Advances in structural understanding of lipases.
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Amino Acid Sequence↗
Biomedical subjects
M Cygler
Publications and source records attributed to M Cygler.
The alpha/beta hydrolase fold.
We have identified a new protein fold--the alpha/beta hydrolase fold--that is common to several hydrolytic enzymes of widely differing phylogenetic origin and catalytic function. The core of each enzyme is similar: an alpha/beta sheet, not barrel, of eight beta-sheets connected by alpha-helices. These enzymes have diverged from a common ancestor so as to preserve the arrangement of the catalytic residues, not the binding site. They all have a catalytic triad, the elements of which are borne on loops which are the best-conserved structural features in the fold. Only the histidine in the nucleophile-histidine-acid catalytic triad is completely conserved, with the nucleophile and acid loops accommodating more than one type of amino acid. The unique topological and sequence arrangement of the triad residues produces a catalytic triad which is, in a sense, a mirror-image of the serine protease catalytic triad. There are now four groups of enzymes which contain catalytic triads and which are related by convergent evolution towards a stable, useful active site: the eukaryotic serine proteases, the cysteine proteases, subtilisins and the alpha/beta hydrolase fold enzymes.
Acetylcholinesterase↗
Multiple crystal forms of lipases from Geotrichum candidum.
Multiple stable crystal forms of two lipases from the fungus Geotrichum candidum have been obtained. The diffraction pattern extends to beyond 2.0 A resolution. Similarity of the cell dimensions of various forms suggested similar packing of molecules in different crystals. This was confirmed by rotation function results. Four heavy-atoms derivatives have been identified.
Crystallization↗
Recognition of a cell-surface oligosaccharide of pathogenic Salmonella by an antibody Fab fragment.
The 2.05 angstrom (A) resolution crystal structure of a dodecasaccharide-Fab complex revealed an unusual carbohydrate recognition site, defined by aromatic amino acids and a structured water molecule, rather than the carboxylic acid and amide side chains and a structured water molecule, rather than the carboxylic acid and amide side chains that are features of transport and other carbohydrate binding proteins. A trisaccharide epitope of a branched bacterial lipopolysaccharide fills this hydrophobic pocket (8 A deep by 7 A wide) in an entropy-assisted association (association constant = 2.05 x 10(5) liters per mole, enthalpy = -20.5 +/- 1.7 kilojoules per mole, and temperature times entropy = +10.0 +/- 2.9 kilojoules per mole). The requirement for the complementarity of van der Waals surfaces and the requirements of saccharide-saccharide and protein-saccharide hydrogen-bonding networks determine the antigen conformation adopted in the bound state.
Amino Acid Sequence↗
Ser-His-Glu triad forms the catalytic site of the lipase from Geotrichum candidum.
The Ser-His-Asp triad is a well known structural feature of the serine proteases. It has also been directly observed in the catalytic sites of two lipases, whose high-resolution three-dimensional structures have been determined 1,2. Lipases show a wide variety of sizes, substrate and positional specificities, and catalytic rates 3. They achieve maximal catalytic rates at oil-water interfaces. The fungus Geotrichum candidum produces several different forms of lipases, two of which have been purified to homogeneity 4,5. Two lipase genes have been identified, cloned and sequenced 6,7. Both code for proteins of 544 amino acids with a total relative molecular mass of about 60,000 (Mr 60K). The two forms are 86% identical. Their isoelectric points differ slightly, being between 4.3 and 4.6. About 7% of the total Mr is carbohydrate. Until now, only a low resolution structure of GCL has been reported 8, but no high resolution structure has followed. We now report the three-dimensional structure of a lipase from G. candidum (GCL) at 2.2 A resolution. Unlike the other lipases and serine proteases, the catalytic triad of GCL is Ser-His-Glu, with glutamic acid replacing the usual aspartate. Although the sequence similarity with the other two lipases is limited to the region near the active-site serine, there is some similarity in their three-dimensional structures. The GCL is also an alpha/beta protein with a central mixed beta sheet whose topology is similar to that of the N-terminal domain of human pancreatic lipase. As in the other lipases 1,2, the catalytic site is buried under surface loops. Sequence comparisons with proteins from the cholinesterase family suggest that they also contain the Ser-His-Glu triad.
Amino Acid Sequence↗
Preliminary crystal structure analysis of an Fab specific for a Salmonella O-polysaccharide antigen.
