Crystal structures of two human pyrophosphorylase isoforms in complexes with UDPGlc(Gal)NAc: role of the alternatively spliced insert in the enzyme oligomeric assembly and active site architecture.

The recently published human genome with its relatively modest number of genes has highlighted the importance of post-transcriptional and post-translational modifications, such as alternative splicing or glycosylation, in generating the complexities of human biology. The human UDP-N-acetylglucosamine (UDPGlcNAc) pyrophosphorylases AGX1 and AGX2, which differ in sequence by an alternatively spliced 17 residue peptide, are key enzymes synthesizing UDPGlcNAc, an essential precursor for protein glycosylation. To better understand the catalytic mechanism of these enzymes and the role of the alternatively spliced segment, we have solved the crystal structures of AGX1 and AGX2 in complexes with UDPGlcNAc (at 1.9 and 2.4 A resolution, respectively) and UDPGalNAc (at 2.2 and 2.3 A resolution, respectively). Comparison with known structures classifies AGX1 and AGX2 as two new members of the SpsA-GnT I Core superfamily and, together with mutagenesis analysis, helps identify residues critical for catalysis. Most importantly, our combined structural and biochemical data provide evidence for a change in the oligomeric assembly accompanied by a significant modification of the active site architecture, a result suggesting that the two isoforms generated by alternative splicing may have distinct catalytic properties.

Mannose-binding plant lectins: different structural scaffolds for a common sugar-recognition process.

Mannose-specific lectins are widely distributed in higher plants and are believed to play a role in recognition of high-mannose type glycans of foreign micro-organisms or plant predators. Structural studies have demonstrated that the mannose-binding specificity of lectins is mediated by distinct structural scaffolds. The mannose/glucose-specific legume (e.g., Con A, pea lectin) exhibit the canonical twelve-stranded beta-sandwich structure. In contrast to legume lectins that interact with both mannose and glucose, the monocot mannose-binding lectins (e.g., the Galanthus nivalis agglutinin or GNA from bulbs) react exclusively with mannose and mannose-containing N-glycans. These lectins possess a beta-prism structure. More recently, an increasing number of mannose-specific lectins structurally related to jacalin (e.g., the lectins from the Jerusalem artichoke, banana or rice), which also exhibit a beta-prism organization, were characterized. Jacalin itself was re-defined as a polyspecific lectin which, in addition to galactose, also interacts with mannose and mannose-containing glycans. Finally the B-chain of the type II RIP of iris, which has the same beta-prism structure as all other members of the ricin-B family, interacts specifically with mannose and galactose. This structural diversity associated with the specific recognition of high-mannose type glycans highlights the importance of mannose-specific lectins as recognition molecules in higher plants.

The crystal structures of Apo and complexed Saccharomyces cerevisiae GNA1 shed light on the catalytic mechanism of an amino-sugar N-acetyltransferase.

The yeast enzymes involved in UDP-GlcNAc biosynthesis are potential targets for antifungal agents. GNA1, a novel member of the Gcn5-related N-acetyltransferase (GNAT) superfamily, participates in UDP-GlcNAc biosynthesis by catalyzing the formation of GlcNAc6P from AcCoA and GlcN6P. We have solved three crystal structures corresponding to the apo Saccharomyces cerevisiae GNA1, the GNA1-AcCoA, and the GNA1-CoA-GlcNAc6P complexes and have refined them to 2.4, 1.3, and 1.8 A resolution, respectively. These structures not only reveal a stable, beta-intertwined, dimeric assembly with the GlcNAc6P binding site located at the dimer interface but also shed light on the catalytic machinery of GNA1 at an atomic level. Hence, they broaden our understanding of structural features required for GNAT activity, provide structural details for related aminoglycoside N-acetyltransferases, and highlight the adaptability of the GNAT superfamily members to acquire various specificities.

Immunocytochemical localization and crystal structure of human frequenin (neuronal calcium sensor 1).

