Preparation and crystal structure of the recombinant alpha(1)/alpha(2) catalytic heterodimer of bovine brain platelet-activating factor acetylhydrolase Ib.

The intracellular form of mammalian platelet activating factor acetylhydrolase found in brain (PAF-AH Ib) is thought to play a critical role in control in neuronal migration during cortex development. This oligomeric complex consists of a homodimer of the 45 kDa (beta) LIS1 protein, the product of the causative gene for type I lissencephaly, and, depending on the developmental stage and species, one of three possible pairs of two homologous approximately 26 kDa alpha-subunits, which harbor all of the catalytic activity. The exact composition of this complex depends on the expression patterns of the alpha(1) and alpha(2) genes, exhibiting tissue specificity and developmental control. All three possible dimers (alpha(1)/alpha(1), alpha(1)/alpha(2) and alpha(2)/alpha(2)) were identified in tissues. The alpha(1)/alpha(2) heterodimer is thought to play an important role in fetal brain. The structure of the alpha(1)/alpha(1) homodimer was solved earlier in our laboratory at 1.7 A. We report here the preparation of recombinant alpha(1)/alpha(2) heterodimers using a specially constructed bi-cistronic expression vector. The approach may be useful in studies of other systems where pure heterodimers of recombinant proteins are required. The alpha(1)/alpha(2) dimer has been crystallized and its structure was solved at 2.1 A resolution by molecular replacement. These results set the stage for a detailed characterization of the PAF-AH Ib complex.

Structure of the RGS-like domain from PDZ-RhoGEF: linking heterotrimeric g protein-coupled signaling to Rho GTPases.

BACKGROUND: The multidomain PDZ-RhoGEF is one of many known guanine nucleotide exchange factors that upregulate Rho GTPases. PDZ-RhoGEF and related family members play a critical role in a molecular signaling pathway from heterotrimeric G protein-coupled receptors to Rho proteins. A approximately 200 residue RGS-like (RGSL) domain in PDZ-RhoGEF and its homologs is responsible for the direct association with Galpha12/13 proteins. To better understand structure-function relationships, we initiated crystallographic studies of the RGSL domain from human PDZ-RhoGEF. RESULTS: A recombinant construct of the RGSL domain was expressed in Escherichia coli and purified, but it did not crystallize. Alternative constructs were designed based on a novel strategy of targeting lysine and glutamic acid residues for mutagenesis to alanine. A triple-point mutant functionally identical to the wild-type protein was crystallized, and its structure was determined by the MAD method using Se-methionine (Se-Met) incorporation. A molecular model of the RGSL domain was refined at 2.2 A resolution, revealing an all-helical tertiary fold with the mutations located at intermolecular lattice contacts. CONCLUSIONS: The first nine helices adopt a fold similar to that observed for RGS proteins, although the sequence identity with other such known structures is below 20%. The last three helices are an integral extension of the RGS fold, packing tightly against helices 3 and 4 with multiple hydrophobic interactions. Comparison with RGS proteins suggests features that are likely relevant for interaction with G proteins. Finally, we conclude that the strategy used to produce crystals was beneficial and might be applicable to other proteins resistant to crystallization.

Expression, purification, and crystallization of the RGS-like domain from the Rho nucleotide exchange factor, PDZ-RhoGEF, using the surface entropy reduction approach.

Lsc-homology domains are found in several eukaryotic nucleotide exchange factors which act on Rho-family GTPases. They show limited amino acid sequence similarity to RGS proteins, which down-regulate the cellular signaling by the alpha-subunits of trimeric G-proteins and have been shown to interact with Galpha12 and Galpha13. It is believed that the RGS-like (RGSL) domain constitutes the functional link between G-protein-coupled receptors and cytosolic Rho-GTPases. We report here the expression, purification, and crystallization of the RGSL domain from the PDZ-RhoGEF. To obtain X-ray-grade crystals we have used the recently proposed approach of crystallization by mutational surface entropy reduction, in which selected Lys --> Ala, Glu --> Ala, and/or combined point mutations are introduced into the target protein to reduce the cumulative conformational entropy of surface residues. Of the five mutants that were designed and prepared, the second one tried (K463A, E465A, E466A) yielded crystals suitable for further analysis and diffracted X-rays to 2.8 A resolution on a home source. The crystals exhibit hexagonal symmetry, space group P6(1) 22 or P6(5) 22, with unit cell parameters a = b = 63.1 A, c = 202.1 A, and contain one molecule in the asymmetric unit.

