Identification of metal-binding residues in the Klebsiella aerogenes urease nickel metallochaperone, UreE

The urease accessory protein encoded by ureE from Klebsiella aerogenes is proposed to bind intracellular Ni(II) for transfer to urease apoprotein. While native UreE possesses a histidine-rich region at its carboxyl terminus that binds several equivalents of Ni, the Ni-binding sites associated with urease activation are internal to the protein as shown by studies involving truncated H144UreE [Brayman and Hausinger (1996) J. Bacteriol. 178, 5410-5416]. Nine potential Ni-binding residues (five His, two Cys, one Asp, and one Tyr) within H144UreE were independently substituted by mutagenesis to determine their roles in metal binding and urease activation. In vivo effects of these substitutions on urease activity were measured in Escherichia coli strains containing the K. aerogenes urease gene cluster with the mutated ureE genes. Several mutational changes led to reductions in specific activity, with substitution of His96 producing urease activity below the level obtained from a ureE deletion mutant. The metal-binding properties of purified variant UreE proteins were characterized by a combination of equilibrium dialysis and UV/visible, EPR, and hyperfine-shifted 1H NMR spectroscopic methods. Ni binding was unaffected for most H144UreE variants, but mutant proteins substituted at His110 or His112 exhibited greatly reduced affinity for Ni and bound one, rather than two, metal ions per dimer. Cys79 was identified as the Cu ligand responsible for the previously observed charge-transfer transition at 370 nm, and His112 also was shown to be associated with this chromophoric site. NMR spectroscopy provided clear evidence that His96 and His110 serve as ligands to Ni or Co. The results from these and other studies, in combination with prior spectroscopic findings for metal-substituted UreE [Colpas et al. (1998) J. Biol. Inorg. Chem. 3, 150-160], allow us to propose that the homodimeric protein possesses two nonidentical metal-binding sites, each symmetrically located at the dimer interface. The first equivalent of added Ni or Co binds via His96 and His112 residues from each subunit of the dimer, and two other N or O donors. Asp111 either functions as a ligand or may affect this site by secondary interactions. The second equivalent of Ni or Co binds via the symmetric pair of His110 residues as well as four other N or O donors. In contrast, the first equivalent of Cu binds via the His110 pair and two other N/O donors, while the second equivalent of Cu binds via the His112 pair and at least one Cys79 residue. UreE sequence comparisons among urease-containing microorganisms reveal that residues His96 and Asp111, associated with the first site of Ni binding, are highly conserved, while the other targeted residues are missing in many cases. Our data are most compatible with one Ni-binding site per dimer being critical for UreE's function as a metallochaperone.     

Formation, crystal structure, and rearrangement of a cytochrome P-450cam iron-phenyl complex

Cytochrome P-450cam reacts with phenyldiazene (PhN = NH), or less efficiently with phenylhydrazine, to give a catalytically inactive complex with an absorption maximum at 474 nm. The prosthetic group extracted anaerobically from the inactivated protein has the spectroscopic properties of a sigma phenyl-iron complex and rearranges, on exposure to air and acid, to an approximately equal mixture of the four N-phenylprotoporphyrin IX regioisomers. The crystal structure of the intact protein complex, refined at 1.9-A resolution to an R factor of 20%, confirms that the phenyl group is directly bonded through one of its carbons to the iron atom. The phenyl ring is tilted from the heme normal by about 10 degrees in the opposite direction from that in which carbon monoxide tilts when bound to P-450cam. Camphor, the natural substrate for P-450cam, is larger than a phenyl group and hydrogen bonds to Tyr 96, the only hydrophilic residue near the active site. Electron density in the active site in addition to that contributed by the phenyl group suggests that two water molecules occupy part of the camphor binding site but are not within hydrogen-bonding distance of Tyr 96. As observed in a previous crystallographic study of inhibitor-P-450cam complexes [Poulos, T.L., & Howard, A.J. (1987) Biochemistry 26, 8165-8174], there are large changes in both the atomic positions and mobilities of the residues in the proposed substrate access channel region of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation, characterization, sequencing and crystal structure of charybdin, a type 1 ribosome-inactivating protein from Charybdis maritima agg

