Detection of native peptides as potent inhibitors of enzymes. Crystal structure of the complex formed between treated bovine alpha-chymotrypsin and an autocatalytically produced fragment, IIe-Val-Asn-Gly-Glu-Glu-Ala-Val-Pro-Gly-Ser-Trp-Pro-Trp, at 2.2 angstroms resolution

Chymotrypsin is a prominent member of the family of serine proteases. The present studies demonstrate the presence of a native fragment containing 14 residues from Ile16 to Trp29 in alpha-chymotrypsin that binds to chymotrypsin at the active site with an exceptionally high affinity of 2.7 +/- 0.3 x 10(-11) M and thus works as a highly potent competitive inhibitor. The commercially available alpha-chymotrypsin was processed through a three phase partitioning system (TPP). The treated enzyme showed considerably enhanced activity. The 14 residue fragment was produced by autodigestion of a TPP-treated alpha-chymotrypsin during a long crystallization process that lasted more than four months. The treated enzyme was purified and kept for crystallization using vapour the diffusion method at 295 K. Twenty milligrams of lyophilized protein were dissolved in 1 mL of 25 mM sodium acetate buffer, pH 4.8. It was equilibrated against the same buffer containing 1.2 M ammonium sulfate. The rectangular crystals of small dimensions of 0.24 x 0.15 x 0.10 mm(3) were obtained. The X-ray intensity data were collected at 2.2 angstroms resolution and the structure was refined to an R-factor of 0.192. An extra electron density was observed at the binding site of alpha-chymotrypsin, which was readily interpreted as a 14 residue fragment of alpha-chymotrypsin corresponding to Ile-Val-Asn-Gly-Glu-Glu-Ala-Val-Pro-Gly-Ser-Trp-Pro-Trp(16-29). The electron density for the eight residues of the C-terminus, i.e. Ala22-Trp29, which were completely buried in the binding cleft of the enzyme, was of excellent quality and all the side chains of these eight residues were clearly modeled into it. However, the remaining six residues from the N-terminus, Ile16-Glu21 were poorly defined although the backbone density was good. There was a continuous electron density at 3.0 sigma between the active site Ser195 Ogamma and the carbonyl carbon atom of Trp29 of the fragment. The final refined coordinates showed a distance of 1.35 angstroms between Ser195 Ogamma and Trp29 C indicating the presence of a covalent linkage between the enzyme and the native fragment. This meant that the enzyme formed an acyl intermediate with the autodigested fragment Ile16-Trp29. In addition to the O-C covalent bond, there were several hydrogen bonds and hydrophobic interactions between the enzyme and the native fragment. The fragment showed a high complementarity with the binding site of alpha-chymotrypsin and the buried part of the fragment matched excellently with the corresponding buried part of Turkey ovomucoid inhibitor of alpha-chymotrypsin.     

Atomic resolution analysis of the catalytic site of an aspartic proteinase and an unexpected mode of binding by short peptides

The X-ray structures of native endothiapepsin and a complex with a hydroxyethylene transition state analog inhibitor (H261) have been determined at atomic resolution. Unrestrained refinement of the carboxyl groups of the enzyme by using the atomic resolution data indicates that both catalytic aspartates in the native enzyme share a single negative charge equally; that is, in the crystal, one half of the active sites have Asp 32 ionized and the other half have Asp 215 ionized. The electron density map of the native enzyme refined at 0.9 A resolution demonstrates that there is a short peptide (probably Ser-Thr) bound noncovalently in the active site cleft. The N-terminal nitrogen of the dipeptide interacts with the aspartate diad of the enzyme by hydrogen bonds involving the carboxyl of Asp 215 and the catalytic water molecule. This is consistent with classical findings that the aspartic proteinases can be inhibited weakly by short peptides and that these enzymes can catalyze transpeptidation reactions. The dipeptide may originate from autolysis of the N-terminal Ser-Thr sequence of the enzyme during crystallization.     