Protein crystallization by rational mutagenesis of surface residues: Lys to Ala mutations promote crystallization of RhoGDI.

Crystallization is a unique process that occurs at the expense of entropy, including the conformational entropy of surface residues, which become ordered in crystal lattices during formation of crystal contacts. It could therefore be argued that epitopes free of amino acids with high conformational entropy are more thermodynamically favorable for crystal formation. For a protein recalcitrant to crystallization, mutation of such surface amino acids to residues with no conformational entropy might lead to enhancement of crystallization. This paper reports the results of experiments with an important cytosolic regulator of GTPases, human RhoGDI, in which lysine residues were systematically mutated to alanines. Single and multiple mutations were introduced into two different variants of RhoGDI, NDelta23 and NDelta66, in which the first 23 and 66 residues, respectively, were removed by recombinant methods. In total, 13 single and multiple mutants were prepared and assessed for crystallization and all were shown to crystallize using the Hampton Research Crystal Screens I and II, in contrast to wild-type NDelta23 and NDelta66 RhoGDI which did not crystallize. Four crystal structures were solved (the triple mutants NDelta23:K135,138,141A and NDelta66:K135,138,141A, and two single mutants NDelta66:K113A and NDelta66:K141A) and in three cases the crystal contacts of the new lattices were found precisely at the sites of mutations. These results support the notion that it is, in principle, possible to rationally design mutations which systematically enhance proteins' ability to crystallize.

Crystal structure of the human acyl protein thioesterase I from a single X-ray data set to 1.5 A.

BACKGROUND: Many proteins undergo posttranslational modifications involving covalent attachment of lipid groups. Among them is palmitoylation, a dynamic, reversible process that affects trimeric G proteins and Ras and constitutes a regulatory mechanism for signal transduction pathways. Recently, an acylhydrolase previously identified as lysophospholipase has been shown to function as an acyl protein thioesterase, which catalyzes depalmitoylation of Galpha proteins as well as Ras. Its amino acid sequence suggested that the protein is evolutionarily related to neutral lipases and other thioesterases, but direct structural information was not available. RESULTS: We have solved the crystal structure of the human putative Galpha-regulatory protein acyl thioesterase (hAPT1) with a single data set collected from a crystal containing the wild-type protein. The phases were calculated to 1.8 A resolution based on anomalous scattering from Br(-) ions introduced in the cryoprotectant solution in which the crystal was soaked for 20 s. The model was refined against data extending to a resolution of 1.5 A to an R factor of 18.6%. The enzyme is a member of the ubiquitous alpha/beta hydrolase family, which includes other acylhydrolases such as the palmitoyl protein thioesterase (PPT1). CONCLUSIONS: The human APT1 is closely related to a previously described carboxylesterase from Pseudomonas fluorescens. The active site contains a catalytic triad of Ser-114, His-203, and Asp-169. Like carboxylesterase, hAPT1 appears to be dimeric, although the mutual disposition of molecules in the two dimers differs. Unlike carboxylesterase, the substrate binding pocket and the active site of hAPT1 are occluded by the dimer interface, suggesting that the enzyme must dissociate upon interaction with substrate.

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.

Substrate specificity in glycoside hydrolase family 10. Structural and kinetic analysis of the Streptomyces lividans xylanase 10A.

Endoxylanases are a group of enzymes that hydrolyze the beta-1, 4-linked xylose backbone of xylans. They are predominantly found in two discrete sequence families known as glycoside hydrolase families 10 and 11. The Streptomyces lividans xylanase Xyl10A is a family 10 enzyme, the native structure of which has previously been determined by x-ray crystallography at a 2.6 A resolution (Derewenda, U., Swenson, L., Green, R., Wei, Y., Morosoli, R., Shareck, F., Kluepfel, D., and Derewenda, Z. S. (1994) J. Biol. Chem. 269, 20811-20814). Here, we report the native structure of Xyl10A refined at a resolution of 1.2 A, which reveals many features such as the rare occurrence of a discretely disordered disulfide bond between residues Cys-168 and Cys-201. In order to investigate substrate binding and specificity in glycoside hydrolase family 10, the covalent xylobiosyl enzyme and the covalent cellobiosyl enzyme intermediates of Xyl10A were trapped through the use of appropriate 2-fluoroglycosides. The alpha-linked intermediate with the nucleophile, Glu-236, is in a (4)C(1) chair conformation as previously observed in the family 10 enzyme Cex from Cellulomonas fimi (Notenboom, V., Birsan, C., Warren, R. A. J., Withers, S. G., and Rose, D. R. (1998) Biochemistry 37, 4751-4758). The different interactions of Xyl10A with the xylobiosyl and cellobiosyl moieties, notably conformational changes in the -2 and -1 subsites, together with the observed kinetics on a range of aryl glycosides, shed new light on substrate specificity in glycoside hydrolase family 10.

