Inhibition of enolase: the crystal structures of enolase-Ca2(+)- 2-phosphoglycerate and enolase-Zn2(+)-phosphoglycolate complexes at 2.2-A resolution

Enolase is a metalloenzyme which catalyzes the elimination of H2O from 2-phosphoglyceric acid (PGA) to form phosphoenolpyruvate (PEP). Mg2+ and Zn2+ are cofactors which strongly bind and activate the enzyme. Ca2+ also binds strongly but does not produce activity. Phosphoglycolate (PG) is a competitive inhibitor of enolase. The structures of two inhibitory ternary complexes: yeast enolase-Ca2(+)-PGA and yeast enolase-Zn2(+)-PG, were determined by X-ray diffraction to 2.2-A resolution and were refined by crystallographic least-squares to R = 14.8% and 15.7%, respectively, with good geometries of the models. These structures are compared with the structure of the precatalytic ternary complex enolase-Mg2(+)-PGA/PEP (Lebioda & Stec, 1991). In the complex enolase-Ca2(+)-PGA, the PGA molecule coordinates to the Ca2+ ion with the hydroxyl group, as in the precatalytic complex. The conformation of the PGA molecule is however different. In the active complex, the organic part of the PGA molecule is planar, similar to the product. In the inhibitory complex, the carboxylic group is in an orthonormal conformation. In the inhibitory complex enolase-Zn2(+)-PG, the PG molecule coordinates with the carboxylic group in a monodentate mode. In both inhibitory complexes, the conformational changes in flexible loops, which were observed in the precatalytic complex, do not take place. The lack of catalytic metal ion binding suggests that these conformational changes are necessary for the formation of the catalytic metal ion binding site.     

Structure of Plasmodium falciparum triose-phosphate isomerase-2-phosphoglycerate complex at 1.1-A resolution

Triose-phosphate isomerase, a key enzyme of the glycolytic pathway, catalyzes the isomerization of dihydroxy acetone phosphate and glyceraldehyde 3-phosphate. In this communication we report the crystal structure of Plasmodium falciparum triose-phosphate isomerase complexed to the inhibitor 2-phosphoglycerate at 1.1-A resolution. The crystallographic asymmetric unit contains a dimeric molecule. The inhibitor bound to one of the subunits in which the flexible catalytic loop 6 is in the open conformation has been cleaved into two fragments presumably due to radiation damage. The cleavage products have been tentatively identified as 2-oxoglycerate and meta-phosphate. The intact 2-phosphoglycerate bound to the active site of the other subunit has been observed in two different orientations. The active site loop in this subunit is in both open and "closed" conformations, although the open form is predominant. Concomitant with the loop closure, Phe-96, Leu-167, and residues 208-211 (YGGS) are also observed in dual conformations in the B-subunit. Detailed comparison of the active-site geometry in the present case to the Saccharomyces cerevisiae triose-phosphate isomerase-dihydroxy acetone phosphate and Leishmania mexicana triose-phosphate isomerase-phosphoglycolate complexes, which have also been determined at atomic resolution, shows that certain interactions are common to the three structures, although 2-phosphoglycerate is neither a substrate nor a transition state analogue.     

