Three-dimensional structure of cat muscle pyruvate kinase at 6 Angstrom resolution.

A 6 Å resolution electron density map of cat muscle pyruvate kinase has been calculated. From this map it has been possible to isolate a single molecule and to assign subunit boundaries. The binding of substrates, products and the divalent metal cation has been studied.     

Phosphofructokinase: structure and control

Phosphofructokinase from Bacillus stearothermophilus shows cooperative kinetics with respect to the substrate fructose-6-phosphate (F6P), allosteric activation by ADP, and inhibition by phosphoenolpyruvate. The crystal structure of the active conformation of the enzyme has been solved to 2.4 A resolution, and three ligand-binding sites have been located. Two of these form the active site and bind the substrates F6P and ATP. The third site binds both allosteric activator and inhibitor. The complex of the enzyme with F6P and ADP has been partly refined at 2.4 A resolution, and a model of ATP has been built into the active site by using the refined model of ADP and a 6 A resolution map of bound 5'-adenylylimidodiphosphate (AMPPNP). The gamma-phosphate of ATP is close to the 1-hydroxyl of F6P, in a suitable position for in-line phosphoryl transfer. The binding of the phosphate of F6P involves two arginines from a neighbouring subunit in the tetramer, which suggests that a rearrangement of the subunits could explain the cooperativity of substrate binding. The activatory ADP is also bound by residues from two subunits.     

8-N(3)-3'-biotinyl-ATP, a novel monofunctional reagent: differences in the F(1)- and V(1)-ATPases by means of the ATP analogue

A novel photoaffinity label, 8-N(3)-3'-biotinyl-ATP, has been synthesized. The introduction of an additional biotin residue is advantageous for easy detection of labeled proteins. This could be first tested by reaction with the F(1)-ATPase from the thermophilic bacterium PS3 (TF(1)). UV irradiation of TF(1) in the presence of 8-N(3)-3'-biotinyl-ATP results in a nucleotide-dependent binding of the analogue in the noncatalytic alpha and the catalytic beta subunits of TF(1), demonstrating the suitability of this analogue as a potential photoaffinity label. Reaction with the V(1)-ATPase, however, led to labeling of subunit E, which has been suggested as a structural and functional homologue of the gamma subunit of the F-ATPases. MALDI-TOF mass spectrometry has been used to map the regions of subunit E involved in the binding of 8-N(3)-3'-biotinyl-ATP.     

Emerging structure of the nicotinic acetylcholine receptors

The conversion of acetylcholine binding into ion conduction across the membrane is becoming more clearly understood in terms of the structure of the receptor and its transitions. A high-resolution structure of a protein that is homologous to the extracellular domain of the receptor has revealed the binding sites and subunit interfaces in great detail. Although the structures of the membrane and cytoplasmic domains are less well determined, the channel lining and the determinants of selectivity have been mapped. The location and structure of the gates, and the coupling between binding sites and gates, remain to be established.     

Identification of ADP in the iron-sulfur flavoprotein trimethylamine dehydrogenase

Analysis of the 2.4-A resolution electron density map of trimethylamine dehydrogenase has revealed the unexpected presence of one molecule of ADP/subunit. This binding has been confirmed chemically. The binding site is located at the analogous position of the ADP moiety of FAD in glutathione reductase, the FAD and NADPH binding domains of which resemble two of the domains of trimethylamine dehydrogenase. Comparison of the environments of the ADP moieties in the two proteins indicates that 32 residues in 6 peptides are in equivalent positions with a root mean square deviation for C alpha positions of 1.11 A. Twelve of these amino acids are identical, based on the electron density-derived "x-ray" sequence of trimethylamine dehydrogenase. Detailed analysis of the environment of the ADP moiety indicates that most of the conserved residues are not in direct contact with the cofactor. Some of them probably represent the "fingerprint" of the beta alpha beta binding fold found in dinucleotide binding proteins, but the remaining conserved residues may indicate a closer evolutionary relationship between these two proteins.     