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.

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.

The functional implications of the dimerization of the catalytic subunits of the mammalian brain platelet-activating factor acetylhydrolase (Ib).

The mammalian brain contains significant amounts of the cytosolic isoform Ib of the platelet-activating factor acetylhydrolase (PAF-AH), a unique type of PLA2. This oligomeric protein complex contains three types of subunits: two homologous (63% identity) 26 kDa catalytic subunits (alpha(1) and alpha(2)) which harbor all the PAF-AH activity, and the 45 kDa beta-subunit (LIS1), a product of the causal gene for Miller-Dieker lissencephaly. During fetal development, the preferentially expressed alpha(1)-subunit forms a homodimer, which binds to a homodimer of LIS1, whereas in adult organisms alpha(1)/alpha(2) and alpha(2)/alpha(2) dimers, also bound to dimeric LIS1, are the prevailing species. The consequences of this "switching" are not understood, but appear to be of physiological significance. The alpha(1)- and alpha(2)-subunits readily associate with very high affinity to form homodimers. The nature of the interface has been elucidated by the 1.7 A resolution crystal structure of the alpha(1)/alpha(1) homodimer (Ho et al., 1997). Here, we examined the functional consequences of the dimerization in both types of alpha-subunits. We obtained monomeric protein in the presence of high concentrations (>50 mM) of Ca2+ ions, and we show that it is catalytically inactive and less stable than the wild type. We further show that Arg29 and Arg22 in one monomer contribute to the catalytic competence of the active site across the dimer interface, and complement the catalytic triad of Ser47, Asp192 and His195, in the second monomer. These results indicate that the brain PAF-acetylhydrolase is a unique PLA2 in which dimerization is essential for both stability and catalytic activity.

PAF-acetylhydrolases.

Platelet-activating factor acetylhydrolases (PAF-AHs, EC 3.1.1.47) constitute a unique subfamily of phospholipases A(2), specific for short acyl chains in the sn-2 position of the phospholipid. Their primary substrate is the platelet-activating factor, PAF, from which they cleave an acetyl moiety with concomitant release of lysoPAF. However, some acetylhydrolase will also hydrolyze other polar phospholipids with up to 6-carbons long acyl chains in the sn-2 position. PAF-acetylhydrolases are diverse enzymes, and the well-characterized isoforms are serine-dependent hydrolases, which do not require Ca(2+) for activity. Given the existence of two pools of PAF, intra- and extracellular, the acetylhydrolases can be divided into two subclasses: those found in the cytosol and enzymes secreted to blood plasma or other body fluids. Recent crystallographic studies shed new light on the complex structure-function relationships in PAF-AHs.

Probing the substrate specificity of the intracellular brain platelet-activating factor acetylhydrolase.

Platelet-activating factor acetylhydrolases (PAF-AHs) are unique PLA2s which hydrolyze the sn-2 ester linkage in PAF-like phospholipids with a marked preference for very short acyl chains, typically acetyl. The recent solution of the crystal structure of the alpha(1) catalytic subunit of isoform Ib of bovine brain intracellular PAF-AH at 1.7 A resolution paved the way for a detailed examination of the molecular basis of substrate specificity in this enzyme. The crystal structure suggests that the side chains of Thr103, Leu48 and Leu194 are involved in substrate recognition. Three single site mutants (L48A, T103S and L194A) were overexpressed and their structures were solved to 2.3 A resolution or better by X-ray diffraction methods. Enzyme kinetics showed that, compared with wild-type protein, all three mutants have higher relative activity against phospholipids with sn-2 acyl chains longer than an acetyl. However, for each of the mutants we observed an unexpected and substantial reduction in the V(max) of the reaction. These results are consistent with the model in which residues Leu48, Thr103 and Leu194 indeed contribute to substrate specificity and in addition suggest that the integrity of the specificity pocket is critical for the expression of full catalytic function, thus conferring very high substrate selectivity on the enzyme.