The Fab from an IgG1, lambda murine monoclonal antibody with specificity for the O-polysaccharide antigen of Salmonella typhimurium has been crystallized in the absence and presence of hapten. The conditions for crystal growth were vapor diffusion equilibration with 16 to 23% polyethylene glycol 8000 solutions. Data have been collected from crystals of the complex in space group P212121, a = 60.6 A, b = 111.3 A, c = 61.1 A, and refinement of a molecular replacement solution is underway.
Animals↗
Preliminary studies on the crystallization of beef heart cytochrome c oxidase by vapor diffusion.
Beef heart cytochrome c oxidase has been crystallized in its native and delipidated forms, using the hanging drop vapor diffusion method. The delipidated oxidase, which is subunit III deficient, can be crystallized in rosettes using this method from a medium containing zinc chloride as well as ammonium sulfate. The crystals give a protein 'powder' pattern on preliminary X-ray analysis. The crystals initially grow rapidly but stop growing at a small size probably as a result of addition of heterogeneous material at the crystal surface.
Animals↗
A full-symmetry translation function based on electron density.
A method for positioning an oriented fragment within the unit cell is presented. It is based on a correlation between a model and observed data which is performed in Fourier rather than Patterson space. Symmetry-related molecules are located in the electron density map calculated in space group P1, with the phases derived from a model that is correctly oriented but arbitrarily positioned in the unit cell. It is shown that considering all symmetry elements simultaneously substantially increases the sensitivity of the method and makes it less susceptible to the errors in the model. The procedure also automatically incorporates a penalty for the overlap of symmetry-related molecules, and the stringency of this requirement is easily modified. The method has been tested on two different proteins and the results compare favorably with other translation functions.
Crystallography↗
Sindbis virus core protein crystals.
The core protein of Sindbis virus has been crystallized. Three different crystal forms have been observed. They diffract variously from 2.5 A to 3.5 A resolution.
Crystallization↗
Application of the molecular replacement method to multidomain proteins. 2. Comparison of various methods for positioning an oriented fragment in the unit cell.
The capabilities of several different methods to determine the correct translation of a model for the application of the molecular replacement method of structure determination to multidomain proteins have been analyzed. The structure of the Fab fragment of the autoimmune anti-poly(dT)-specific antibody HED10 was determined using molecular replacement and provides an example for comparing different methods of determining the correct translation of the model and for evaluating the importance of the parameters used. Expansion to space group P1 and phasing with a correctly oriented randomly positioned model was found to be superior to either the Crowther-Blow translation function [Crowther & Blow (1967). Acta Cryst. 23, 544-548] or a brute-force search when only a small part of the molecule was used as a model.
Antibodies↗
Application of the molecular replacement method to multidomain proteins. 1. Determination of the orientation of an immunoglobulin Fab fragment.
Multidomain proteins provide special problems in the application of the molecular replacement method of structure determination. The structure of the Fab fragment from the autoimmune poly(dT)-specific antibody HED10 has been determined using molecular replacement. An analysis of the effects of varying the model and the parameters used in the rotation function indicates that dividing the molecule into individual relatively rigid domains simplifies interpretation of the results, and that the optimal parameters depend on the molecule under study.
Antibodies↗
Crystallization and structure determination of an autoimmune anti-poly(dT) immunoglobulin Fab fragment at 3.0 A resolution.
HED10 is an autoimmune antibody (IgG) which shows considerable specificity for the single-stranded DNA poly(dT). Production of Fab fragments by papain digestion resulted in heterogeneity as judged by isoelectric focusing gels, which had a marked negative effect on crystallization. However, a single species of Fab with a pI of 7.6 could be isolated in good yield by DEAE-cellulose chromatography, and good crystals were produced by the hanging drop vapor diffusion method. The space group was P21 with cell dimensions, a = 64.2, b = 90.0, c = 42.3 A, and beta = 96.7 degrees. These crystals diffract to about 2.2 A resolution. The structure of Fab HED10 was solved by the molecular replacement method using the known structure of McPC603 and is refined to R = 27.2% at 3.0 A resolution. Fab HED10 is more extended than McPC603 and has an elbow angle (between the variable and constant domains) of 162 degrees, very similar to that observed in Fab KOL. The majority of the hypervariable regions are visible in the model.
Animals↗
Prediction of 3-D structure of the Cro protein from phage 434.