Frequenin, a member of a large family of myristoyl-switch calcium-binding proteins, functions as a calcium-ion sensor to modulate synaptic activity and secretion. We show that human frequenin colocalizes with ARF1 GTPase in COS-7 cells and occurs in similar cellular compartments as the phosphatidylinositol-4-OH kinase PI4Kbeta, the mammalian homolog of the yeast kinase PIK1. In addition, the crystal structure of unmyristoylated, calcium-bound human frequenin has been determined and refined to 1.9 A resolution. The overall fold of frequenin resembles those of neurocalcin and the photoreceptor, recoverin, of the same family, with two pairs of calcium-binding EF hands and three bound calcium ions. Despite the similarities, however, frequenin displays significant structural differences. A large conformational shift of the C-terminal region creates a wide hydrophobic crevice at the surface of frequenin. This crevice, which is unique to frequenin and distinct from the myristoyl-binding box of recoverin, may accommodate a yet unknown protein ligand.

Crystal structure of Streptococcus pneumoniae N-acetylglucosamine-1-phosphate uridyltransferase bound to acetyl-coenzyme A reveals a novel active site architecture.

The bifunctional bacterial enzyme N-acetyl-glucosamine-1-phosphate uridyltransferase (GlmU) catalyzes the two-step formation of UDP-GlcNAc, a fundamental precursor in bacterial cell wall biosynthesis. With the emergence of new resistance mechanisms against beta-lactam and glycopeptide antibiotics, the biosynthetic pathway of UDP-GlcNAc represents an attractive target for drug design of new antibacterial agents. The crystal structures of Streptococcus pneumoniae GlmU in unbound form, in complex with acetyl-coenzyme A (AcCoA) and in complex with both AcCoA and the end product UDP-GlcNAc, have been determined and refined to 2.3, 2.5, and 1.75 A, respectively. The S. pneumoniae GlmU molecule is organized in two separate domains connected via a long alpha-helical linker and associates as a trimer, with the 50-A-long left-handed beta-helix (LbetaH) C-terminal domains packed against each other in a parallel fashion and the C-terminal region extended far away from the LbetaH core and exchanged with the beta-helix from a neighboring subunit in the trimer. AcCoA binding induces the formation of a long and narrow tunnel, enclosed between two adjacent LbetaH domains and the interchanged C-terminal region of the third subunit, giving rise to an original active site architecture at the junction of three subunits.

Dissection of the bifunctional Escherichia coli N-acetylglucosamine-1-phosphate uridyltransferase enzyme into autonomously functional domains and evidence that trimerization is absolutely required for glucosamine-1-phosphate acetyltransferase activity and cell growth.

The bifunctional N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) enzyme catalyzes both the acetylation of glucosamine 1-phosphate and the uridylation of N-acetylglucosamine 1-phosphate, two subsequent steps in the pathway for UDP-N-acetylglucosamine synthesis in bacteria. In our previous work describing its initial characterization in Escherichia coli, we proposed that the 456-amino acid (50.1 kDa) protein might possess separate uridyltransferase (N-terminal) and acetyltransferase (C-terminal) domains. In the present study, we confirm this hypothesis by expression of the two independently folding and functional domains. A fragment containing the N-terminal 331 amino acids (Tr331, 37.1 kDa) has uridyltransferase activity only, with steady-state kinetic parameters similar to the full-length protein. Further deletion of 80 amino acid residues at the C terminus results in a 250-amino acid fragment (28.6 kDa) still exhibiting significant uridyltransferase activity. Conversely, a fragment containing the 233 C-terminal amino acids (24.7 kDa) exhibits acetyltransferase activity exclusively. None of these individual domains could complement a chromosomal glmU mutation, indicating that each of the two activities is essential for cell viability. Analysis of truncated GlmU proteins by gel filtration further localizes regions of the protein involved in its trimeric organization. Interestingly, overproduction of the truncated Tr331 protein in a wild-type strain results in a rapid depletion of endogenous acetyltransferase activity, an arrest of peptidoglycan synthesis and cell lysis. It is shown that the acetyltransferase activity of the full-length protein is abolished once trapped within heterotrimers formed in presence of the truncated protein, suggesting that this enzyme activity absolutely requires a trimeric organization and that the catalytic site involves regions of contact between adjacent monomers. Data are discussed in connection with the recently obtained crystal structure of the truncated Tr331 protein.