The crystal structure of ribonuclease B at 2.5-A resolution

The glycosylated form of bovine pancreatic ribonuclease, RNase B, was crystallized from polyethylene glycol 4000 at low ionic strength in space group C2 with unit cell dimensions of a = 101.81 A, b = 33.36 A, c = 73.60 A, and beta = 90.4 degrees. The crystals, which contained two independent molecules of RNase B as the asymmetric unit, were solved by a combination of multiple isomorphous replacement and molecular replacement approaches. The structures of the two molecules were refined to 2.5-A resolution and a conventional R factor of 0.22 using a constrained-restrained least squares procedure (CORELS). Complexes were also investigated of RNase B plus ruthenium pentaamine and between RNase B and a substrate analogue iodouridine. The polypeptide backbones of the two molecules of RNase B in the asymmetric unit were found to be statistically identical and their differences from RNase A to be statistically insignificant. The carbohydrate chains of both molecules extended into solvent cavities in the crystal lattice and appear to be disordered for the most part. The oligosaccharides appear to exert no influence on the structure of the protein. Iodouridine was observed to bind identically in the pyrimidine site of both RNase B molecules and in a way apparently the same as that previously observed for RNase A. Ruthenium pentaamine bound at histidine 105 of both RNase B molecules in the asymmetric unit, but at a number of secondary sites as well. An array of bound ions was observed by Fo-Fc difference Fourier syntheses. These ions were proximal to lysine and arginine residues at the surface of the proteins while a pair of strong ion binding sites were seen to fall exactly in the active site clefts of both RNase B molecules in the asymmetric unit.     

Draft crystal structure of the vault shell at 9-A resolution

Vaults are the largest known cytoplasmic ribonucleoprotein structures and may function in innate immunity. The vault shell self-assembles from 96 copies of major vault protein and encapsulates two other proteins and a small RNA. We crystallized rat liver vaults and several recombinant vaults, all among the largest non-icosahedral particles to have been crystallized. The best crystals thus far were formed from empty vaults built from a cysteine-tag construct of major vault protein (termed cpMVP vaults), diffracting to about 9-A resolution. The asymmetric unit contains a half vault of molecular mass 4.65 MDa. X-ray phasing was initiated by molecular replacement, using density from cryo-electron microscopy (cryo-EM). Phases were improved by density modification, including concentric 24- and 48-fold rotational symmetry averaging. From this, the continuous cryo-EM electron density separated into domain-like blocks. A draft atomic model of cpMVP was fit to this improved density from 15 domain models. Three domains were adapted from a nuclear magnetic resonance substructure. Nine domain models originated in ab initio tertiary structure prediction. Three C-terminal domains were built by fitting poly-alanine to the electron density. Locations of loops in this model provide sites to test vault functions and to exploit vaults as nanocapsules.     

The crystal structure of erabutoxin a at 2.0-A resolution

The three-dimensional structure of erabutoxin a, a single-chain, 62-residue protein neurotoxin from snake venom, has been determined to 2.0-A resolution by x-ray crystal structure analysis. Molecular replacement methods were used, and the structure refined to a residual R = 0.17. The sites of 62 water molecules and 1 sulfate ion have been located and refined. The structure of erabutoxin a is very similar to that established earlier for erabutoxin b. These toxins from venom of the same snake differ in sequence only at residue 26, which is Asn in erabutoxin a and His in erabutoxin b. The substitution leads to only minor variations in intramolecular hydrogen bonding. Furthermore, the distribution of thermal parameters and the implied regional mobilities are similar in the two structures. In particular, the highly mobile character of the peripheral segment Pro44-Gly49 in both structures supports the specific role proposed for this segment in neurotoxin binding to the acetylcholine receptor. Forty-eight of the solvent sites determined are first surface positions; approximately one-half of these are equivalent to solvent sites in erabutoxin b.     

The crystal structure of pea lectin at 3.0-A resolution

The structure of pea lectin has been determined to 3.0-A resolution based on multiple isomorphous replacement phasing to 6.0-A resolution and a combination of single isomorphous replacement, anomalous scattering, and density modification to 3.0-A resolution. The pea lectin model has been optimized by restrained least squares refinement against the data between 7.0- and 3.0-A resolution. The final model at 3.0 A gives an R factor of 0.24 and a root mean square deviation from ideal bond distances of 0.02 A. The two monomers in the asymmetric unit are related by noncrystallographic 2-fold symmetry to form a dimer. Monomers were treated independently in modeling and refinement, but are found to be virtually identical at this resolution. The molecular structure of the pea lectin monomer is very similar to that of concanavalin A, the lectin from the jack bean. Similarities extend from secondary and tertiary structures to the occurrence of a cis-peptide bond and the pattern of coordination of the Ca2+ and Mn2+ ions. Differences between the two lectin structures are confined primarily to the loop regions and to the chain termini, which are different and give rise to the unusual permuted relationship between the pea lectin and concanavalin A protein sequences.     

