjorkling, F., Huge-Jensen, B., Patkar, S. A., & Thim, L. (1991) Nature 351, 491-494], where the conformational change was originally observed. The higher resolution of the present study allowed for a detailed analysis of the stereochemistry of the change observed in the inhibited enzyme. The movement of a 15 amino acid long "lid" (residues 82-96) is a hinge-type rigid-body motion which transports some of the atoms of a short alpha-helix (residues 85-91) by over 12 A. There are two hinge regions (residues 83-84 and 91-95) within which pronounced transitions of secondary structure between alpha and beta conformations are caused by dramatic changes of specific conformational dihedral angles (phi and psi). As a result of this change a hydrophobic area of ca. 800 A2 (8% of the total molecule surface) becomes exposed. Other triglyceride lipases are also known to have "lids" similar to the one observed in the R. miehei enzyme, and it is possible that the general stereochemistry of lipase activation at the oil-water interfaces inferred from the present X-ray study is likely to apply to the entire family of lipases.

Expression, purification and crystallization of a BH domain from the GTPase regulatory protein associated with focal adhesion kinase.

Signaling by small GTPases is down-regulated by GTPase activating proteins (GAPs) which enhance the rate of GTP hydrolysis. The activity of GAPs specific for Rho GTPases resides in the BH domain, many homologues of which are found in any mammalian genome. One of them was identified in the GTPase regulator associated with focal-adhesion kinase (GRAF). It shares approximately 20% sequence identity with p50RhoGAP. This GAP activates RhoA and Cdc42Hs, but not Rac. In order to dissect the molecular basis of this specificity, a 231-residue-long fragment corresponding to the BH domain of GRAF has been expressed, purified and crystallized. Trigonal crystals, of space group P3(1)21 or P3(2)21, with unit-cell dimensions a = b = 63.5, c = 90.38 A were grown from solutions of PEG 6000. Data to 2.15 A were collected from a flash-frozen sample on an R-AXIS IV imaging-plate detector mounted on a rotating anode X-ray generator.

Crystal structure, at 2.6-A resolution, of the Streptomyces lividans xylanase A, a member of the F family of beta-1,4-D-glycanases.

The crystal structure of the 32-kDa catalytic domain of the Streptomyces lividans xylanase A was solved by molecular isomorphous replacement methods and subsequently refined at 2.6-A resolution to a conventional crystallographic R factor of 0.21. This is the first successful structure determination of a member of the F family of endo-beta-1,4-D-glycanases. Unlike the recently determined xylanases of the G family (Wakarchuk, W. W., Campbell, R. L., Sung, W. L., Davoodi, J., and Yaguchi, M. (1994) Protein Sci. 3, 467-475), where the catalytic domains have a unique beta-sheet structure, the 32-kDa domain of the S. lividans xylanase A is folded into a complete (alpha/beta)8 barrel, the first such fold observed among beta-1,4-D-glycanases. The active site is located at the carbonyl end of the beta barrel. The crystal structure supports the earlier assignment of Glu-128 and Glu-236 as the catalytic amino acids (Moreau, A., Roberge, M., Manin, C., Shareck, F., Kluepfel, D., and Morosoli, R. (1994) Biochem. J., in press).

Current progress in crystallographic studies of new lipases from filamentous fungi.

Lipases from filamentous fungi have been studied extensively over many years. They exhibit properties attractive for industrial applications, e.g. in laundry detergents, tanning and paper industries and stereospecific organic synthesis. Enzymes from the fungi Rhizomucor miehei and Geotrichum candidum have been among the first neutral lipases to be characterized structurally by X-ray diffraction methods. In this paper we report a preliminary account of crystallographic studies of three other fungal lipases homologous to that from R. miehei and obtained from Humicola lanuginosa, Penicillium camembertii and Rhizopus delemar. These newly characterized structures have important implications for our understanding of structure-function relationships in lipases in general and the molecular basis of interfacial activation.

Sequence polymorphisms and antibody binding to the group 2 dust mite allergens.

BACKGROUND: The group 2 allergens Der p 2, Der f 2 and Eur m 2 are 14-kD proteins with > 80% sequence identity. Isoforms within each genus have been identified which differ by 3-4 amino acids. The aim of this study was to investigate the importance of these substitutions to antibody binding. METHODS: Recombinant allergens were expressed and purified from Escherichia coli. ELISA and skin testing were used to evaluate antibody binding. Molecular modeling of the tertiary structure was preformed to examine the location of substitutions. RESULTS: The three Der f 2 isoforms and two of three of the Der p 2 isoforms reacted with all monoclonal antibodies (mAb). Der p 2.0101, the isoform with aspartate at position 114, bound all mAb except 1D8. Substitution of asparagine for aspartate restored binding of rDer p 2.0101 to mAb 1D8 and increased the correlation coefficient for IgE binding from 0.72 to 0.77. The three Der p 2 isoforms showed comparable skin test reactivity to nDer p 2 and commercial extract. rEur m 2.0101 bound to all mAb except 7A1 and when compared with rDer p 2 for IgE binding, r(2) = of 0.58 (n = 72). Lep d 2 did not react with mAb or with Dermatophagoides spp. allergic sera. Modeling revealed that Eur m 2, Lep d 2 and Tyr p 2 retain the tertiary fold of Der p 2 and the substitutions are on the surface. CONCLUSIONS: mAb could distinguish isoform substitutions. IgE binding showed a good correlation among all isoforms, thus the recombinant allergens are useful for diagnosis.