Glycoside hydrolases and glycosyltransferases: families and functional modules.

The past year has witnessed the expected increase in the number of solved structures of glycoside hydrolases and glycosyltransferases, and their constitutive modules. These structures show that, while glycoside hydrolases display an extraordinary variety of folds, glycosyltransferases and carbohydrate-binding modules appear to belong to a much smaller number of folding families.

Crystal structure and mutational analysis of the Saccharomyces cerevisiae cell cycle regulatory protein Cks1: implications for domain swapping, anion binding and protein interactions.

BACKGROUND: The Saccharomyces cerevisiae protein Cks1 (cyclin-dependent kinase subunit 1) is essential for cell-cycle progression. The biological function of Cks1 can be modulated by a switch between two distinct molecular assemblies: the single domain fold, which results from the closing of a beta-hinge motif, and the intersubunit beta-strand interchanged dimer, which arises from the opening of the beta-hinge motif. The crystal structure of a cyclin-dependent kinase (Cdk) in complex with the human Cks homolog CksHs1 single-domain fold revealed the importance of conserved hydrophobic residues and charged residues within the beta-hinge motif. RESULTS: The 3.0 A resolution Cks1 structure reveals the strict structural conservation of the Cks alpha/beta-core fold and the beta-hinge motif. The beta hinge identified in the Cks1 structure includes a novel pivot and exposes a cluster of conserved tyrosine residues that are involved in Cdk binding but are sequestered in the beta-interchanged Cks homolog suc1 dimer structure. This Cks1 structure confirms the conservation of the Cks anion-binding site, which interacts with sidechain residues from the C-terminal alpha helix of another subunit in the crystal. CONCLUSIONS: The Cks1 structure exemplifies the conservation of the beta-interchanged dimer and the anion-binding site in evolutionarily distant yeast and human Cks homologs. Mutational analyses including in vivo rescue of CKS1 disruption support the dual functional roles of the beta-hinge residue Glu94, which participates in Cdk binding, and of the anion-binding pocket that is located 22 A away and on an opposite face to Glu94. The Cks1 structure suggests a biological role for the beta-interchanged dimer and the anion-binding site in targeting Cdks to specific phosphoproteins during cell-cycle progression.

Active and inhibited human catalase structures: ligand and NADPH binding and catalytic mechanism.

Human catalase is an heme-containing peroxisomal enzyme that breaks down hydrogen peroxide to water and oxygen; it is implicated in ethanol metabolism, inflammation, apoptosis, aging and cancer. The 1. 5 A resolution human enzyme structure, both with and without bound NADPH, establishes the conserved features of mammalian catalase fold and assembly, implicates Tyr370 as the tyrosine radical, suggests the structural basis for redox-sensitive binding of cognate mRNA via the catalase NADPH binding site, and identifies an unexpectedly substantial number of water-mediated domain contacts. A molecular ruler mechanism based on observed water positions in the 25 A-long channel resolves problems for selecting hydrogen peroxide. Control of water-mediated hydrogen bonds by this ruler selects for the longer hydrogen peroxide and explains the paradoxical effects of mutations that increase active site access but lower catalytic rate. The heme active site is tuned without compromising peroxide binding through a Tyr-Arg-His-Asp charge relay, arginine residue to heme carboxylate group hydrogen bonding, and aromatic stacking. Structures of the non-specific cyanide and specific 3-amino-1,2, 4-triazole inhibitor complexes of human catalase identify their modes of inhibition and help reveal the catalytic mechanism of catalase. Taken together, these resting state and inhibited human catalase structures support specific, structure-based mechanisms for the catalase substrate recognition, reaction and inhibition and provide a molecular basis for understanding ethanol intoxication and the likely effects of human polymorphisms.