Three-dimensional structure of the ribonuclease T1 2'-GMP complex at 1.9-A resolution

The complex formed between the enzyme ribonuclease T1 (EC 3.1.27.3) and its specific inhibitor 2'-guanylic acid (2'-GMP) has been refined to R = 0.180 using x-ray diffraction data to 1.9-A resolution. The protein molecule displays a compact fold; a 4.5 turn alpha-helix packed over an antiparallel beta-pleated sheet shields most of the hydrophobic interior of the protein against the solvent. The extended pleated sheet structure of ribonuclease T1 is composed of three long and four short strands building up a two-stranded minor beta-sheet near the amino terminus and a five-stranded major sheet in the interior of the protein molecule. In the complex with ribonuclease T1, the inhibitor 2'-guanylic acid adopts the syn-conformation and C2'-endo sugar pucker. Binding of the nucleotide is mainly achieved through amino acid residues 38-46 of the protein. The catalytically active amino acid residues of ribonuclease T1 (His40, Glu58, Arg77, and His92) are located within the major beta-sheet which, as evident from the analysis of atomic temperature factors, provides an environment of minimal local mobility. The geometry of the active site is consistent with a mechanism for phosphodiester hydrolysis where, in the transesterification step, His40 and/or Glu58 act as a general base toward the ribose 2'-hydroxyl group and His92, as a general acid, donates a proton to the leaving 5'-hydroxyl group.     

Crystal structure of the extracellular domain of mouse RANK ligand at 2.2-A resolution.

Bone remodeling involves the resorption of bone by osteoclasts and the synthesis of bone matrix by osteoblasts. Receptor activator of NF-kappa B ligand (RANKL, also known as ODF and OPGL), a member of the tumor necrosis factor (TNF) family, triggers osteoclastogenesis by forming a complex with its receptor, RANK. We have determined the crystal structure of the extracellular domain of mouse RANKL at 2.2-A resolution. The structure reveals that the RANKL extracellular domain is trimeric, which was also shown by analytical ultracentrifugation, and each subunit has a beta-strand jellyroll topology like the other members of the TNF family. A comparison of RANKL with TNF beta and TNF-related apoptosis-inducing ligand (TRAIL), whose structures were determined to be in the complex form with their respective receptor, reveals conserved and specific features of RANKL in the TNF superfamily and suggests the presence of key residues of RANKL for receptor binding.     

The crystal structure of mercury-substituted poplar plastocyanin at 1.9-A resolution

The crystal structure of Hg(II)-plastocyanin has been determined and refined at a resolution of 1.9 A. The crystals were prepared by soaking crystals of Cu(II)-plastocyanin from poplar leaves (Populus nigra var. italica) in a solution of a mercuric salt. Replacement of the Cu(II) atom in plastocyanin by Hg(II) causes only minor changes in the geometry of the metal site, and there are few significant changes elsewhere in the molecule. It is concluded that, as in the case of the native protein, the geometry of the metal site is determined by the polypeptide. The weak metal-S(methionine) bond found in Cu(II)-plastocyanin remains weak in Hg(II)-plastocyanin. The "flip" of a proline side chain close to the metal site from a C gamma-exo conformation in Cu(II)-plastocyanin to a C gamma-endo conformation in Hg(II)-plastocyanin suggests that this region of the molecule is particularly flexible. Crystallographic evidence for the close similarity of the Hg(II)- and Cu(II)-plastocyanin structures was originally obtained from electron density difference maps at 2.5-A resolution. The refinement of the structure was begun with a set of atomic coordinates taken from the structure of Cu(II)-plastocyanin. A Hg(II) atom was substituted for the Cu(II) atom, and the side chains of 6 residues in the vicinity of the metal site were omitted. Three series of stereochemically restrained least-squares refinement calculations were interspersed with two stages of model adjustment followed by phase extension. Fifty-nine water molecules were located. The final structure has a crystallographic residual R = 0.16.     