Stereochemistry of binding of the tetrapeptide acetyl-Pro-Ala-Pro-Tyr-NH2 to porcine pancreatic elastase. Combined use of two-dimensional transferred nuclear Overhauser enhancement measurements, restrained molecular dynamics, X-ray crystallography and molecular modelling

A nuclear magnetic resonance study of the conformation of the tetrapeptide acetyl-Pro-Ala-Pro-Tyr-NH2 bound to porcine pancreatic elastase is presented. From two-dimensional transferred nuclear Overhauser enhancement measurements, a set of 23 approximate distance restraints between pairs of bound ligand protons, indicative of an extended type structure, is derived. The structure of the bound tetrapeptide is then refined from two different starting structures (an extended beta-strand and a polyproline helix) by restrained molecular dynamics, in which the interproton distances are incorporated into the total energy of the system in the form of effective potentials. Convergence to essentially the same average restrained dynamics structures is achieved. The refined structures are then modelled into the active site of elastase by interactive molecular graphics. The determination of the anchor point of the bound tetrapeptide on the enzyme was aided by a simultaneous crystallographic study which, despite the fact that only electron density for a Pro-X dipeptide fragment was visible, enabled both the approximate position and orientation of binding to be determined. It is found that the tetrapeptide is bound in the S' binding site in the reverse orientation found in other serine protease-inhibitor complexes and is stabilized both by hydrogen-bonding and by van der Waals' interactions.     

Structure of gamma-chymotrypsin in the range pH 2.0 to pH 10.5 suggests that gamma-chymotrypsin is a covalent acyl-enzyme adduct at low pH

Crystals of gamma-chymotrypsin (gamma-CHT) grown at pH 7.0 are stable from pH 2.0 to 11.0. Crystalline gamma-CHT therefore provides an unusually favourable system to observe the structure of a protein and its bound solvent over a broad range of pH. In this report we describe the high-resolution refined structure of gamma-CHT at pH values of 2.0, 7.0 and 10.5. The apparent tetrapeptide seen bound in the active site of gamma-CHT at pH 7.0 is also present at pH 2.0 and 10.5 although it is better defined at low pH. A comparison of the respective structures shows that there is additional electron density in the low pH structure at the point where the side-chain of Ser 195 approaches most closely to the presumptive inhibitor. This suggests that the adduct is most likely to be covalently linked to the enzyme at low pH and to be non-covalent at higher pH. As the pH is lowered from 7.0 to 2.0, the side-chain of His 40 rotates approximately 120 degrees about its C alpha-C beta bond and, in concert, the side-chain of Gln 34 also rotates approximately 140 degrees about its C alpha-C beta bond. Apart from these localized rearrangements in the vicinity of His 40, the structure of gamma-CHT at pH 2.0 is very similar to that at neutral pH. The structure of gamma-CHT at pH 10.5 is also seen to be almost identical with that at neutral pH. There is no indication that the internal salt bridge between Asp 194 and the alpha-amino group of lle 16 begins to dissociate at pH 10.5. With the exception of the vicinity of His 40, the structure of the bound solvent in the crystal structures at low, neutral and high pH is very similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polarization of cinnamoyl-CoA substrates bound to enoyl-CoA hydratase: correlation of (13)C NMR with quantum mechanical calculations and calculation of electronic strain energy