A novel, type 1 ribosome-inactivating protein designated charybdin was isolated from bulbs of Charybdis maritima agg. The protein, consisting of a single polypeptide chain with a molecular mass of 29 kDa, inhibited translation in rabbit reticulocytes with an IC50 of 27.2 nm. Plant genomic DNA extracted from the bulb was amplified by PCR between primers based on the N-terminal and C-terminal sequence of the protein from dissolved crystals. The complete mature protein sequence was derived by partial DNA sequencing and terminal protein sequencing, and was confirmed by high-resolution crystal structure analysis. The protein contains Val at position 79 instead of the conserved Tyr residue of the ribosome-inactivating proteins known to date. To our knowledge, this is the first observation of a natural substitution of a catalytic residue at the active site of a natural ribosome-inactivating protein. This substitution in the active site may be responsible for the relatively low in vitro translation inhibitory effect compared with other ribosome-inactivating proteins. Single crystals were grown in the cold room from PEG6000 solutions. Diffraction data collected to 1.6 A resolution were used to determine the protein structure by the molecular replacement method. The fold of the protein comprises two structural domains: an alpha + beta N-terminal domain (residues 4-190) and a mainly alpha-helical C-terminal domain (residues 191-257). The active site is located in the interface between the two domains and comprises residues Val79, Tyr117, Glu167 and Arg170.     

Removing a hydrogen bond in the dimer interface of Escherichia coli manganese superoxide dismutase alters structure and reactivity

Among manganese superoxide dismutases, residues His30 and Tyr174 are highly conserved, forming part of the substrate access funnel in the active site. These two residues are structurally linked by a strong hydrogen bond between His30 NE2 from one subunit and Tyr174 OH from the other subunit of the dimer, forming an important element that bridges the dimer interface. Mutation of either His30 or Tyr174 in Escherichia coli MnSOD reduces the superoxide dismutase activity to 30--40% of that of the wt enzyme, which is surprising, since Y174 is quite remote from the active site metal center. The 2.2 A resolution X-ray structure of H30A-MnSOD shows that removing the Tyr174-->His30 hydrogen bond from the acceptor side results in a significant displacement of the main-chain segment containing the Y174 residue, with local rearrangement of the protein. The 1.35 A resolution structure of Y174F-MnSOD shows that disruption of the same hydrogen bond from the donor side has much greater consequences, with reorientation of F174 having a domino effect on the neighboring residues, resulting in a major rearrangement of the dimer interface and flipping of the His30 ring. Spectroscopic studies on H30A, H30N, and Y174F mutants show that (like the previously characterized Y34F mutant of E. coli MnSOD) all lack the high pH transition of the wt enzyme. This observation supports assignment of the pH sensitivity of MnSOD to coordination of hydroxide ion at high pH rather than to ionization of the phenolic group of Y34. Thus, mutations near the active site, as in the Y34F mutant, as well as at remote positions, as in Y174F, similarly affect the metal reactivity and alter the effective pK(a) for hydroxide ion binding. These results imply that hydrogen bonding of the H30 imidazole N--H group plays a key role in substrate binding and catalysis.     

Biochemical, mutational and in silico structural evidence for a functional dimeric form of the ornithine decarboxylase from Entamoeba histolytica

Background

Entamoeba histolytica is responsible for causing amoebiasis. Polyamine biosynthesis pathway enzymes are potential drug targets in parasitic protozoan diseases. The first and rate-limiting step of this pathway is catalyzed by ornithine decarboxylase (ODC). ODC enzyme functions as an obligate dimer. However, partially purified ODC from E. histolytica (EhODC) is reported to exist in a pentameric state.