Crystal structure of allophycocyanin from red algae Porphyra yezoensis at 2.2-A resolution.

The crystal structure of allophycocyanin from red algae Porphyra yezoensis (APC-PY) at 2.2-A resolution has been determined by the molecular replacement method. The crystal belongs to space group R32 with cell parameters a = b = 105.3 A, c = 189.4 A, alpha = beta = 90 degrees, gamma = 120 degrees. After several cycles of refinement using program X-PLOR and model building based on the electron density map, the crystallographic R-factor converged to 19.3% (R-free factor is 26.9%) in the range of 10.0 to 2.2 A. The r.m.s. deviations of bond length and angles are 0.015 A and 2.9 degrees, respectively. In the crystal, two APC-PY trimers associate face to face into a hexamer. The assembly of two trimers within the hexamer is similar to that of C-phycocyanin (C-PC) and R-phycoerythrin (R-PE) hexamers, but the assembly tightness of the two trimers to the hexamer is not so high as that in C-PC and R-PE hexamers. The chromophore-protein interactions and possible pathway of energy transfer were discussed. Phycocyanobilin 1alpha84 of APC-PY forms 5 hydrogen bonds with 3 residues in subunit 2beta of another monomer. In R-PE and C-PC, chromophore 1alpha84 only forms 1 hydrogen bond with 2beta77 residue in subunit 2beta. This result may support and explain great spectrum difference exists between APC trimer and monomer.     

Refined crystal structure of calcium-liganded carp parvalbumin 4.25 at 1.5-A resolution

The crystal structure of carp parvalbumin (pI = 4.25) has been refined by restrained least-squares analysis employing X-ray diffractometer data to 1.5-A resolution. The final residual for 12,653 reflections between 10 and 1.5 A with I(hkl) greater than 2 sigma(I) is 0.215. A total of 74 solvent molecules were included in the least-squares analysis. The root mean square deviation from ideality of bond lengths is 0.024 A. The model has a root mean square difference of 0.59 A from the positions of the main-chain atoms in a previously reported structure [Moews, P. C., & Kretsinger, R. H. (1975) J. Mol. Biol. 91, 201-228], which was refined by difference Fourier syntheses using data collected by film to 1.9 A. Although the overall features of the two models are very similar, there are significant differences in the amino-terminal region, which was extensively refit, and in the number of oxygen atoms liganding calcium in the CD and EF sites, which increased from six to seven in the CD site and decreased from eight to seven in the EF site.     

Ribonuclease structure and catalysis: crystal structure of sulfate-free native ribonuclease A at 1.5-A resolution

The structure of native bovine pancreatic ribonuclease A, without the inhibitory sulfate anion normally bound at the active site, has been determined by X-ray diffraction at 1.53-A resolution. Treatment of a crystal of ribonuclease containing sulfate with an alkaline buffer released most of the sulfate anions. On return to active pH, few of the side chains moved, and the backbone structure remained unchanged. The active site conformation was essentially unchanged except for the replacement of the sulfate anion by a water molecule, which is hydrogen-bonded to histidine-12 and to another water, and for a small movement of the side chain of lysine-41. Histidines-12 and -119, the catalytic basic and acidic residues, have not moved. Thus the distance between them, and the presence of an intervening water, prohibits the possibility of their being hydrogen-bonded together. The structure has been refined by restrained least squares to an R factor of 0.17. Analysis of individual atomic temperature factors indicates that the molecule has become less rigid in general but that some regions were particularly affected by loss of the sulfate, while others were relatively unaffected. The active site geometry of native ribonuclease A supports the original in-line mechanism of Rabin and co-workers and is in disagreement with the adjacent mechanism of Witzel and co-workers.     