When alpha,beta-unsaturated substrates bind to the active site of enoyl-CoA hydratase, large spectral changes can be observed [D'Ordine, R. L., et al. (1994) Biochemistry 33, 12635-12643]. The differences in the isotropic magnetic shieldings of the free and active site-bound forms of the carbonyl, alpha-, and beta-carbons of the substrates, hexadienoyl-CoA, cinnamoyl-CoA, and (N,N-dimethyl-p-amino)cinnamoyl-CoA have been experimentally determined. The carbonyl and beta-carbons are all deshielded, while the alpha-carbons show increased shielding. These chemical shift perturbations are interpreted to suggest that the pi-electrons of the enoyl thiolester are polarized when bound at the active site. Using the crystal structure of (N,N-dimethyl-p-amino)cinnamoyl-CoA bound at the enzyme active site, the shielding tensors were calculated at three different levels of theory, up to a density functional theory model that included all of the contiguous active site residues. These calculations successfully reproduced the observed spectral changes and permitted the electronic polarization of the substrate to be quantified as an electron density difference map. The calculated electron density difference confirms the loss of electrons at the electrophilic beta-carbon and carbonyl carbon, while a slight increase in electron density at the alpha-carbon where proton donation occurs during the hydration reaction and a larger increase in electron density at the carbonyl oxygen are predicted. The energy required to polarize the electrons to the observed extent was calculated to be 3.2 kcal/mol. The force that provides the requisite energy for the polarization is the interaction of the electric field generated by the protein at the enzyme active site with the polarizable electrons of the substrate. Because the induced electronic polarization is along the predicted reaction pathway, the extent of substrate activation by the induced electronic strain is catalytically relevant.     

Two structures of alliinase from Alliium sativum L.: apo form and ternary complex with aminoacrylate reaction intermediate covalently bound to the PLP cofactor

Alliinase (alliin lyase EC 4.4.1.4), a PLP-dependent alpha, beta-eliminating lyase, constitutes one of the major protein components of garlic (Alliium sativum L.) bulbs. The enzyme is a homodimeric glycoprotein and catalyzes the conversion of a specific non-protein sulfur-containing amino acid alliin ((+S)-allyl-L-cysteine sulfoxide) to allicin (diallyl thiosulfinate, the well known biologically active component of freshly crushed garlic), pyruvate and ammonia. The enzyme was crystallized in the presence of (+S)-allyl-L-cysteine, forming dendrite-like monoclinic crystals. In addition, intentionally produced apo-enzyme was crystallized in tetragonal form. These structures of alliinase with associated glycans were resolved to 1.4 A and 1.61 A by molecular replacement. Branched hexasaccharide chains N-linked to Asn146 and trisaccharide chains N-linked to Asn328 are seen. The structure of hexasaccharide was found similar to "short chain complex vacuole type" oligosaccharide most commonly seen in plant glycoproteins. An unexpected state of the enzyme active site has been observed in the present structure. The electron density in the region of the cofactor made it possible to identify the cofactor moiety as aminoacrylate intermediate covalently bound to the PLP cofactor. It was found in the present structure to be stabilized by large number of interactions with surrounding protein residues. Moreover, the existence of the expected internal aldimine bond between the epsilon-amino group of Lys251 and the aldehyde of the PLP is ruled out on the basis of a distinct separation of electron density of Lys251. The structure of the active site cavity in the apo-form is nearly identical to that seen in the holo-form, with two sulfate ions, an acetate and several water molecules from crystallization conditions that replace and mimic the PLP cofactor.     

On the mechanism of biological methane formation: structural evidence for conformational changes in methyl-coenzyme M reductase upon substrate binding

Methyl-coenzyme M reductase (MCR) catalyzes the final reaction of the energy conserving pathway of methanogenic archaea in which methylcoenzyme M and coenzyme B are converted to methane and the heterodisulfide CoM-S-S-CoB. It operates under strictly anaerobic conditions and contains the nickel porphinoid F430 which is present in the nickel (I) oxidation state in the active enzyme. The known crystal structures of the inactive nickel (II) enzyme in complex with coenzyme M and coenzyme B (MCR-ox1-silent) and in complex with the heterodisulfide CoM-S-S-CoB (MCR-silent) were now refined at 1.16 A and 1.8 A resolution, respectively. The atomic resolution structure of MCR-ox1-silent describes the exact geometry of the cofactor F430, of the active site residues and of the modified amino acid residues. Moreover, the observation of 18 Mg2+ and 9 Na+ ions at the protein surface of the 300 kDa enzyme specifies typical constituents of binding sites for either ion. The MCR-silent and MCR-ox1-silent structures differed in the occupancy of bound water molecules near the active site indicating that a water chain is involved in the replenishment of the active site with water molecules. The structure of the novel enzyme state MCR-red1-silent at 1.8 A resolution revealed an active site only partially occupied by coenzyme M and coenzyme B. Increased flexibility and distinct alternate conformations were observed near the active site and the substrate channel. The electron density of the MCR-red1-silent state aerobically co-crystallized with coenzyme M displayed a fully occupied coenzyme M-binding site with no alternate conformations. Therefore, the structure was very similar to the MCR-ox1-silent state. As a consequence, the binding of coenzyme M induced specific conformational changes that postulate a molecular mechanism by which the enzyme ensures that methylcoenzyme M enters the substrate channel prior to coenzyme B as required by the active-site geometry. The three different enzymatically inactive enzyme states are discussed with respect to their enzymatically active precursors and with respect to the catalytic mechanism.     