Conformational flexibility of the acetylcholinesterase tetramer suggested by x-ray crystallography.

Acetylcholinesterase, a polymorphic enzyme, appears to form amphiphilic and nonamphiphilic tetramers from a single splice variant; this suggests discrete tetrameric arrangements where the amphipathic carboxyl-terminal sequences can be either buried or exposed. Two distinct, but related crystal structures of the soluble, trypsin-released tetramer of acetylcholinesterase from Electrophorus electricus were solved at 4.5 and 4.2 A resolution by molecular replacement. Resolution at these levels is sufficient to provide substantial information on the relative orientations of the subunits within the tetramer. The two structures, which show canonical homodimers of subunits assembled through four-helix bundles, reveal discrete geometries in the assembly of the dimers to form: (a) a loose, pseudo-square planar tetramer with antiparallel alignment of the two four-helix bundles and a large space in the center where the carboxyl-terminal sequences may be buried or (b) a compact, square nonplanar tetramer that may expose all four sequences on a single side. Comparison of these two structures points to significant conformational flexibility of the tetramer about the four-helix bundle axis and along the dimer-dimer interface. Hence, in solution, several conformational states of a flexible tetrameric arrangement of acetylcholinesterase catalytic subunits may exist to accommodate discrete carboxyl-terminal sequences of variable dimensions and amphipathicity.

Crystal structure of the bifunctional N-acetylglucosamine 1-phosphate uridyltransferase from Escherichia coli: a paradigm for the related pyrophosphorylase superfamily.

N-acetylglucosamine 1-phosphate uridyltransferase (GlmU) is a cytoplasmic bifunctional enzyme involved in the biosynthesis of the nucleotide-activated UDP-GlcNAc, which is an essential precursor for the biosynthetic pathways of peptidoglycan and other components in bacteria. The crystal structure of a truncated form of GlmU has been solved at 2.25 A resolution using the multiwavelength anomalous dispersion technique and its function tested with mutagenesis studies. The molecule is composed of two distinct domains connected by a long alpha-helical arm: (i) an N-terminal domain which resembles the dinucleotide-binding Rossmann fold; and (ii) a C-terminal domain which adopts a left-handed parallel beta-helix structure (LbetaH) as found in homologous bacterial acetyltransferases. Three GlmU molecules assemble into a trimeric arrangement with tightly packed parallel LbetaH domains, the long alpha-helical linkers being seated on top of the arrangement and the N-terminal domains projected away from the 3-fold axis. In addition, the 2.3 A resolution structure of the GlmU-UDP-GlcNAc complex reveals the structural bases required for the uridyltransferase activity. These structures exemplify a three-dimensional template for the development of new antibacterial agents and for studying other members of the large family of XDP-sugar bacterial pyrophosphorylases.

Crystal structures of the bovine beta4galactosyltransferase catalytic domain and its complex with uridine diphosphogalactose.

beta1,4-galactosyltransferase T1 (beta4Gal-T1, EC 2.4.1.90/38), a Golgi resident membrane-bound enzyme, transfers galactose from uridine diphosphogalactose to the terminal beta-N-acetylglucosamine residues forming the poly-N-acetyllactosamine core structures present in glycoproteins and glycosphingolipids. In mammals, beta4Gal-T1 binds to alpha-lactalbumin, a protein that is structurally homologous to lyzozyme, to produce lactose. beta4Gal-T1 is a member of a large family of homologous beta4galactosyltransferases that use different types of glycoproteins and glycolipids as substrates. Here we solved and refined the crystal structures of recombinant bovine beta4Gal-T1 to 2.4 A resolution in the presence and absence of the substrate uridine diphosphogalactose. The crystal structure of the bovine substrate-free beta4Gal-T1 catalytic domain showed a new fold consisting of a single conical domain with a large open pocket at its base. In the substrate-bound complex, the pocket encompassed residues interacting with uridine diphosphogalactose. The structure of the complex contained clear regions of electron density for the uridine diphosphate portion of the substrate, where its beta-phosphate group was stabilized by hydrogen-bonding contacts with conserved residues including the Asp252ValAsp254 motif. These results help the interpretation of engineered beta4Gal-T1 point mutations. They suggest a mechanism possibly involved in galactose transfer and enable identification of the critical amino acids involved in alpha-lactalbumin interactions.