Methodology and Results

In present study, the oligomeric state of EhODC was re-investigated. The enzyme was over-expressed in Escherichia coli and purified. Pure protein was used for determination of secondary structure content using circular dichroism spectroscopy. The percentages of α-helix, β-sheets and random coils in EhODC were estimated to be 39%, 25% and 36% respectively. Size-exclusion chromatography and mass spectrophotometry analysis revealed that EhODC enzyme exists in dimeric form. Further, computational model of EhODC dimer was generated. The homodimer contains two separate active sites at the dimer interface with Lys57 and Cys334 residues of opposite monomers contributing to each active site. Molecular dynamic simulations were performed and the dimeric structure was found to be very stable with RMSD value ∼0.327 nm. To gain insight into the functional role, the interface residues critical for dimerization and active site formation were identified and mutated. Mutation of Lys57Ala or Cys334Ala completely abolished enzyme activity. Interestingly, partial restoration of the enzyme activity was observed when inactive Lys57Ala and Cys334Ala mutants were mixed confirming that the dimer is the active form. Furthermore, Gly361Tyr and Lys157Ala mutations at the dimer interface were found to abolish the enzyme activity and destabilize the dimer.

Conclusion

To our knowledge, this is the first report which demonstrates that EhODC is functional in the dimeric form. These findings and availability of 3D structure model of EhODC dimer opens up possibilities for alternate enzyme inhibition strategies by targeting the dimer disruption.     

p38α MAP kinase dimers with swapped activation segments and a novel catalytic loop conformation

Many protein kinase functions, including autophosphorylation in trans, require dimerization, possibly by activation segment exchange. Such dimers have been reported for a few autophosphorylating protein kinases, but not for mitogen-activated protein kinases (MAPKs). Activation of MAPKs proceeds not only via the well-characterized action of dual T/Y specificity MAPK kinases, phosphorylating both residues of the MAPK TxY activation loop motif, but also via a noncanonical activation pathway triggered by phosphorylation at Tyr323 and homodimerization. Here, we report the 2. 7-Å-resolution structure of p38α MAPK from Salmo salar in a novel domain-swapped homodimeric form. The tyrosines of the conserved sequence YxAPE anchor the swapped activation segments in a configuration suitable for autophosphorylation in trans and provide a model for the noncanonical pathway. In the dimer, a structural unit containing Tyr323 is formed at a dimerization contact region that stabilizes the HRD catalytic loop in a unique inactive geometry. This feature is consistent with the requirement of Tyr323 phosphorylation for the initiation of the noncanonical pathway. Despite the interacting surface of more than 2600 Å², the dimer is not obligate, as gel filtration shows the dimerization to occur only at relatively high concentrations. The transition from monomer to dimer involves a relatively simple hinged displacement of helix EF and adjacent residues. Thus, dimer formation is likely to be transient, compatible with functional requirements for autophosphorylation, allowing further modulation, for example, by scaffolding mechanisms.     

Trapped tyrosyl radical populations in modified reaction centers from Rhodobacter sphaeroides

The photosynthetic reaction center from the purple bacterium Rhodobacter sphaeroides has been modified such that the bacteriochlorophyll dimer, when it becomes oxidized after light excitation, is capable of oxidizing tyrosine residues. One factor in this ability is a high oxidation-reduction midpoint potential for the dimer, although the location and protein environment of the tyrosine residue appear to be critical as well. These factors were tested in a series of mutants, each of which contains changes, at residues L131, M160, M197, and M210, that give rise to a bacteriochlorophyll dimer with a midpoint potential of at least 800 mV. The protein environment was altered near tyrosine residues that are either present in the wild type or introduced by mutagenesis, focusing on residues that could act as acceptors for the phenolic proton of the tyrosine upon oxidation. These mutations include Ser M190 to His, which is near Tyr L162, the combination of His M193 to Tyr and Arg M164 to His, which adds a Tyr-His pair, and the combinations of Arg L135 to Tyr with Tyr L164 to His, Arg L135 to Tyr with Tyr L144 to Glu, and Arg L135 to Tyr with Tyr L164 to Phe. Radicals were produced in the mutants by using light to initiate electron transfer. The radicals were trapped by freezing the samples, and the relative populations of the oxidized dimer and tyrosyl radicals were determined by analysis of low-temperature electron paramagnetic resonance spectra. The mutants all showed evidence of tyrosyl radical formation at high pH, and the extent of radical formation at Tyr L135 with pH differed depending on the identity of L144 and L164. The results show that tyrosine residues within approximately 10 A of the dimer can become oxidized when provided with a suitable protein environment.     