Evidence for major structural changes in subunit C of the vacuolar ATPase due to nucleotide binding

The ability of subunit C of eukaryotic V-ATPases to bind ADP and ATP is demonstrated by photoaffinity labeling and fluorescence correlation spectroscopy (FCS). Quantitation of the photoaffinity and the FCS data indicate that the ATP-analogues bind more weakly to subunit C than the ADP-analogues. Site-directed mutagenesis and N-terminal sequencing of subunit C from Arabidopsis (VHA-C) and yeast (Vma5p) have been used to map the C-terminal region of subunit C as the nucleotide-binding site. Tryptophan fluorescence quenching and decreased susceptibility to tryptic digestion of subunit C after binding of different nucleotides provides evidence for structural changes in this subunit caused by nucleotide-binding.     

Mapping the Gbetagamma-binding sites in GIRK1 and GIRK2 subunits of the G protein-activated K+ channel

G protein-activated K+ channels (Kir3 or GIRK) are activated by direct binding of Gbetagamma. The binding sites of Gbetagamma in the ubiquitous GIRK1 (Kir3.1) subunit have not been unequivocally charted, and in the neuronal GIRK2 (Kir3.2) subunit the binding of Gbetagamma has not been studied. We verified and extended the map of Gbetagamma-binding sites in GIRK1 by using two approaches: direct binding of Gbetagamma to fragments of GIRK subunits (pull down), and competition of these fragments with the Galphai1 subunit for binding to Gbetagamma. We also mapped the Gbetagamma-binding sites in GIRK2. In both subunits, the N terminus binds Gbetagamma. In the C terminus, the Gbetagamma-binding sites in the two subunits are not identical; GIRK1, but not GIRK2, has a previously unrecognized Gbetagamma-interacting segments in the first half of the C terminus. The main C-terminal Gbetagamma-binding segment found in both subunits is located approximately between amino acids 320 and 409 (by GIRK1 count). Mutation of C-terminal leucines 262 or 333 in GIRK1, recognized previously as crucial for Gbetagamma regulation of the channel, and of the corresponding leucines 273 and 344 in GIRK2 dramatically altered the properties of K+ currents via GIRK1/GIRK2 channels expressed in Xenopus oocytes but did not appreciably reduce the binding of Gbetagamma to the corresponding fusion proteins, indicating that these residues are mainly important for the regulation of Gbetagamma-induced changes in channel gating rather than Gbetagamma binding.     

Low resolution structure of human erythrocyte glutathione reductase

Glutathione reductase from human erythrocytes is a dimeric flavoenzyme with a molecular weight of 100,000. X-ray diffraction analysis using the isomorphous replacement technique with four heavy-atom derivatives yielded an electron density map at 6 Å resolution with a figure of merit of 0.88. Only minor cuts had to be made in the electron density map to isolate one molecule. The dimer interface is on a crystallographic 2-fold axis. Each subunit can be subdivided into three domains: I, II and III, which are aggregated in such a way that deep clefts are formed on opposite sides of the subunit. These clefts accommodate the substrate glutathione, binding to domain III, and the oxidized cofactor NADP, binding to domain I in a similar extended conformation as NAD binds to the dehydrogenases. The shortest connection between the centres of the nicotinamide ring of NADP and the cystine of oxidized glutathione is 18 Å long and goes along the interface between domains II and III right through the centre of the subunit. Presumably, FAD binds to domain II and its isoalloxazine ring bridges the gap between NADP and glutathione.     

The structural subunit of glucose-6-phosphate dehydrogenase (baker's yeast).

The subunit molecular weight of glucose-6-phosphate dehydrogenase (G6PD) from baker's yeast has been evaluated. The subunit molecular weight value is shown to be 25,500 daltons by analytical ultracentrifugation, SDS-polyacrylamide gel electrophoresis, and the number of peptides produced by CNBr cleavage. The number of NADP binding sites was determined to be one per 25,500 dalton unit.     

Effects of beta subunit acylation on lutropin receptor site binding.