A comparative model building process has been utilized to predict the three-dimensional structure of the bacteriophage 434 Cro protein. Amino acid sequence similarities between the 434 Cro protein and other bacteriophage repressor and Cro proteins have been used, in conjunction with secondary structure prediction and the known structures of other base sequence specific DNA binding proteins, to derive the model. From this model the interactions between the 434 Cro protein and its operator DNA have been deduced. These proposed interactions are consistent with the known properties of the bacteriophage 434 Cro protein.
Amino Acid Sequence↗
Computer modeling studies of the structure of a repressor.
Due to advances in molecular biology the DNA sequences of structural genes coding for proteins are often known before a protein is characterized or even isolated. The function of a protein whose amino acid sequence has been deduced from a DNA sequence may not even be known. This has created greater interest in the development of methods to predict the tertiary structures of proteins. The a priori prediction of a protein's structure from its amino acid sequence is not yet possible. However, since proteins with similar amino acid sequences are observed to have similar three-dimensional structures, it is possible to use an analogy with a protein of known structure to draw some conclusions about the structure and properties of an uncharacterized protein. The process of predicting the tertiary structure of a protein relies very much upon computer modeling and analysis of the structure. The prediction of the structure of the bacteriophage 434 cro repressor is used as an example illustrating current procedures.
Amino Acid Sequence↗
Crystallographic data for complexes of the Cro repressor with DNA.
Complexes of the bacteriophage lambda Cro repressor with two DNA duplexes have been crystallized. The DNA sequences are equimolar mixtures of ApTpCpApCpC and its complementary strand and ApCpCpGpCpApApGpG and its complementary strand, which are both parts of the lambda OR3 operator. The space group of both co-crystals is C2221 with cell dimensions a = 81.1 A, b = 89.2 A, and c = 80.0 A. Analysis of dissolved crystals shows that they respectively contain approximately two hexamers per Cro dimer and one nonamer per dimer. The co-crystals diffract to about 3 A resolution and appear suitable for structural studies.
DNA↗
Structure and conformation of the potent antiherpes agent 9-(2-hydroxyethoxymethyl) guanine (acycloguanosine).
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Acyclovir↗
Comparison of solid state and solution conformations of R and S epimers of 8,5'-cycloadenosine and their relevance to some enzymatic reactions.
The C(5')-R epimer of 8,5'-cycloadenosine crystallizes in the monoclonic space group P2(1) (Z = 2) with unit cell dimensions a = 5.755 (1), b = 16.895 (1), and c = 5.511 (1) A and beta = 104.16 (1) degree. X-ray intensity data were measured on a diffractometer, and the crystal structure was determined by direct methods. Least-squares refinement converged at R = 0.037 for 1008 reflections. The conformation about the glycosyl bond is anti, as imposed by the 8,5' cyclization, with XCN = 29.8 degrees. The ribose ring adopts the unusual C(1')endo-O(4')exp (o1T) conformation with pseudo-rotation parameters P = 289.0 degrees and tau m = 490 degrees. The six-membered ring formed by the 8,5' linkage is approximately a half-chair with C(4') and O(4'), respectively, above and below a plane defined by the other four atoms. These results are compared with those previously reported for the corresponding S epimer [Haromy, T. P., Raleigh, J., & Sundaralingam, M. (1980) Biochemistry 19, 1718-1722]. The conformations of the sugar rings and the exocyclic groups of both epimers in the solid state are compared to the conformations in solution, as determined by analysis of the systems of proton-proton vicinal coupling constants from the 1H NMR spectra. The foregoing findings are employed to examine the role of the conformational parameters of adenosine and 5'-AMP in reactions catalyzed by the appropriate enzymes.
AMP Deaminase↗
Crystallization of human oxyhaemoglobin from poly(ethylene glycol) solutions.
Crystals of human oxyhaemoglobin were obtained from poly(ethylene glycol) solutions. Spectroscopic and spectrophotometric measurements on the solutions during crystallization and on the dissolved crystals indicate that the method yields stable crystals of oxyhaemoglobin. Preliminary X-ray studies showed that the crystals obtained are isomorphous with those of deoxyhaemoglobin obtained from poly(ethylene glycol) solutions [Ward, Wishner, Lattman & Love (1975) J. Mol. Biol. 98, 161-177].
Chemical Phenomena↗