In silico structural characterization and molecular docking studies of first glucuronoxylan-xylanohydrolase (Xyn30A) from family 30 glycosyl hydrolase (GH30) from Clostridium thermocellum

CtXynGH30 is a carbohydrate active modular enzyme and component of cellulosome of Clostridium thermocellum. The full length CtXynGH30 contains an N-terminal catalytic module named as Xyn30A and a family 6 carbohydrate binding module (CBM6) at C-terminus. Xyn30A was modeled by computer program Modeller9v8 taking crystal structure of XynC from B. subtilis as a template to generate the molecular model. Model refinement was done using energy minimization by implementing steepest descent algorithm with GROMOS96 43a1 force field. Quality assessment by Ramachandran plot showed that 91% amino acids lie in most favourable region and 9% in additional allowed region. Structural analysis depicted that Xyn30A has a (β/α)₈ barrel fold. Additionally, it had a β-strand rich structure called ‘side β-structure’ attached with main catalytic core. Structural superimposition reflected that Glu136 act as a catalytic acid/base while Glu225 act as a catalytic nucleophile. Multiple sequence alignment showed that these catalytic residues are conserved within the family. The docking results showed that these residues display polar interaction with linear and substituted xylo-oligosaccharides. The binding interaction of ligands depicted that aromatic amino acids Trp81, Tyr139, Trp143, Phe172, His198, Tyr200, Tyr227, Trp264 and Tyr265 create binding site pocket around the active site. We report overall structural feature, conserved active site residues and enzyme-ligand docking of first glucuronoxylan-xylanohydrolase (Xyn30A) of family 30 glycosyl hydrolase (GH30) from Clostridium thermocellum.     

Optically detected magnetic resonance study of tyrosine residues in point-mutated bacteriophage T4 lysozyme

Two spectroscopically distinct types of tyrosine (Tyr) residues in triply point mutated bacteriophage T4 lysozyme, which contains no tryptophan (Trp), have been detected by optical detection of triplet-state magnetic resonance (ODMR) spectroscopy. Their triplet states are characterized by similar E but different D values. The Tyr site which exhibits the lower D value and has the red-shifted phosphorescence origin is quenched by energy transfer to Trp and has D and E values comparable to previously studied Tyr residues. The blue-shifted Tyr site, which is not quenched by Trp, exhibits a larger D value that has been found previously. Calculation of energy-transfer efficiencies of Tyr-Trp pairs based on the crystal structure of the native enzyme provides a possible assignment of Tyr sites to the two different spectral types.     

Conserved residues and their role in the structure, function, and stability of acyl-coenzyme A binding protein