The effects of various modifications on the beta subunit of lutropin have been studied using the binding characteristics of the reconstituted hormone in the rat testicular radioligand assay. Conditions for iodinating lutropin and lutropin derivatives were determined which resulted in 15 per cent specific binding when tested immediately and retention of 6 to 7 per cent specific binding even after storage for 6 months. Acetimidinyl, acetyl, and carbamyl derivatives of the beta subunit were prepared and combined with unmodified alpha subunit to form reconstituted lutropin. Modification of the beta subunit was shown to have no effect on the time course of binding to testicular receptors or, with one exception, on the extent of receptor saturation. Very high concentrations of lutropin reconstituted with acetylated beta subunit showed an anomalous binding behavior. Scatchard plots of the binding data support the view that the native hormone has a unique receptor affinity which is irreversibly disrupted by separation of subunits and that derivatization of the beta subunit does not alter this parameter further. These data also suggest that there are no significant differences in the amino groups modified on the beta subunit. Competition and preincubation tests for receptor sites that reacted only with modified lutropin and not with the native hormone were negative.     

Predicted structures of cAMP binding domains of type I and II regulatory subunits of cAMP-dependent protein kinase

The mammalian cAMP-dependent protein kinases have regulatory (R) subunits that show substantial homology in amino acid sequence with the catabolite gene activator protein (CAP), a cAMP-dependent gene regulatory protein from Escherichia coli. Each R subunit has two in-tandem cAMP binding domains, and the structure of each of these domains has been modeled by analogy with the crystal structure of CAP. Both the type I and II regulatory subunits have been considered, so that four cAMP binding domains have been modeled. The binding of cAMP in general is analogous in all the structures and has been correlated with previous results based on photolabeling and binding of cAMP analogues. The model predicts that the first cAMP binding domain correlates with the previously defined fast dissociation site, which preferentially binds N6-substituted analogues of cAMP. The second domain corresponds to the slow dissociation site, which has a preference for C8-substituted analogues. The model also is consistent with cAMP binding in the syn conformation in both sites. Finally, this model has targeted specific regions that are likely to be involved in interdomain contacts. This includes contacts between the two cAMP binding domains as well as contacts with the amino-terminal region of the R subunit and with the catalytic subunit.     

Meanderings of the mRNA through the ribosome

A map of how mRNA travels through the ribosome is critical for any detailed understanding of the process of translation. This feat has recently been achieved using X-ray crystallography. The structure reveals, for the first time, details of the interactions between the mRNA and the 30S subunit beyond those at the tRNA binding sites. Elements of both 16S rRNA and ribosomal proteins contribute to mRNA binding. This work also identifies two tunnels that the mRNA passes through as it wraps around the 30S subunit. The mechanisms and mechanics of reading frame selection, translational fidelity, and translocation can now be informed by the structure.     

Structure of yeast hexokinase. II. A 6 angstrom resolution electron density map showing molecular shape and heterologous interaction of subunits

An electron density map of yeast hexokinase has been calculated at 6 Å resolution using six heavy atom derivatives. The map shows each of the enzyme's two 51,000 molecular weight subunits to consist of two separate lobes connected by a narrow bridge of density. Furthermore, these two subunits are related to each other in the asymmetric unit of the crystal by a quasi-2-fold rather than a true 2-fold axis. That is, they are related by a rotation of 180 ° plus a relative translation of 3.6 Å along the symmetry axis. This gives rise to a heterologous subunit interaction and a possibility of non-identical structure and function for these chemically identical subunits. The molecule is quite asymmetric, having dimensions of 150 Å × 45 Å × 55 Å. Each subunit is about 80 Å × 40 Å × 50 Å.A portion of an electron density map at 3 Å resolution has been also calculated, based on phases from two heavy atom derivatives. Polypeptide backbone and side chains are visible in this map.     