Metal substitution in transferrins: the crystal structure of human copper-lactoferrin at 2.1-A resolution.

The structural consequences of binding a metal other than iron to a transferrin have been examined by crystallographic analysis of human copper-lactoferrin, Cu2Lf. X-ray diffraction data were collected from crystals of Cu2Lf, using a diffractometer, to 2.6-A resolution, and oscillation photography on a synchrotron source, to 2.1-A resolution. The structure was refined crystallographically, by restrained least-squares methods, starting with a model based on the isomorphous diferric structure from which the ligands, metal ions, anions, and solvent molecules had been deleted. The final model, comprising 5321 protein atoms (691 residues), 2 Cu2+ ions, 2 (bi)carbonate ions, and 308 solvent molecules has good stereochemistry (rms deviation of bond lengths from standard values of 0.018 A) and gives a crystallographic R value of 0.196 for 43,525 reflections in the range 7.5-2.1-A resolution. The copper coordination is different in the two binding sites. In the N-terminal site, the geometry is square pyramidal, with equatorial bonds to Asp 60, Tyr 192, His 253, and a monodentate anion and a longer apical bond to Tyr 92. In the C-terminal site, the geometry is distorted octahedral, with bonds to Asp 395, Tyr 435, Tyr 528, and His 597 and an asymmetrically bidentate anion. The protein structure is the same as for the diferric protein, Fe2Lf, demonstrating that the closure of the protein domains over the metal is the same in each case irrespective of whether Fe3+ or Cu2+ is bound and that copper could be transported and delivered to cells equally well as iron. The differences in metal coordination are achieved by small movements of the metal ion and anion within each binding site, which do not affect the protein structure.     

X-ray structure of a serine protease acyl-enzyme complex at 0.95-A resolution

Kinetic analyses led to the discovery that N-acetylated tripeptides with polar residues at P3 are inhibitors of porcine pancreatic elastase (PPE) that form unusually stable acyl-enzyme complexes. Peptides terminating in a C-terminal carboxylate were more potent than those terminating in a C-terminal amide, suggesting recognition by the oxy-anion hole is important in binding. X-ray diffraction data were recorded to 0.95-A resolution for an acyl-enzyme complex formed between PPE and N-acetyl-Asn-Pro-Ile-CO2H at approximately pH 5. The accuracy of the crystallographic coordinates allows structural issues concerning the mechanism of serine proteases to be addressed. Significantly, the ester bond of the acyl-enzyme showed a high level of planarity, suggesting geometric strain of the ester link is not important during catalysis. Several hydrogen atoms could be clearly identified and were included within the model. In keeping with a recent x-ray structure of subtilisin at 0.78 A (1), limited electron density is visible consistent with the putative location of a hydrogen atom approximately equidistant between the histidine and aspartate residues of the catalytic triad. Comparison of this high resolution crystal structure of the acyl-enzyme complex with that of native elastase at 1.1 A (2) showed that binding of the N-terminal part of the substrate can be accommodated with negligible structural rearrangements. In contrast, comparison with structures obtained as part of "time-resolved" studies on the reacting acyl-enzyme complex at >pH 7 (3) indicate small but significant structural differences, consistent with the proposed synchronization of ester hydrolysis and substrate release.     