In the family of acyl-coenzyme A binding proteins, a subset of 26 sequence sites are identical in all eukaryotes and conserved throughout evolution of the eukaryotic kingdoms. In the context of the bovine protein, the importance of these 26 sequence positions for structure, function, stability, and folding has been analyzed using single-site mutations. A total of 28 mutant proteins were analyzed which covered 17 conserved sequence positions and three nonconserved positions. As a first step, the influence of the mutations on the protein folding reaction has been probed, revealing a folding nucleus of eight hydrophobic residues formed between the N- and C-terminal helices [Kragelund, B. B., et al. (1999) Nat. Struct. Biol. (In press)]. To fully analyze the role of the conserved residues, the function and the stability have been measured for the same set of mutant proteins. Effects on function were measured by the extent of binding of the ligand dodecanoyl-CoA using isothermal titration calorimetry, and effects on protein stability were measured with chemical denaturation followed by intrinsic tryptophan and tyrosine fluorescence. The sequence sites that have been conserved for direct functional purposes have been identified. These are Phe5, Tyr28, Tyr31, Lys32, Lys54, and Tyr73. Binding site residues are mainly polar or charged residues, and together, four of these contribute approximately 8 kcal mol-1 of the total free energy of binding of 11 kcal mol-1. The sequence sites conserved for stability of the structure have likewise been identified and are Phe5, Ala9, Val12, Leu15, Leu25, Tyr28, Lys32, Gln33, Tyr73, Val77, and Leu80. Essentially, all of the conserved residues that maintain the stability are hydrophobic residues at the interface of the helices. Only one conserved polar residue, Gln33, is involved in stability. The results indicate that conservation of residues in homologous proteins may result from a summed optimization of an effective folding reaction, a stable native protein, and a fully active binding site. This is important in protein design strategies, where optimization of one of these parameters, typically function or stability, may influence any of the others markedly.     

Three-dimensional structure of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum at 2.9 A resolution

The three-dimensional structure of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Rhodospirillum rubrum has been determined at 2.9 Å resolution by X-ray crystallographic methods. The MIR-electron density map was substantially improved by two-fold non-crystallographic symmetry averaging. The polypeptide chains in the dimer were traced using a graphics display system with the help of the BONES option in FRODO. The dimer has approximate dimensions of 50 x 72 x 105 Å. The enzyme subunit is a typical two-domain protein. The smaller, N-terminal domain consists of 137 amino acid residues and forms a central, mixed five-stranded β-sheet with α-helices on both sides of the sheet. The larger C-terminal domain consists of 329 amino acid residues. This domain has an eight-stranded parallel α/β barrel structure as found in triosephosphate isomerase and a number of other functionally non-related proteins. The active site in Rubisco determined by difference Fourier techniques and fitting of active site residues to the electron density map, is located at the carboxy-end of the β-strands in the α/β barrel of the C-terminal domain. There are few domain–domain interactions within the subunit. The interactions at the interface between the two subunits of the dimer are tight and extensive. There are tight contacts between the two C-terminal domains, which build up the core of the molecule. There are also interactions between the N-terminal domain of one subunit and the C-terminal domain of the second subunit, close to the active site.     

The molecular structure of a dimer composed of the variable portions of the Bence-Jones protein REI refined at 2.0-A resolution.

The structure of the variable portions of a K-type Bence-Jones protein REI forming a dimer has been determined by X-ray diffraction to a resolution of 2.0 A. The structure has been refined using a constrained crystallographic refinement procedure. The final R value is 0.24 for 15000 significantly measured reflections; the estimated standard deviation of atomic positions is 0.09 A. A more objective assessment of the error in the atomic positions is possible by comparing the two independently refined monomers. The mean deviation of main-chain atoms of the two chains in internal segments in 0.22 A, of main-chain dihedral angles 6.3 degrees for these segments. The unrefined molecular structure of the VREI dimer has been published (Epp, O., Colman, P., Fehlhammer, H., Bode, W., Schiffer, M., Huber, R., and Palm, W. (1974), Eur. J. Biochem. 45, 513). Now a detailed analysis is presented in terms of hydrogen bonds and conformational angles. Secondary structural elements (antiparallel beta structure, reverse turns) are defined. A more precise atomic arrangement of the amino acid residues forming the contact region and the hapten binding site is given as well as the localization of solvent molecules. Two cis-prolines (Pro-8 and Pro-95) were detected. The intrachain disulfide bridge (Cys-23-Cys-88) occurs statistically in two alternative conformations. The structure suggests reasons for strong conservation of several amino acid residues. The knowledge of the refined molecular structure enables crystal structure analyses of related molecules to be made by Patterson search techniques. The calculated phases based on the refined structure are much improved compared to isomorphous phases. Therefore the effects of hapten binding on the molecular structure can be analyzed by the difference Fourier technique with more reliability. Hapten binding studies have been started.     