Structures of partridge egg-white lysozyme with and without tri-N-acetylchitotriose inhibitor at 1.9 A resolution.

The three-dimensional structures of native partridge egg-white lysozyme (PEWL) and PEWL complexed with tri-N-acetylchitotriose inhibitor have been determined crystallographically and refined at 1.9 A resolution. Crystals of native and complexed protein are isomorphous and have space group and cell dimensions that are identical to those of hen egg-white lysozyme (HEWL) under similar crystallization conditions. Full occupancy of the trisaccharide in the inhibitor complex has allowed definitive modeling and refinement of all three sugar residues, located at subsites A, B, and C in the PEWL active site. A comparison has been made with HEWL/inhibitor complexes in which coordinates were either not refined (Blake CCF, et al., 1967, Proc R Soc B 167:378-388) or were refined at partial occupancy (Cheetham JC, Artymiuk PJ, Phillips DC, 1992, J Mol Biol 224:613-628). Although the loop comprising residues 70-75 is located on the surface of the protein and not near the active site, it appears to be affected indirectly by trisaccharide binding such that the loop shifts toward the active site and becomes relatively immobilized. The source of this loop movement appears to be the anchoring of Trp62, located in the active site cleft, as it forms a hydrogen bond with O6 of the N-acetylglucosamine at site C. Good electron density for the trisaccharide in the PEWL complex structure shows that Asp 101 is involved in hydrogen bonding interactions with the terminal sugar residue.     

Structural comparison suggests that thermolysin and related neutral proteases undergo hinge-bending motion during catalysis.

Crystal structures are known for three members of the bacterial neutral protease family: thermolysin from Bacillus thermoproteolyticus (TLN), the neutral protease from Bacillus cereus (NEU), and the elastase of Pseudomonas aeruginosa (PAE), both in free and ligand-bound forms. Each enzyme consists of an N-terminal and C-terminal domain with the active site formed at the junction of the two domains. Comparison of the different molecules reveals that the structure within each domain is well conserved, but there are substantial hinge-bending displacements (up to 16 degrees) of one domain relative to the other. These domain motions can be correlated with the presence or absence of bound inhibitor, as was previously observed in the specific example of PAE [Thayer, M.M., Flaherty, K.M., & McKay, D.B. (1991) J. Biol. Chem. 266, 2864-2871]. The binding of inhibitor appears to be associated with a reduction of the domain hinge-bending angle by 6-14 degrees and a closure of the "jaws" of the active site cleft by about 2 A. Crystallographic refinement of the structure of thermolysin suggests that electron density seen in the active site of the enzyme in the original structure determination probably corresponds to a bound dipeptide. Thus, the crystal structure appears to correspond to an enzyme-inhibitor or enzyme-product complex, rather than the free enzyme, as has previously been assumed.     