Crystal structure of mouse acetylcholinesterase. A peripheral site-occluding loop in a tetrameric assembly.

The crystal structure of mouse acetylcholinesterase at 2.9-A resolution reveals a tetrameric assembly of subunits with an antiparallel alignment of two canonical homodimers assembled through four-helix bundles. In the tetramer, a short Omega loop, composed of a cluster of hydrophobic residues conserved in mammalian acetylcholinesterases along with flanking alpha-helices, associates with the peripheral anionic site of the facing subunit and sterically occludes the entrance of the gorge leading to the active center. The inverse loop-peripheral site interaction occurs within the second pair of subunits, but the peripheral sites on the two loop-donor subunits remain freely accessible to the solvent. The position and complementarity of the peripheral site-occluding loop mimic the characteristics of the central loop of the peptidic inhibitor fasciculin bound to mouse acetylcholinesterase. Tetrameric forms of cholinesterases are widely distributed in nature and predominate in mammalian brain. This structure reveals a likely mode of subunit arrangement and suggests that the peripheral site, located near the rim of the gorge, is a site for association of neighboring subunits or heterologous proteins with interactive surface loops.

Helianthus tuberosus lectin reveals a widespread scaffold for mannose-binding lectins.

BACKGROUND: Heltuba, a tuber lectin from the Jerusalem artichoke Helianthus tuberosus, belongs to the mannose-binding subgroup of the family of jacalin-related plant lectins. Heltuba is highly specific for the disaccharides Man alpha 1-3Man or Man alpha 1-2Man, two carbohydrates that are particularly abundant in the glycoconjugates exposed on the surface of viruses, bacteria and fungi, and on the epithelial cells along the gastrointestinal tract of lower animals. Heltuba is therefore a good candidate as a defense protein against plant pathogens or predators. RESULTS: The 2.0 A resolution structure of Heltuba exhibits a threefold symmetric beta-prism fold made up of three four-stranded beta sheets. The crystal structures of Heltuba in complex with Man alpha 1-3Man and Man alpha 1-2Man, solved at 2.35 A and 2.45 A resolution respectively, reveal the carbohydrate-binding site and the residues required for the specificity towards alpha 1-3 or alpha 1-2 mannose linkages. In addition, the crystal packing reveals a remarkable, donut-shaped, octahedral assembly of subunits with the mannose moieties at the periphery, suggesting possible cross-linking interactions with branched oligomannosides. CONCLUSIONS: The structure of Heltuba, which is the prototype for an extended family of mannose-binding agglutinins, shares the carbohydrate-binding site and beta-prism topology of its galactose-binding counterparts jacalin and Maclura pomifera lectin. However, the beta-prism elements recruited to form the octameric interface of Heltuba, and the strategy used to forge the mannose-binding site, are unique and markedly dissimilar to those described for jacalin. The present structure highlights a hitherto unrecognized adaptability of the beta-prism building block in the evolution of plant proteins.

Inhibition of mouse acetylcholinesterase by fasciculin: crystal structure of the complex and mutagenesis of fasciculin.