Proposed amino acid sequence and the 1.63 A X-ray crystal structure of a plant cysteine protease,ervatamin B: some insights into the structural basis of its stability and substrate specificity

The crystal structure of a cysteine protease ervatamin B, isolated from the medicinal plant Ervatamia coronaria, has been determined at 1.63 A. The unknown primary structure of the enzyme could also be traced from the high-quality electron density map. The final refined model, consisting of 215 amino acid residues, 208 water molecules, and a thiosulfate ligand molecule, has a crystallographic R-factor of 15.9% and a free R-factor of 18.2% for F > 2sigma(F). The protein belongs to the papain superfamily of cysteine proteases and has some unique properties compared to other members of the family. Though the overall fold of the structure, comprising two domains, is similar to the others, a few natural substitutions of conserved amino acid residues at the interdomain cleft of ervatamin B are expected to increase the stability of the protein. The substitution of a lysine residue by an arginine (residue 177) in this region of the protein may be important, because Lys --> Arg substitution is reported to increase the stability of proteins. Another substitution in this cleft region that helps to hold the domains together through hydrogen bonds is Ser36, replacing a conserved glycine residue in the others. There are also some substitutions in and around the active site cleft. Residues Tyr67, Pro68, Val157, and Ser205 in papain are replaced by Trp67, Met68, Gln156, and Leu208, respectively, in ervatamin B, which reduces the volume of the S2 subsite to almost one-fourth that of papain, and this in turn alters the substrate specificity of the enzyme.     

Manganese superoxide dismutase from Thermus thermophilus. A structural model refined at 1.8 A resolution

The structure of Mn(III) superoxide dismutase (Mn(III)SOD) from Thermus thermophilus, a tetramer of chains 203 residues in length, has been refined by restrained least-squares methods. The R-factor [formula: see text] for the 54,056 unique reflections measured between 10.0 and 1.8 A (96% of all possible reflections) is 0.176 for a model comprising the protein dimer and 180 bound solvents, the asymmetric unit of the P4(1)2(1)2 cell. The monomer chain forms two domains as determined by distance plots: the N-terminal domain is dominated by two long antiparallel helices (residues 21 to 45 and 69 to 89) and the C-terminal domain (residues 100 to 203) is an alpha + beta structure including a three-stranded sheet. Features that may be important for the folding and function of this MnSOD include: (1) a cis-proline in a turn preceding the first long helix; (2) a residue inserted at position 30 that distorts the helix near the first Mn ligand; and (3) the locations of glycine and proline residues in the domain connector (residues 92 to 99) and in the vicinity of the short cross connection (residues 150 to 159) that links two strands of the beta-sheet. Domain-domain contacts include salt bridges between arginine residues and acidic side chains, an extensive hydrophobic interface, and at least ten hydrogen-bonded interactions. The tetramer possesses 222 symmetry but is held together by only two types of interfaces. The dimer interface at the non-crystallographic dyad is extensive (1000 A2 buried surface/monomer) and incorporates 17 trapped or structural solvents. The dimer interface at the crystallographic dyad buries fewer residues (750 A2/monomer) and resembles a snap fastener in which a type I turn thrusts into a hydrophobic basket formed by a ring of helices in the opposing chain. Each of the metal sites is fully occupied, with the Mn(III) five-co-ordinate in trigonal bipyramidal geometry. One of the axial ligands is solvent; the four protein ligands are His28, His83, Asp166 and His170. Surrounding the metal-ligand cluster is a shell of predominantly hydrophobic residues from both chains of the asymmetric unit (Phe86A, Trp87A, Trp132A, Trp168A, Tyr183A, Tyr172B, Tyr173B), and both chains collaborate in the formation of a solvent-lined channel that terminates at Tyr36 and His32 near the metal ion and is presumed to be the path by which substrate or other inner-sphere ligands reach the metal.(ABSTRACT TRUNCATED AT 400 WORDS)     