Role of N-Terminal Myristylation in the Structure and Regulation of cAMP-Dependent Protein Kinase

The catalytic (C) subunit of cAMP-dependent protein kinase [protein kinase A (PKA)] is a major target of cAMP signaling, and its regulation is of fundamental importance to biological processes. One mode of regulation is N-myristylation, which has eluded structural and functional characterization so far because most crystal structures are of the non-myristylated enzyme, are phosphorylated on Ser10, and generally lack electron density for the first 13 residues. We crystallized myristylated wild-type (WT) PKA and a K7C mutant as binary (bound to a substrate peptide) and ternary [bound to a substrate peptide and adenosine-5′-(β,γ-imido)triphosphate] complexes. There was clear electron density for the entire N-terminus in the binary complexes, both refined to 2.0 Å, and K7C ternary complex, refined to 1.35 Å. The N-termini in these three structures display a novel conformation with a previously unseen helix from residues 1 to 7. The K7C mutant appears to have a more stable N-terminus, and this correlated with a significant decrease in the B-factors for the N-terminus in the myr-K7C complexes compared to the WT binary complex. The N-terminus of the myristylated WT ternary complex, refined to 2.0 Å, was disordered as in previous structures. In addition to a more ordered N-terminus, the myristylated K7C mutant exhibited a 53% increase in k_cat. The effect of nucleotide binding on the structure of the N-terminus in the WT protein and the kinetic changes in the K7C protein suggest that myristylation or occupancy of the myristyl binding pocket may serve as a site for allosteric regulation in the C-subunit.     

Crystal structure of Methanobacterium thermoautotrophicum phosphoribosyl-AMP cyclohydrolase HisI

The metabolic pathway for histidine biosynthesis is interesting from an evolutionary perspective because of the diversity of gene organizations and protein structures involved. Hydrolysis of phosphoribosyl-AMP, the third step in the histidine biosynthetic pathway, is carried out by PR-AMP cyclohydrolase, the product of the hisI gene. The three-dimensional structure of PR-AMP cyclohydrolase from Methanobacterium thermoautotrophicum was solved and refined to 1.7 A resolution. The enzyme is a homodimer. The position of the Zn(2+)-binding site that is essential for catalysis was inferred from the positions of bound Cd(2+) ions, which were part of the crystallization medium. These metal binding sites include three cysteine ligands, two from one monomer and the third from the second monomer. The enzyme remains active when Cd(2+) is substituted for Zn(2+). The likely binding site for Mg(2+), also necessary for activity in a homologous cyclohydrolase, was also inferred from Cd(2+) positions and is comprised of aspartic acid side chains. The putative substrate-binding cleft is formed at the interface between the two monomers of the dimer. This fact, combined with the localization of the Zn(2+)-binding site, indicates that the enzyme is an obligate dimer.     

Crystal structures of open and closed forms of d-serine deaminase from Salmonella typhimurium - implications on substrate specificity and catalysis

Metabolism of D-amino acids is of considerable interest due to their key importance in cell structure and function. Salmonella typhimuriumd-serine deaminase (StDSD) is a pyridoxal 5' phosphate (PLP) dependent enzyme that catalyses degradation of D-Ser to pyruvate and ammonia. The first crystal structure of d-serine deaminase described here reveals a typical Foldtype II or tryptophan synthase β subunit fold of PLP-dependent enzymes. Although holoenzyme was used for crystallization of both wild-type StDSD (WtDSD) and selenomethionine labelled StDSD (SeMetDSD), significant electron density was not observed for the cofactor, indicating that the enzyme has a low affinity for the cofactor under crystallization conditions. Interestingly, unexpected conformational differences were observed between the two structures. The WtDSD was in an open conformation while SeMetDSD, crystallized in the presence of isoserine, was in a closed conformation suggesting that the enzyme is likely to undergo conformational changes upon binding of substrate as observed in other Foldtype II PLP-dependent enzymes. Electron density corresponding to a plausible sodium ion was found near the active site of the closed but not in the open state of the enzyme. Examination of the active site and substrate modelling suggests that Thr166 may be involved in abstraction of proton from the Cα atom of the substrate. Apart from the physiological reaction, StDSD catalyses α, β elimination of D-Thr, D-Allothr and L-Ser to the corresponding α-keto acids and ammonia. The structure of StDSD provides a molecular framework necessary for understanding differences in the rate of reaction with these substrates.     