Wolinella succinogenes quinol:fumarate reductase-2.2-A resolution crystal structure and the E-pathway hypothesis of coupled transmembrane proton and electron transfer

The structure of the respiratory membrane protein complex quinol:fumarate reductase (QFR) from Wolinella succinogenes has been determined by X-ray crystallography at 2.2-A resolution [Nature 402 (1999) 377]. Based on the structure of the three protein subunits A, B, and C and the arrangement of the six prosthetic groups (a covalently bound FAD, three iron-sulfur clusters, and two haem b groups), a pathway of electron transfer from the quinol-oxidising dihaem cytochrome b in the membrane to the site of fumarate reduction in the hydrophilic subunit A has been proposed. The structure of the membrane-integral dihaem cytochrome b reveals that all transmembrane helical segments are tilted with respect to the membrane normal. The "four-helix" dihaem binding motif is very different from other dihaem-binding transmembrane four-helix bundles, such as the "two-helix motif" of the cytochrome bc(1) complex and the "three-helix motif" of the formate dehydrogenase/hydrogenase group. The gamma-hydroxyl group of Ser C141 has an important role in stabilising a kink in transmembrane helix IV. By combining the results from site-directed mutagenesis, functional and electrochemical characterisation, and X-ray crystallography, a residue was identified which was found to be essential for menaquinol oxidation [Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 13051]. The distal location of this residue in the structure indicates that the coupling of the oxidation of menaquinol to the reduction of fumarate in dihaem-containing succinate:quinone oxidoreductases could in principle be associated with the generation of a transmembrane electrochemical potential. However, it is suggested here that in W. succinogenes QFR, this electrogenic effect is counterbalanced by the transfer of two protons via a proton transfer pathway (the "E-pathway") in concert with the transfer of two electrons via the membrane-bound haem groups. According to this "E-pathway hypothesis", the net reaction catalysed by W. succinogenes QFR does not contribute directly to the generation of a transmembrane electrochemical potential.     

X-ray crystal structure of the bovine alpha-chymotrypsin/eglin c complex at 2.6 A resolution

The crystal structure of the molecular complex formed by bovine alpha-chymotrypsin and the recombinant serine proteinase inhibitor eglin c from Hirudo medicinalis has been solved using monoclinic crystals of the complex, reported previously. Four circle diffractometer data at 3.0 A resolution were employed to determine the structure by molecular replacement techniques. Bovine alpha-chymotrypsin alone was used as the search model; it allowed us to correctly orient and translate the enzyme in the unit cell and to obtain sufficient electron density for positioning the eglin c molecule. After independent rigid body refinement of the two complex components, the molecular model yielded a crystallographic R factor of 0.39. Five iterative cycles of restrained crystallographic refinement and model building were conducted, gradually increasing resolution. The current R factor at 2.6 A resolution (diffractometer data) is 0.18. The model includes 56 solvent molecules. Eglin c binds to bovine alpha-chymotrypsin in a manner consistent with other known serine proteinase/inhibitor complex structures. The reactive site loop shows the expected conformation for productive binding and is in tight contact with bovine alpha-chymotrypsin between subsites P3 and P'2; Leu 451 acts as the P1 residue, located in the primary specificity S1 site of the enzyme. Hydrogen bonds equivalent to those observed in complexes of trypsin(ogen) with the pancreatic basic- and secretory-inhibitors are found around the scissile peptide bond.     

X-ray structure of a biantennary octasaccharide-lectin complex refined at 2.3-A resolution.

The structure and flexibility of saccharides have a profound and specific influence in several biological processes such as protein protection and the maintenance of conformational integrity, and in recognition events involving viruses, enzymes, and lectins. To establish the structural bases of these phenomena, we describe herein the extensively refined 2.3-A resolution x-ray structure of a biantennary octasaccharide of the N-acetyllactosamine type, complexed to isolectin I from Lathyrus ochrus. The two octasaccharides are located in clefts at each end of the long axis of the lectin. The conformations of both the lectin and the saccharide are slightly modified upon binding. The complex is stabilized by numerous hydrogen bonds, many of them involving water molecules. It is also stabilized by van der Waals interactions, including some with aromatic residues. A more general model of a possible lectin-glycoprotein interaction is also proposed.     