Fasciculins are members of the superfamily of three-fingered peptidic toxins from Elapidae venoms. They selectively inhibit mammalian and electric fish acetylcholinesterases (AChE) with Ki values in the pico- to nanomolar range. Kinetic studies performed in solution indicate that fasciculin does not totally occlude ligand access to the active site of AChE, but rather binds to a peripheral site of the enzyme to inhibit catalysis, perhaps allosterically. The crystal structure of the Fas2-mouse AChE complex delineated a large contact area consistent with the low dissociation constant of the complex; the Fas2 and AChE residues participating in the binding interface were unambiguously established, and major hydrophobic interactions were identified. The structure however suggests that fasciculin totally occludes substrate entry into the catalytic site of AChE, and does not reveal to what extent each contact between Fas2 and AChE contributes to the overall binding energy. New probes, designed to delineate the individual contributions of the fasciculin residues to the complex formation and conformation, were generated by site-directed mutagenesis of a synthetic Fas2 gene. A fully processed recombinant fasciculin, rFas2, that is undistinguishable from the natural, venom-derived Fas2, was expressed in a mammalian system; fourteen mutants, encompassing 16 amino acid residues distributed among the three loops (fingers) of Fas2, were developed from both the kinetic and structural data and analyzed for inhibition of mouse AChE. The determinants identified by the structural and the functional approaches do coincide. However, only a few of the many residues which make up the overall interactive site of the Fas2 molecule provide the strong interactions required for high affinity binding and enzyme inhibition. Potential drug design from the fasciculin molecule is discussed.

Barley alpha-amylase bound to its endogenous protein inhibitor BASI: crystal structure of the complex at 1.9 A resolution.

BACKGROUND: Barley alpha-amylase is a 45 kDa enzyme which is involved in starch degradation during barley seed germination. The released sugars provide the plant embryo with energy for growth. The major barley alpha-amylase isozyme (AMY2) binds with high affinity to the endogenous inhibitor BASI (barley alpha-amylase/subtilisin inhibitor) whereas the minor isozyme (AMY1) is not inhibited. BASI is a 19.6 kDa bifunctional protein that can simultaneously inhibit AMY2 and serine proteases of the subtilisin family. This inhibitor may therefore prevent degradation of the endosperm starch during premature sprouting and protect the seed from attack by pathogens secreting proteases. RESULTS: The crystal structure of AMY2 in complex with BASI was determined and refined at 1.9 A resolution. BASI consists of a 12-stranded beta-barrel structure which belongs to the beta-trefoil fold family and inhibits AMY2 by sterically occluding access of the substrate to the active site of the enzyme. The AMY2-BASI complex is characterized by an unusual completely solvated calcium ion located at the protein-protein interface. CONCLUSIONS: The AMY2-BASI complex represents the first reported structure of an endogenous protein-protein complex from a higher plant. The structure of the complex throws light on the strict specificity of BASI for AMY2, and shows that domain B of AMY2 contributes greatly to the specificity of enzyme-inhibitor recognition. In contrast to the three-dimensional structures of porcine pancreatic alpha-amylase in complex with proteinaceous inhibitors, the AMY2-BASI structure reveals that the catalytically essential amino acid residues of the enzyme are not directly bound to the inhibitor. Binding of BASI to AMY2 creates a cavity, exposed to the external medium, that is ideally shaped to accommodate an extra calcium ion. This feature may contribute to the inhibitory effect, as the key amino acid sidechains of the active site are in direct contact with water molecules which are in turn ligated to the calcium ion.

Novel dimeric interface and electrostatic recognition in bacterial Cu,Zn superoxide dismutase.