Crystal structure of UDP-N-acetylmuramoyl-L-alanyl-D-glutamate: meso-diaminopimelate ligase from Escherichia coli

UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-diaminopimelate ligase is a cytoplasmic enzyme that catalyzes the addition of meso-diaminopimelic acid to nucleotide precursor UDP-N-acetylmuramoyl-l-alanyl-d-glutamate in the biosynthesis of bacterial cell-wall peptidoglycan. The crystal structure of the Escherichia coli enzyme in the presence of the final product of the enzymatic reaction, UDP-MurNAc-l-Ala-gamma-d-Glu-meso-A(2)pm, has been solved to 2.0 A resolution. Phase information was obtained by multiwavelength anomalous dispersion using the K shell edge of selenium. The protein consists of three domains, two of which have a topology reminiscent of the equivalent domain found in the already established three-dimensional structure of the UDP-N-acetylmuramoyl-l-alanine: D-glutamate-ligase (MurD) ligase, which catalyzes the immediate previous step of incorporation of d-glutamic acid in the biosynthesis of the peptidoglycan precursor. The refined model reveals the binding site for UDP-MurNAc-l-Ala-gamma-d-Glu-meso-A(2)pm, and comparison with the six known MurD structures allowed the identification of residues involved in the enzymatic mechanism. Interestingly, during refinement, an excess of electron density was observed, leading to the conclusion that, as in MurD, a carbamylated lysine residue is present in the active site. In addition, the structural determinant responsible for the selection of the amino acid to be added to the nucleotide precursor was identified.     

Crystal Structure of the Pig Pancreatic α-Amylase Complexed with Malto-Oligosaccharides

The structural X-ray map of a pig pancreatic α-amylase crystal soaked (and flash-frozen) with a maltopentaose substrate showed a pattern of electron density corresponding to the binding of oligosaccharides at the active site and at three surface binding sites. The electron density region observed at the active site, filling subsites ?3 through ?1, was interpreted in terms of the process of enzyme-catalyzed hydrolysis undergone by maltopentaose. Because the expected conformational changes in the “flexible loop” that constitutes the surface edge of the active site were not observed, the movement of the loop may depend on aglycone site being filled. The crystal structure was refined at 2.01 å resolution to an R factor of 17.0% (R _free factor of 19.8%). The final model consists of 3910 protein atoms, one calcium ion, two chloride ions, 103 oligosaccharide atoms, 761 atoms of water molecules, and 23 ethylene glycol atoms.     

Stable copper-nitrosyl formation by nitrite reductase in either oxidation state

Nitrite reductase (NiR) is an enzyme that uses type 1 and type 2 copper sites to reduce nitrite to nitric oxide during bacterial denitrification. A copper-nitrosyl intermediate is a proposed, yet poorly characterized feature of the NiR catalytic cycle. This intermediate is formally described as Cu(I)-NO+ and is proposed to be formed at the type 2 copper site after nitrite binding and electron transfer from the type 1 copper site. In this study, copper-nitrosyl complexes were formed by prolonged exposure of exogenous NO to crystals of wild-type and two variant forms of NiR from Alcaligenes faecalis (AfNiR), and the structures were determined to 1.8 A or better resolution. Exposing oxidized wild-type crystals to NO results in the reverse reaction and formation of nitrite that remains bound at the active site. In a type 1 copper site mutant (H145A) that is incapable of electron transfer to the type 2 site, the reverse reaction is not observed. Instead, in both oxidized and reduced H145A crystals, NO is observed bound in a side-on manner to the type 2 copper. In AfNiR, Asp98 forms hydrogen bonds to both substrate and product bound to the type 2 Cu. In the D98N variant, NO is bound side-on but is more disordered when observed for the wild-type enzyme. The solution EPR spectra of the crystallographically characterized NiR-NO complexes indicate the presence of an oxidized type 2 copper site and thus are interpreted as resulting from stable copper-nitrosyls and formally assigned as Cu(II)-NO-. A reaction scheme in which a second NO molecule is oxidized to nitrite can account for the formation of a Cu(II)-NO- species after exposure of the oxidized H145A variant to NO gas.     