Crystal structures of Streptococcus pyogenes Dpr reveal a dodecameric iron-binding protein with a ferroxidase site

DNA-binding protein from starved cells (Dps)-like proteins are key factors involved in oxidative stress protection in bacteria. They bind and oxidize iron, thus preventing the formation of harmful reactive oxygen species that can damage biomolecules, particularly DNA. Dps-like proteins are composed of 12 identical subunits assembled in a spherical structure with a hollow central cavity. The iron oxidation occurs at 12 intersubunit sites located at dimer interfaces. Streptococcus pyogenes Dps-like peroxide resistance protein (Dpr) has been previously found to protect the catalase-lacking S. pyogenes bacterium from oxidative stress. We have determined the crystal structure of S. pyogenes Dpr, the second Dpr structure from a streptococcal bacterium, in iron-free and iron-bound forms at 2.0- and 1.93-Å resolution, respectively. The iron binds to well-conserved sites at dimer interfaces and is coordinated directly to Asp77 and Glu81 from one monomer, His50 from a twofold symmetry-related monomer, a glycerol molecule, and a water molecule. Upon iron binding, Asp77 and Glu81 change conformation. Site-directed mutagenesis of active-site residues His50, His62, Asp66, Asp77, and Glu81 to Ala revealed a dramatic decrease in iron incorporation. A short helix at the N-terminal was found in a different position compared with other Dps-like proteins. Two types of pores were identified in the dodecamer. Although the N-terminal pore was found to be similar to that of other Dps-like proteins, the C-terminal pore was found to be blocked by bulky Tyr residues instead of small residues present in other Dps-like proteins.     

Purification of crystallization of transaldolase isozyme I and evidence for different genetic origin of isozymes I and III in Candida utilis

A modified procedure for the purification and crystallization of isozymes I and III of transaldolase from extracts of Candida utilis has been developed which makes both enzymes available in sufficient quantity for structural studies. Each is composed of a pair of identical subunits, but the molecular weight of isozyme I is somewhat larger than that of isozyme III. An important difference is in the number of histidine residues: one per subunit in isozyme III and two per subunit in isozyme I. A nonapeptide containing both histidine residues has now been isolated from isozyme I; its sequence is identical to that of the corresponding segment from isozyme III, except that tyrosine is replaced by histidine: His (in place of Tyr)-Gly-Ile-His-Cys-Asx-Thr-Leu-Leu. This amino acid substitution establishes that two different genes code for the two isozymes.     

The structure of the bovine protein tyrosine phosphatase dimer reveals a potential self-regulation mechanism.

The bovine protein tyrosine phosphatase (BPTP) is a member of the class of low-molecular weight protein tyrosine phosphatases (PTPases) found to be ubiquitous in mammalian cells. The catalytic site of BPTP contains a CX(5)R(S/T) phosphate-binding motif or P-loop (residues 12-19) which is the signature sequence for all PTPases. Ser19, the final residue of the P-loop motif, interacts with the catalytic Cys12 and participates in stabilizing the conformation of the active site through interactions with Asn15, also in the P-loop. Mutations at Ser19 result in an enzyme with altered kinetic properties with changes in the pK(a) of the neighboring His72. The X-ray structure of the S19A mutant enzyme shows that the general conformation of the P-loop is preserved. However, changes in the loop containing His72 result in a displacement of the His72 side chain that may explain the shift in the pK(a). In addition, it was found that in the crystal, the protein forms a dimer in which Tyr131 and Tyr132 from one monomer insert into the active site of the other monomer, suggesting a dual-tyrosine motif on target sites for this enzyme. Since the activity of this PTPase is reportedly regulated by phosphorylation at Tyr131 and Tyr132, the structure of this dimer may provide a model of a self-regulation mechanism for the low-molecular weight PTPases.     

