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PubMed2003

Two crystal structures demonstrate large conformational changes in the eukaryotic ribosomal translocase.

Two crystal structures of yeast translation elongation factor 2 (eEF2) were determined: the apo form at 2.9 A resolution and eEF2 in the presence of the translocation inhibitor sordarin at 2.1 A resolution. The overall conformation of apo eEF2 is similar to that of its prokaryotic homolog elongation factor G (EF-G) in complex with GDP. Upon sordarin binding, the three tRNA-mimicking C-terminal domains undergo substantial conformational changes, while the three N-terminal domains containing the nucleotide-binding site form an almost rigid unit. The conformation of eEF2 in complex with sordarin is entirely different from known conformations observed in crystal structures of EF-G or from cryo-EM studies of EF-G-70S complexes. The domain rearrangements induced by sordarin binding and the highly ordered drug-binding site observed in the eEF2-sordarin structure provide a high-resolution structural basis for the mechanism of sordarin inhibition. The two structures also emphasize the dynamic nature of the ribosomal translocase.

Amino Acid Sequence↗
PubMed1996

Parallel and antiparallel (G.GC)2 triple helix fragments in a crystal structure.

Nucleic acid triplexes are formed by sequence-specific interactions between single-stranded polynucleotides and the double helix. These triplexes are implicated in genetic recombination in vivo and have application to areas that include genome analysis and antigene therapy. Despite the importance of the triple helix, only limited high-resolution structural information is available. The x-ray crystal structure of the oligonucleotide d(GGCCAATTGG) is described; it was designed to contain the d(G middle dotGC)2 fragment and thus provide the basic repeat unit of a DNA triple helix. Parameters derived from this crystal structure have made it possible to construct models of both parallel and antiparallel triple helices.

Base Composition↗
PubMed2006

Evaluation of 27Al and 51V electric field gradients and the crystal structure for aluminum orthovanadate (AlVO4) by density functional theory calculations.

Three sets of crystal-structure data reported for AlVO(4) from two powder-XRD studies and a density functional theory (DFT) investigation, employing the Vienna ab initio simulation package (VASP), have been examined and refined using the DFT structure-optimization scheme implemented in the WIEN2k software. The crystal structures are evaluated on the basis of (27)Al and (51)V quadrupole coupling parameters recently reported for AlVO(4), employing the corresponding electric-field gradient (EFG) tensor elements obtained from the DFT calculations. The DFT calculations provide a reliable assignment of the (27)Al/(51)V resonances from three distinct Al and three V environments to the specific crystallographic sites in the asymmetric unit for AlVO(4). An improved agreement between experimental quadrupole tensor elements and calculated EFG tensors is achieved after the DFT structure optimizations and consistent results are obtained using the three different structures as starting points. The improvement of the structural data is also supported by an evaluation of the Al-O and V-O bond lengths before and after DFT structure optimization. The (51)V nuclear quadrupole moment, |Q((51)V)| = 4.8 +/- 0.1 fm(2), derived from the present analysis, represents a value of higher accuracy than earlier reported Q((51)V) values. The origin of the (27)Al and (51)V EFGs are investigated by an evaluation of the orientations of the EFG tensors in the crystal frame and by an examination of the individual contributions from the valence electrons and the surrounding lattice. The latter investigation shows that the magnitude and orientation of the tensors are largely determined by the p-p((27)Al) and p-p, d-d((51)V) orbital contributions to the valence electrons, while the lattice part only gives a minor contribution for both nuclei.

Journal Article↗
PubMed2004

Crystal structure of Mycobacterium tuberculosis catalase-peroxidase.