Structures of D-xylose isomerase from Arthrobacter strain B3728 containing the inhibitors xylitol and D-sorbitol at 2.5 A and 2.3 A resolution, respectively

The structures of D-xylose isomerase from Arthrobacter strain B3728 containing the polyol inhibitors xylitol and D-sorbitol have been solved at 2.5 A and 2.3 A, respectively. The structures have been refined using restrained least-squares refinement methods. The final crystallographic R-factors for the D-sorbitol (xylitol) bound molecules, for 43,615 (32,989) reflections are 15.6 (14.7). The molecule is a tetramer and the asymmetric unit of the crystal contains a dimer, the final model of which, incorporates a total of 6086 unique protein, inhibitor and magnesium atoms together with 535 bound solvent molecules. Each subunit of the enzyme contains two domains: the main domain is a parallel-stranded alpha-beta barrel, which has been reported in 14 other enzymes. The C-terminal domain is a loop structure consisting of five helical segments and is involved in intermolecular contacts between subunits that make up the tetramer. The structures have been analysed with respect to molecular symmetry, intersubunit contacts, inhibitor binding and active site geometry. The refined model shows the two independent subunits to be similar apart from local deviations due to solvent contacts in the solvent-exposed helices. The enzyme is dependent on a divalent cation for catalytic activity. Two metal ions are required per monomer, and the high-affinity magnesium(II) site has been identified from the structural results presented here. The metal ion is complexed, at the high-affinity site, by four carboxylate side-chains of the conserved residues, Glu180, Glu216, Asp244 and Asp292. The inhibitor polyols are bound in the active site in an extended open chain conformation and complete an octahedral co-ordination shell for the magnesium cation via their oxygen atoms O-2 and O-4. The active site lies in a deep pocket near the C-terminal ends of the beta-strands of the barrel domain and includes residues from a second subunit. The tetrameric molecule can be considered to be a dimer of "active" dimers, the active sites being composed of residues from both subunits. The analysis has revealed the presence of several internal salt-bridges stabilizing the tertiary and quaternary structure. One of these, between Asp23 and Arg139, appears to play a key role in stabilizing the active dimer and is conserved in the known sequences of this enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Three-dimensional structure of bovine pancreatic DNase I at 2.5 A resolution

The three-dimensional structure of bovine pancreatic deoxyribonuclease I (DNase I) has been determined at 2.5 A resolution by X-ray diffraction from single crystals. An atomic model was fitted into the electron density using a graphics display system. DNase I is an alpha, beta-protein with two 6-stranded beta-pleated sheets packed against each other forming the core of a 'sandwich'-type structure. The two predominantly anti-parallel beta-sheets are flanked by three longer alpha-helices and extensive loop regions. The carbohydrate side chain attached to Asn 18 is protruding by approximately 15 A from the otherwise compact molecule of approximate dimensions 45 A X 40 A. The binding site of CA2+-deoxythymidine-3',5'-biphosphate (Ca-pdTp) has been determined by difference Fourier techniques confirming biochemical results that the active centre is close to His 131. Ca-pdTp binds at the surface of the enzyme between the two beta-pleated sheets and seems to interact with several charged amino acid side chains. Active site geometry and folding pattern of DNase I are quite different from staphylococcal nuclease, the only other Ca2+-dependent deoxyribonuclease whose structure is known at high resolution. The electron density map indicates that two Ca2+ ions are bound to the enzyme under crystallization conditions.     