The crystal structure of a novel,inactive, lysine 49 PLA2 from Agkistrodon acutus venom: an ultrahigh resolution,AB initio structure determination

The crystal structure of acutohaemolysin, a lysine 49 phospholipase A2 protein with 1010 non-hydrogen protein atoms and 232 water molecules, has been determined ab initio using the program SnB at an ultrahigh resolution of 0.8 A. The lack of catalytic activity appears to be related to the presence of Phe102, which prevents the access of substrate to the active site. The substitution of tryptophan for leucine at residue 10 interferes with dimer formation and may be responsible for the additional loss of hemolytic activity. The ultrahigh resolution of the experimental diffraction data permits alternative conformations to be modeled for disordered residues, many hydrogen atoms to be located, the protonation of the Nepsilon2 atom in the catalytic residue His48 to be observed experimentally, and the density of the bonding electrons to be analyzed in detail.     

Structural Analysis of Sindbis Virus Capsid Mutants Involving Assembly and Catalysis

Sindbis virus core protein (SCP) has been isolated from virus and crystallized. The X-ray crystallographic structure showed that the amino-terminal 113 residues appeared to be either disordered or truncated during crystallization and that the carboxy-terminal residues 114 to 264 had a chymotrypsin-like structure. The carboxy-terminal residues 106 to 264 and 106 to 266 of SCP have now been expressed inEscherichia coli. Most crystal forms of the truncated proteins were isomorphous with those of the virally extracted protein. There are only small structural differences between the truncated recombinant protein and the ordered part of the wild-type virus-extracted protein. Hence,E. coli-expressed SCP can be used to study proteolytic properties and the contribution of SCP to nucleocapsid assembly, interaction with the E2 glycoprotein and interaction with RNA.The same dimer that was found in two different crystal forms of the virus-extracted SCP was present also in some of the crystals of the truncated recombinant protein. The monomer – monomer interface is maintained by two pairs of hydrogen bonds and by hydrophobic interactions. Removal of the hydrogen bonds by single substitutions did not prevent dimer formation. However, a mutation that reduced the hydrophobic contacts did inhibit dimer formation.The wild-type truncated SCP is active inE. coli, as evidenced by proteolytic processing of a series of progressively longer precursors that extend beyond residue 264. Unlike the virus-extracted capsid protein, theE. coli-expressed SCP described here is terminated following the carboxy-terminal residue and, therefore, does not require autocatalysis. Nevertheless, theE. coli-expressed protein folds with the carboxy-terminal tryptophan residue in the specificity pocket. Two crystallographically independent molecules of SCP(106 to 266), which had two additional downstream residues and had the essential S215 mutated to alanine, showed two distinct modes of binding the uncleaved carboxy-terminal residues. These may represent successive steps of binding substrate prior to catalytic cleavage.Refinement of the various crystal structures of SCP showed that the amino-terminal arm from residues 107 to 113 was not disordered, but is associated with neighboring molecules. Residues 108 to 111 bind into a hydrophobic pocket composed primarily of Y180, W247 and F166. It had been shown that the double mutant (Y180S; E183G), with the Y180S substitution in this pocket, produced a large number of non-infectious virions, possibly because of modification in the interaction of the glycoprotein spikes with core proteins. The crystal structure of this double mutant showed that there was a large positional change in the side-chain of W247, which moved into the space created by the replacement of Y180 with serine. These conformational changes may alter the stability of the virion and, thus, regulate its functional requirements during cell entry.