Homologs of the alpha- and beta-subunits of mammalian brain platelet-activating factor acetylhydrolase Ib in the Drosophila melanogaster genome.

The mammalian intracellular brain platelet-activating factor acetylhydrolase, implicated in the development of cerebral cortex, is a member of the phospholipase A2 superfamily. It is made up of a homodimer of the 45 kDa LIS1 protein (a product of the causative gene for type I lissencephaly) and a pair of homologous 26-kDa alpha-subunits which account for all the catalytic activity. LIS1 is hypothesized to regulate nuclear movement in migrating neurons through interactions with the cytoskeleton, while the alpha-subunits, whose structure is known, contain a trypsin-like triad within the framework of a unique tertiary fold. The physiological significance of the association of the two types of subunits is not known. In an effort to better understand the function of the complex we turned to genomic data mining in search of related proteins in lower eukaryotes. We found that the Drosophila melanogaster genome contains homologs of both alpha- and beta-subunits, and we cloned both genes. The alpha-subunit homolog has been overexpressed, purified and crystallized. It lacks two of the three active-site residues and, consequently, is catalytically inactive against PAF-AH (Ib) substrates. Our study shows that the beta-subunit homolog is highly conserved from Drosophila to mammals and is able to interact with the mammalian alpha-subunits but is unable to interact with the Drosophila alpha-subunit. Proteins 2000;39:1-8.

Structure of the BH domain from graf and its implications for Rho GTPase recognition.

Cellular signaling by small G-proteins is down-regulated by GTPase-activating proteins (GAPs), which increase the rate of GTP hydrolysis. The GTPase regulator associated with focal adhesion kinase (Graf) exhibits GAP activity toward the RhoA and Cdc42 GTPases, but is only weakly active toward the closely related Rac1. We determined the crystal structure of a 231-residue fragment of Graf (GrafGAP), a domain containing the GAP activity, at 2.4-A resolution. The structure clarifies the boundaries of the functional domain and yields insight to the mechanism of substrate recognition. Modeling its interaction with substrate suggested that a favorable interaction with Glu-95 of Cdc42 (Glu-97 of RhoA) would be absent with the corresponding Ala-95 of Rac1. Indeed, GrafGAP activity is diminished approximately 40-fold toward a Cdc42 E95A mutant, whereas a approximately 10-fold increase is observed for a Rac1 A95E mutant. The GrafGAP epitope that apparently interacts with Glu-95(Glu-97) contains Asn-225, which was recently found mutated in some myeloid leukemia patients. We conclude that position 95 of the GTPase is an important determinant for GrafGAP specificity in cellular function and tumor suppression.

The consequences of engineering an extra disulfide bond in the Penicillium camembertii mono- and diglyceride specific lipase.

The extracellular lipase from Penicillium camembertii has unique substrate specificity restricted to mono- and diglycerides. The enzyme is a member of a homologous family of lipases from filamentous fungi. Four of these proteins, from the fungi Rhizomucor miehei, Humicola lanuginosa, Rhizopus delemar and P. camembertii, have had their structures elucidated by X-ray crystallography. In spite of pronounced sequence similarities the enzymes exhibit significant differences. For example, the thermostability of the P. camembertii lipase is considerably lower than that of the H. lanuginosa enzyme. Since only the P. camembertii enzyme lacks the characteristic long disulfide bridge, corresponding to Cys22-Cys268 in the H. lanuginosa lipase, we have engineered this disulfide into the former enzyme in the hope of obtaining a significantly more stable fold. The properties of the double mutant (Y22C and G269C) were assessed by a variety of biophysical techniques. The extra disulfide link was found to increase the melting temperature of the protein from 51 to 63 degrees C. However, no difference is observed under reducing conditions, indicating an intrinsic instability of the new disulfide. The optimal temperature for catalytic activity decreased by 10 degrees C and the optimum pH was shifted by 0.7 units to more acidic.

Structure of a myristoyl-ACP-specific thioesterase from Vibrio harveyi.

The crystal structure of a myristoyl acyl carrier protein specific thioesterase (C14ACP-TE) from a bioluminescent bacterium, Vibrio harveyi, was solved by multiple isomorphous replacement methods and refined to an R factor of 22% at 2.1-A resolution. This is the first elucidation of a three-dimensional structure of a thioesterase. The overall tertiary architecture of the enzyme resembles closely the consensus fold of the rapidly expanding superfamily of alpha/beta hydrolases, although there is no detectable homology with any of its members at the amino acid sequence level. Particularly striking similarity exists between the C14ACP-TE structure and that of haloalkane dehalogenase from Xanthobacter autotrophicus. Contrary to the conclusions of earlier studies [Ferri, S. R., & Meighen, E. A. (1991) J. Biol. Chem. 266, 12852-12857] which implicated Ser77 in catalysis, the crystal structure of C14ACP-TE reveals a lipase-like catalytic triad made up of Ser114, His241, and Asp211. Surprisingly, the gamma-turn with Ser114 in a strained secondary conformation (phi = 53 degrees, psi = -127 degrees), characteristic of the so-called nucleophilic elbow, does not conform to the frequently invoked lipase/esterase consensus sequence (Gly-X-Ser-X-Gly), as the positions of both glycines are occupied by larger amino acids. Site-directed mutagenesis and radioactive labeling support the catalytic function of Ser114. Crystallographic analysis of the Ser77-->Gly mutant at 2.5-A resolution revealed no structural changes; in both cases the loop containing the residue in position 77 is disordered.(ABSTRACT TRUNCATED AT 250 WORDS)