How RhoGDI binds Rho.

Like all Rho (Ras homology) GTPases, RhoA functions as a molecular switch in cell signaling, alternating between GTP- and GDP-bound states, with its biologically inactive GDP-bound form maintained as a cytosolic complex with RhoGDI (guanine nucleotide-exchange inhibitor). The crystal structures of RhoA-GDP and of the C-terminal immunoglobulin-like domain of RhoGDI (residues 67-203) are known, but the mechanism by which the two proteins interact is not known. The functional human RhoA-RhoGDI complex has been expressed in yeast and crystallized (P6(5)22, unit-cell parameters a = b = 139, c = 253 A, two complexes in the asymmetric unit). Although diffraction from these crystals extends to 3.5 A and is highly anisotropic, the experimentally phased (MAD plus MIR) electron-density map was adequate to reveal the mutual disposition of the two molecules. The result was validated by molecular-replacement calculations when data were corrected for anisotropy. Furthermore, the N-terminus of RhoGDI (the region involved in inhibition of nucleotide exchange) can be identified in the electron-density map: it is bound to the switch I and switch II regions of RhoA, occluding an epitope which binds Dbl-like nucleotide-exchange factors. The entrance of the hydrophobic pocket of RhoGDI is 25 A from the last residue in the RhoA model, with its C-terminus oriented to accommodate the geranylgeranyl group without conformational change in RhoA.

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.

Domain identification of hormone-sensitive lipase by circular dichroism and fluorescence spectroscopy, limited proteolysis, and mass spectrometry.

Structure-function relationship analyses of hormone-sensitive lipase (HSL) have suggested that this metabolically important enzyme consists of several functional and at least two structural domains (Osterlund, T., Danielsson, B., Degerman, E., Contreras, J. A., Edgren, G., Davis, R. C., Schotz, M. C., and Holm, C. (1996) Biochem. J. 319, 411-420; Contreras, J. A., Karlsson, M., Osterlund, T., Laurell, H., Svensson, A., and Holm, C. (1996) J. Biol. Chem. 271, 31426-31430). To analyze the structural domain composition of HSL in more detail, we applied biophysical methods. Denaturation of HSL was followed by circular dichroism measurements and fluorescence spectroscopy, revealing that the unfolding of HSL is a two-step event. Using limited proteolysis in combination with mass spectrometry, several proteolytic fragments of HSL were identified, including one corresponding exactly to the proposed N-terminal domain. Major cleavage sites were found in the predicted hinge region between the two domains and in the regulatory module of the C-terminal, catalytic domain. Analyses of a hinge region cleavage mutant and calculations of the hydropathic pattern of HSL further suggest that the hinge region and regulatory module are exposed parts of HSL. Together, these data support our previous hypothesis that HSL consists of two major structural domains, encoded by exons 1-4 and 5-9, respectively, of which the latter contains an exposed regulatory module outside the catalytic alpha/beta-hydrolase fold core.

Crystal structure of brefeldin A esterase, a bacterial homolog of the mammalian hormone-sensitive lipase.

Brefeldin A esterase (BFAE), a detoxifying enzyme isolated from Bacillus subtilis, hydrolyzes and inactivates BFA, a potent fungal inhibitor of intracellular vesicle-dependent secretory transport and poliovirus RNA replication. We have solved the crystal structure of BFAE and we discovered that the previously reported amino acid sequence was in serious error due to frame shifts in the cDNA sequence. The correct sequence, inferred from the experimentally phased electron density map, revealed that BFAE is a homolog of the mammalian hormone sensitive lipase (HSL). It is a canonical alpha/beta hydrolase with two insertions forming the substrate binding pocket. The enzyme contains a lipase-like catalytic triad, Ser 202, Asp 308 and His 338, consistent with mutational studies that implicate the homologous Ser 424, Asp 693 and His 723 in the catalytic triad in human HSL.

Structure of the gangrene alpha-toxin: the beauty in the beast.

The crystal and molecular structure of the Clostridium perfringens alpha-toxin crowns over a century-long research into the mechanisms of pathogenesis of gas gangrene. The structure reveals a two-domain enzyme, with a catalytic all-helical N-terminal domain, and a C-terminal domain similar in its jelly-roll topology to those found in pancreatic lipase and lipoxygenases.

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.

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.