The Mycobacterium tuberculosis catalase-peroxidase is a multifunctional heme-dependent enzyme that activates the core anti-tuberculosis drug isoniazid. Numerous studies have been undertaken to elucidate the enzyme-dependent mechanism of isoniazid activation, and it is well documented that mutations that reduce activity or inactivate the catalase-peroxidase lead to increased levels of isoniazid resistance in M. tuberculosis. Interpretation of the catalytic activities and the effects of mutations upon the action of the enzyme to date have been limited due to the lack of a three-dimensional structure for this enzyme. In order to provide a more accurate model of the three-dimensional structure of the M. tuberculosis catalase-peroxidase, we have crystallized the enzyme and now report its crystal structure refined to 2.4-A resolution. The structure reveals new information about dimer assembly and provides information about the location of residues that may play a role in catalysis including candidates for protein-based radical formation. Modeling and computational studies suggest that the binding site for isoniazid is located near the delta-meso heme edge rather than in a surface loop structure as currently proposed. The availability of a crystal structure for the M. tuberculosis catalase-peroxidase also permits structural and functional effects of mutations implicated in causing elevated levels of isoniazid resistance in clinical isolates to be interpreted with improved confidence.

Amino Acid Sequence↗
PubMed2002

On the irrelevance of electrostatics for the crystal structures and polymorphism of long even n-alkanes.

It is known that the experimental triclinic crystal structures of even n-alkanes are not well reproduced upon energy minimization with current force fields. The inclusion of electrostatics does not solve this, and, moreover, some charge schemes show unphysical features such as positively charged carbon atoms or charge alternation. The effect of the electrostatics on the energies of the crystal structures of the even n-alkanes, and thereby on their polymorphism, has never been established. A new charge scheme is introduced that yields physically sensible charges without constraints. It will also be shown, however, that electrostatics are relevant neither for the structures of the crystals, nor for their energies.

Alkanes↗
PubMed1997

The rational construction of an antibody against cystatin: lessons from the crystal structure of an artificial Fab fragment.

In a protein design study the artificial antibody M41 was modelled with its binding site complementary to the protease inhibitor cystatin, which was chosen as a structurally well-characterized "antigen". The modelling of M41 took advantage of the crystal structure of the anti-lysozyme antibody HyHEL-10 as a structural template. Its combining site was reshaped by replacing 19 amino acid side-chains in the hypervariable loops. In addition, ten amino acid residues were substituted in the framework regions. The crystal structure of the corresponding antibody model M41, which was produced as an F(ab) fragment in Escherichia coli, was determined at a resolution of 1.95 A. The crystals exhibited symmetry of the space group P2(1)2(1)2(1) (a = 96.5 A; b = 103.5 A; c = 113.6 A) with two F(ab) fragments in the asymmetric unit, which were independently refined (final R-factor 21.7%). The resulting coordinates were used for a detailed comparison with the modelled protein structure. It was found that the mutual arrangement of the six complementarity-determining regions as well as most of their backbone conformation had been correctly predicted. One major difference that was detected for the conformation of a five residue insertion in complementarity-determining region L1 could be explained by an erroneously defined segment in the structure of the antibody 4-4-20, which had been used as a template for this loop. In the light of more recent crystallographic data it appears that this segment adopts a new canonical structure. Apart from this region, most of the side-chains in the antigen-binding site had been properly placed in the M41 model. There was however one important exception concerning Trp H98, whose side-chain conformation had been kept as it appeared in HyHEL-10. The differing orientation of this residue in the model compared with the crystal structure of the artificial F(ab) fragment M41 explains why an antigen affinity could not be detected so far. The detailed analysis of this and other, more subtle deviations suggests how to make this F(ab) fragment function by introducing a few additional amino acid changes into M41.

Amino Acid Sequence↗
PubMed1999

Crystal structure of the EF-hand parvalbumin at atomic resolution (0.91 A) and at low temperature (100 K). Evidence for conformational multistates within the hydrophobic core.