The refined crystal structure of a fully active semisynthetic ribonuclease at 1.8-A resolution

A fully active, semisynthetic analog of bovine ribonuclease A, comprised of residues 1-118 of the molecule in a noncovalent complex with the synthetic peptide analog of residues 111-124, has been crystallized in space group P3(2)21 from a solution of 1.3 M ammonium sulfate and 3.0 M cesium chloride at pH 5.2. The crystallographic structure was determined by rotation and translation searches utilizing the coordinates for ribonuclease A reported by Wlodawer and Sjolin (Wlodawer, A., and Sjolin, L. (1983) Biochemistry 22, 2720-2728) and has been refined at 1.8-A resolution to an agreement factor of 0.204. Most of the structure of the semisynthetic enzyme closely resembles that found in ribonuclease A with the synthetic peptide replacing the C-terminal elements of the naturally occurring enzyme. No redundant structure is seen; residues 114-118 of the larger chain and residues 111-113 of the peptide do not appear in our map. The positions of those residues at or near the active site are very similar to, if not identical with, those previously reported by others, except for histidine 119, which occupies predominantly the B position seen as a minor site by Borkakoti et al. (Borkakoti, N., Moss, D. S., and Palmer, R. A. (1982) Acta Crystallogr. Sect. B Struct. Crystallogr. Cryst. Chem. 38,2210-2217) and not at all by Wlodawer and Sjolin (1983).     

The refined crystal structure of alpha-cobratoxin from Naja naja siamensis at 2.4-A resolution

The crystal structure of the "long" alpha-neurotoxin alpha-cobratoxin was refined to an R-factor of 19.5% using 3271 x-ray data to 2.4-A resolution. The polypeptide chain forms three loops, I, II, III, knotted together by four disulfide bridges, with the most prominent, loop II, containing another disulfide close to its lower tip. Loop I is stabilized by one beta-turn and two beta-sheet hydrogen bonds; loop II by eight beta-sheet hydrogen bonds, with the tip folded into two distorted right-handed helical turns stabilized by two alpha-helical and two beta-turn hydrogen bonds; and loop III by hydrophobic interactions and one beta-turn. Loop II and one strand of loop III form an antiparallel triple-pleated beta-sheet, and tight anchoring of the Asn63 side chain fixes the tail segment. In the crystal lattice, the alpha-cobratoxin molecules dimerize by beta-sheet formation between strands 53 and 57 of symmetry-related molecules. Because such interactions are found also in a cardiotoxin and alpha-bungarotoxin, this could be of importance for interaction with acetylcholine receptor.     

Structure of a recombinant calmodulin from Drosophila melanogaster refined at 2.2-A resolution

The crystal structure of calmodulin (Mr 16,700, 148 residues) from Drosophila melanogaster as expressed in a bacterial system has been determined and refined at 2.2-A resolution. Starting with the structure of mammalian calmodulin, we produced an extensively refitted and refined model with a conventional crystallographic R value of 0.197 for the 5,239 reflections (F greater than or equal to 2 sigma (F)) within the 10.0-2.2-A resolution range. The model includes 1,164 protein atoms, 4 calcium ions, and 78 water molecules and has root mean square deviations from standard values of 0.018 A for bond lengths and 0.043 A for angle distances. The overall structure is similar to mammalian calmodulin, with a seven-turn central helix connecting the two calcium-binding domains. The "dumb-bell" shaped molecule contains seven alpha-helices and four "EF hand" calcium-binding sites. Although the amino acid sequences of mammalian and Drosophila calmodulins differ by only three conservative amino acid changes, the refined model reveals a number of significant differences between the two structures. Superimposition of the structures yields a root mean square deviation of 1.22 A for the 1,120 equivalent atoms. The calcium-binding domains have a root mean square deviation of 0.85 A for the 353 equivalent atoms. There are also differences in the amino terminus, the bend of the central alpha-helix, and the orientations of some of the side chains.