Closed conformation of the active site loop of rabbit muscle triosephosphate isomerase in the absence of substrate: evidence of conformational heterogeneity

The active site loop of triosephosphate isomerase (TIM) exhibits a hinged-lid motion, alternating between the two well defined "open" and "closed" conformations. Until now the closed conformation had only been observed in protein complexes with substrate analogues. Here, we present the first rabbit muscle apo TIM structure, refined to 1.5A resolution, in which the active site loop is either in the open or in the closed conformation in different subunits of the enzyme. In the closed conformation described here, the lid loop residues participate in stabilizing hydrogen bonds characteristic of holo TIM structures, whereas chemical interactions observed in the open loop conformation are similar to those found in the apo structures of TIM. In the closed conformation, a number of water molecules are observed at the projected ligand atom positions that are hydrogen bonded to the active site residues. Additives used during crystallization (DMSO and Tris molecules and magnesium atoms) were modeled in the electron density maps. However, no specific binding of these molecules is observed at, or close to, the active site and the lid loop. To further investigate this unusual closed conformation of the apo enzyme, two more rabbit muscle TIM structures, one in the same and another in a different crystal form, were determined. These structures present the open lid conformation only, indicating that the closed conformation cannot be explained by crystal contact effects. To rationalize why the active site loop is closed in the absence of ligand in one of the subunits, extensive comparison with previously solved TIM structures was carried out, supported by the bulk of available experimental information about enzyme kinetics and reaction mechanism of TIM. The observation of both open and closed lid conformations in TIM crystals might be related to a persistent conformational heterogeneity of this protein in solution.     

Structural studies of E. coli topoisomerase III-DNA complexes reveal a novel type IA topoisomerase-DNA conformational intermediate

Escherichia coli DNA topoisomerase III belongs to the type IA family of DNA topoisomerases, which transiently cleave single-stranded DNA (ssDNA) via a 5' phosphotyrosine intermediate. We have solved crystal structures of wild-type E. coli topoisomerase III bound to an eight-base ssDNA molecule in three different pH environments. The structures reveal the enzyme in three distinct conformational states while bound to DNA. One conformation resembles the one observed previously with a DNA-bound, catalytically inactive mutant of topoisomerase III where DNA binding realigns catalytic residues to form a functional active site. Another conformation represents a novel intermediate in which DNA is bound along the ssDNA-binding groove but does not enter the active site, which remains in a catalytically inactive, closed state. A third conformation shows an intermediate state where the enzyme is still in a closed state, but the ssDNA is starting to invade the active site. For the first time, the active site region in the presence of both the catalytic tyrosine and ssDNA substrate is revealed for a type IA DNA topoisomerase, although there is no evidence of ssDNA cleavage. Comparative analysis of the various conformational states suggests a sequence of domain movements undertaken by the enzyme upon substrate binding.     

The refinement and the structure of the dimer of alpha-chymotrypsin at 1.67-A resolution

The two molecules of the asymmetric unit of the pH 3.5 conformer of alpha-chymotrypsin have been refined at 1.67-A resolution using restrained least squares methods with Hendrickson's program (PROLSQ). The final R factor is 0.179 (including 247 water molecules). The folding of the main chain of the independent molecules is the same within experimental error but the same does not generally apply to the side chain stereochemistry. From this we conclude that the folding of a protein structure is basically independent of most of the detailed stereochemistry of its side chains. The side chains of the interface region between the independent molecules display pronounced asymmetry. This asymmetry suggests that dynamic and asymmetrical structural changes take place at the time of oligomerization leading to more energetically favorable interactions for the dimer. Comparison of the structures of the independent molecules of alpha-chymotrypsin with the structure of monomeric gamma-chymotrypsin revealed that although the folding of the three molecules is essentially the same, numerous and significant differences pervade the side chain stereochemistry attributable to general flexibility. The specificity site of alpha-chymotrypsin is occupied by ordered water molecules in a similar way to gamma-chymotrypsin and other proteins. Some of these water molecules are displaced when substrate binds to the enzyme, while the others appear to help identify and position the aromatic side chain in catalysis.