Eukaryotic Cu,Zn superoxide dismutases (CuZnSODs) are antioxidant enzymes remarkable for their unusually stable beta-barrel fold and dimer assembly, diffusion-limited catalysis, and electrostatic guidance of their free radical substrate. Point mutations of CuZnSOD cause the fatal human neurodegenerative disease amyotrophic lateral sclerosis. We determined and analyzed the first crystallographic structure (to our knowledge) for CuZnSOD from a prokaryote, Photobacterium leiognathi, a luminescent symbiont of Leiognathid fish. This structure, exemplifying prokaryotic CuZnSODs, shares the active-site ligand geometry and the topology of the Greek key beta-barrel common to the eukaryotic CuZnSODs. However, the beta-barrel elements recruited to form the dimer interface, the strategy used to forge the channel for electrostatic recognition of superoxide radical, and the connectivity of the intrasubunit disulfide bond in P. leiognathi CuZnSOD are discrete and strikingly dissimilar from those highly conserved in eukaryotic CuZnSODs. This new CuZnSOD structure broadens our understanding of structural features necessary and sufficient for CuZnSOD activity, highlights a hitherto unrecognized adaptability of the Greek key beta-barrel building block in evolution, and reveals that prokaryotic and eukaryotic enzymes diverged from one primordial CuZnSOD and then converged to distinct dimeric enzymes with electrostatic substrate guidance.

A mutation in the human cyclin-dependent kinase interacting protein, CksHs2, interferes with cyclin-dependent kinase binding and biological function, but preserves protein structure and assembly.

A mutation directing an amino acid substitution in the conserved beta-hinge region of one of the human Cks isoforms, CksHs2, was constructed by site-directed mutagenesis. Replacement of glutamine for glutamate 63 (E63Q) was predicted to stabilize the beta-interchanged dimeric and hexameric assembly of CksHs2. However, such an effect was seen only at high, non-physiological pH. Three-dimensional structures of the E63Q hexameric mutant protein were determined to 2.6 A resolution in a P4(3)2(1)2 space group and 2.1 A in the C2 space group isostructural with wild-type, and both were shown to be virtually identical to the refined 1.7 A wild-type structure. Thus, the E63Q mutation did not alter the wild-type structure and assembly of CksHs2 but, surprisingly, disrupted the essential biological function of the protein and significantly reduced its ability to bind to cyclin-dependent kinases. The Kd of wild-type CksHs2 for CDK2 was 5.05 x 10(-8) M, whereas the affinity of the mutant protein for CDK2 was too low to allow a determination. These data, coupled with the observation that monomeric but not hexameric CksHs2 interacts with cyclin-dependent kinases, suggest that glutamine 63 is likely to be directly involved in cyclin-dependent kinase binding in vitro and in vivo.

Soluble monomeric acetylcholinesterase from mouse: expression, purification, and crystallization in complex with fasciculin.

A soluble, monomeric form of acetylcholinesterase from mouse (mAChE), truncated at its carboxyl-terminal end, was generated from a cDNA encoding the glycophospholipid-linked form of the mouse enzyme by insertion of an early stop codon at position 549. Insertion of the cDNA behind a cytomegalovirus promoter and selection by aminoglycoside resistance in transfected HEK cells yielded clones secreting large quantities of mAChE into the medium. The enzyme sediments as a soluble monomer at 4.8 S. High levels of expression coupled with a one-step purification by affinity chromatography have allowed us to undertake a crystallographic study of the fasciculin-mAChE complex. Complexes of two distinct fasciculins, Fas1-mAChE and Fas2-mAChE, were formed prior to the crystallization and were characterized thoroughly. Single hexagonal crystals, up to 0.6 mm x 0.5 mm x 0.5 mm, grew spontaneously from ammonium sulfate solutions buffered in the pH 7.0 range. They were found by electrophoretic migration to consist entirely of the complex and diffracted to 2.8 A resolution. Analysis of initial X-ray data collected on Fas2-mAChE crystals identified the space group as P6(1)22 or P6(5)22 with unit cell dimensions a = b = 75.5 A, c = 556 A, giving a Vm value of 3.1 A3/Da (or 60% of solvent), consistent with a single molecule of Fas2-AChE complex (72 kDa) per asymmetric unit. The complex Fas1-mAChE crystallizes in the same space group with identical cell dimensions.