Mechanism of action of the wheat germ ribosome dissociation factor: Interaction with the 60 S subunit

Recently a ribosome dissociation factor that stimulates natural mRNA translation has been isolated from extracts of wheat germ. In this investigation, we have studied the subunit site of action of the purified ribosome dissociation factor (eucaryotic initiation), eIF-6. The following evidence strongly indicates that eIF-6 acts as a dissociation factor by binding to the 60 S ribosomal subunit and preventing its interaction with the 40 S subunit. Incubation of 60 S subunits with eIF-6 reduces the formation of 80 S monosomes when 40 S subunits are subsequently added at 5 mm Mg²⁺. The 40 S subunits preincubated with eIF-6 reassociate normally with 60 S subunits. ¹⁴C-labeled eIF-6 binds to 60 S subunits but not to 40 S subunits. Slight binding to 80 S ribosomes is also observed. The interaction of eIF-6 with the 60 S subunit requires an elevated temperature, and occurs rapidly at 37 °C.     

Topological mapping of 13 epitopes on a subunit of Androctonus australis hemocyanin.

A topological localization of epitopes on the surface of the Aa6 subunit of Androctonus australis hemocyanin has been carried out. First, immunocomplex strings composed of native hemocyanin and monoclonal antibodies were examined in the electron microscope and submitted to an image processing by correspondence analysis. The average images were then compared to a three-dimensional model of the 24-mer suggesting that 11 of the 13 epitopes are located in three zones of the subunit surface. Second, the overlaps between the epitopes were then studied by polyacrylamide gel electrophoresis, competitive binding inhibition, and immunoelectron microscopy. Four groups of epitopes were identified. One group was capable of binding exclusively to the free subunit. The other three groups were identical to those found in immunoelectron microscopy. The data are consistent with the existence of a small number of immunodominant regions on the surface of the Aa6 subunit.     

Assembly of the 30S ribosomal subunit: positioning ribosomal protein S13 in the S7 assembly branch

Studies of Escherichia coli 30S ribosomal subunit assembly have revealed a hierarchical and cooperative association of ribosomal proteins with 16S ribosomal RNA; these results have been used to compile an in vitro 30S subunit assembly map. In single protein addition and omission studies, ribosomal protein S13 was shown to be dependent on the prior association of ribosomal protein S20 for binding to the ribonucleoprotein particle. While the overwhelming majority of interactions revealed in the assembly map are consistent with additional data, the dependency of S13 on S20 is not. Structural studies position S13 in the head of the 30S subunit > 100 A away from S20, which resides near the bottom of the body of the 30S subunit. All of the proteins that reside in the head of the 30S subunit, except S13, have been shown to be part of the S7 assembly branch, that is, they all depend on S7 for association with the assembling 30S subunit. Given these observations, the assembly requirements for S13 were investigated using base-specific chemical footprinting and primer extension analysis. These studies reveal that S13 can bind to 16S rRNA in the presence of S7, but not S20. Additionally, interaction between S13 and other members of the S7 assembly branch have been observed. These results link S13 to the 3' major domain family of proteins, and the S7 assembly branch, placing S13 in a new location in the 30S subunit assembly map where its position is in accordance with much biochemical and structural data.     

Glucose-6-phosphate isomerase

Glucose-6-phosphate isomerase (EC 5.3.1.9) is a dimeric enzyme of molecular mass 132000 which catalyses the interconversion of D-glucose-6-phosphate and D-fructose-6-phosphate. The crystal structure of the enzyme from pig muscle has been determined at a nominal resolution of 2.6 A. The structure is of the alpha/beta type. Each subunit consists of two domains and the active site is in both the domain interface and the subunit interface (P.J. Shaw & H. Muirhead (1976), FEBS Lett. 65, 50-55). Each subunit contains 13 methionine residues so that cyanogen bromide cleavage will produce 14 fragments, most of which have been identified and at least partly purified. Sequence information is given for about one-third of the molecule from 5 cyanogen bromide fragments. One of the sequences includes a modified lysine residue. Modification of this residue leads to a parallel loss of enzymatic activity. A tentative fit of two of the peptides to the electron density map has been made. It seems possible that glucose-6-phosphate isomerase, triose phosphate isomerase and pyruvate kinase all contain a histidine and a glutamate residue at the active site.     

Three-dimensional structure of p-cresol methylhydroxylase (flavocytochrome c) from Pseudomonas putida at 3.0-A resolution.

p-Cresol methylhydroxylase (PCMH) isolated from Pseudomonas putida is an alpha 2 beta 2 tetramer of approximate subunit Mr 49,000 and 9,000. It is a flavocytochrome c containing covalently bound FAD in the larger subunit and covalently bound heme in the smaller. Crystals in space group P2(1)2(1)2(1) with unit-cell parameters a = 140.3 A, b = 130.6 A, and c = 74.1 A contain one full molecule per asymmetric unit and diffract anisotropically to about 2.8-A resolution in two directions and to about 3.3-A resolution in the third. An electron density map has been computed at a nominal resolution of 3.0 A by use of area detector data from native crystals and from two derivatives. The phases were improved with the B.C. Wang solvent leveling procedure, and the map was averaged about the noncrystallographic 2-fold axis. The cytochrome subunit, whose amino acid sequence is known, has been fitted to the electron density on a graphics system. The course of the polypeptide chain of the flavoprotein subunit, whose sequence is mostly unknown, has been traced in a minimap and a model of polyalanine fitted to the electron density on the graphics system. The flavoprotein subunit consists of three domains in close contact. The N-terminal domain consists largely of beta-structure and contains most of the FAD binding site. The second domain contains a seven-stranded antiparallel beta-sheet of unusual topology connected by antiparallel alpha-helices on one side. The flavin ring lies at the juncture of the first two domains. The third domain lies against the first domain and helps cover the rest of the FAD chain. The cytochrome subunit resembles other small cytochromes such as c-551 and c5 and fits into a depression on the surface of the large flavoprotein subunit. The flavin and heme planes are nearly perpendicular, the normals to the planes being approximately 65 degrees apart. The two groups are separated by about 8 A, the distance from one of the vinyl methylene carbon atoms of the heme to the 8 alpha-methyl group of the flavin ring.     

Characterization of the nuclear export adaptor protein Nmd3 in association with the 60S ribosomal subunit

The nucleocytoplasmic shuttling protein Nmd3 is an adaptor for export of the 60S ribosomal subunit from the nucleus. Nmd3 binds to nascent 60S subunits in the nucleus and recruits the export receptor Crm1 to facilitate passage through the nuclear pore complex. In this study, we present a cryoelectron microscopy (cryo-EM) reconstruction of the 60S subunit in complex with Nmd3 from Saccharomyces cerevisiae. The density corresponding to Nmd3 is directly visible in the cryo-EM map and is attached to the regions around helices 38, 69, and 95 of the 25S ribosomal RNA (rRNA), the helix 95 region being adjacent to the protein Rpl10. We identify the intersubunit side of the large subunit as the binding site for Nmd3. rRNA protection experiments corroborate the structural data. Furthermore, Nmd3 binding to 60S subunits is blocked in 80S ribosomes, which is consistent with the assigned binding site on the subunit joining face. This cryo-EM map is a first step toward a molecular understanding of the functional role and release mechanism of Nmd3.     

Structure of glycolate oxidase from spinach at a resolution of 5.5 Å

The crystal structure of glycolate oxidase from spinach has been determined to 5.5 Å resolution, using two isomorphous heavy-atom derivatives and their anomalous contributions. In the electron density map the boundaries of the octameric molecules are clearly seen. The subunit molecular weight is 37,000. Two protomers are in very close contact around one of the crystallographic 2-fold axes. Four such dimers are in contact around the 4-fold axis, so that the glycolate oxidase molecules are arranged as octamers with 422 symmetry in the crystal lattice. The roughly spherical octameric molecules have a diameter of approximately 100 Å. These octamers are arranged in a network, such that large solvent channels, approximately 60Å in diameter, pass right through the crystal lattice.The secondary structure of two-thirds of the subunit density has been interpreted in terms of eight consecutive β strand-α-helix units forming a cylinder very similar to the structure of triose phosphate isomerase. This interpretation is based on the very characteristic arrangement of the eight helices which form such a cylinder. The binding site of a substrate analogue, thioglycolate, has been localized in a deep cleft of the subunit at one end of the βα-barrel close to its axis.