Several crystal structures of parvalbumin (Parv), a typical EF-hand protein, have been reported so far for different species with the best resolution achieving 1.5 A. Using a crystal grown under microgravity conditions, cryotechniques (100 K), and synchrotron radiation, it has now been possible to determine the crystal structure of the fully Ca2+-loaded form of pike (component pI 4.10) Parv.Ca2 at atomic resolution (0.91 A). The availability of such a high quality structure offers the opportunity to contribute to the definition of the validation tools useful for the refinement of protein crystal structures determined to lower resolution. Besides a better definition of most of the elements in the protein three-dimensional structure than in previous studies, the high accuracy thus achieved allows the detection of well-defined alternate conformations, which are observed for 16 residues out of 107 in total. Among them, six occupy an internal position within the hydrophobic core and converge toward two small buried cavities with a total volume of about 60 A3. There is no indication of any water molecule present in these cavities. It is probable that at temperatures of physiological conditions there is a dynamic interconversion between these alternate conformations in an energy-barrier dependent manner. Such motions for which the amplitudes are provided by the present study will be associated with a time-dependent remodeling of the void internal space as part of a slow dynamics regime (millisecond timescales) of the parvalbumin molecule. The relevance of such internal dynamics to function is discussed.

Binding Sites↗
PubMed1997

Cis Effects in the Cobalt Corrins. 1. Crystal Structures of 10-Chloroaquacobalamin Perchlorate, 10-Chlorocyanocobalamin, and 10-Chloromethylcobalamin.

The crystal structures of 10-chloroaquacobalamin perchlorate hydrate (10-Cl-H(2)OCbl.ClO(4)) (Mo Kalpha, 0.710 73 Å, monoclinic system, P2(1), a = 11.922(4) Å, b = 26.592(10) Å, c = 13.511(5) Å, beta = 93.05(3) degrees, 10 535 independent reflections, R(1) = 0.0426), 10-chlorocyanocobalamin-acetone hydrate (10-Cl-CNCbl) (Mo Kalpha, 0.710 73 Å, orthorhombic system, P2(1)2(1)2(1), a = 16.24(3) Å, b = 21.85(5) Å, c = 26.75(8) Å, 7699 independent reflections, R(1) = 0.0698), and 10-chloromethylcobalamin-acetone hydrate (10-Cl-MeCbl) (Mo Kalpha, 0.71073 Å, orthorhombic system, P2(1)2(1)2(1), a = 16.041(14) Å, b = 22.13(2) Å, c = 26.75(4) Å, 6792 independent reflections, R(1) = 0.0554), in which the C10 meso H is substituted by Cl, are reported. An unusual feature of the structures is disorder in the C ring, consistent with a two-site occupancy in which the major conformation has the C46 methyl group in the usual position, "upwardly" axial, and the C47 methyl group equatorial, while in the minor conformation both are pseudoequatorial, above and below the corrin ring. (13)C NMR chemical shifts of C46, C47, C12, and C13 suggest that the C ring disorder may persist in solution as a ring flip. Since molecular dynamics simulations fail to reveal any population of the minor conformation, the effect is likely to be electronic rather than steric. The axial bond lengths in 10-Cl-MeCbl are very similar to those in MeCbl (d(Co)(-)(C) = 1.979(7) vs 1.99(2); to 5,6-dimethylbenzimidazole, d(Co)(-)(NB3) = 2.200(7) vs 2.19(2)), but the bonds to the four equatorial N donors, d(Co)(-)(N(eq)), are on average 0.05 Å shorter. In 10-Cl-CNCbl, d(Co)(-)(C) and d(Co)(-)(NB3) are longer (by 0.10(2) and 0.03(1) Å, respectively) than the bond lengths observed in CNCbl itself, while conversely, the C-N bond length is shorter by 0.06(2) Å, but there is little difference in d(Co)(-)(N(eq)). The Co-O bond length to coordinated water in 10-Cl-H(2)OCbl(+) is very similar to that found in H(2)OCbl(+) itself, but the d(Co)(-)(NB3) bond is longer (1.967 vs1.925(2) Å), while the average d(Co)(-)(N(eq)) is very similar. The coordinated water molecule in 10-Cl-H(2)OCbl(+) is hydrogen bonded to the c side chain carbonyl oxygen, as in H(2)OCbl(+). NMR observations indicate that the H bond between coordinated H(2)O and the c side chain amide persists in solution. The equilibrium constant, K(Co), for coordination of bzm to Co(III) is smaller in 10-Cl-MeCbl and 10-Cl-CNCbl than in their C10-unsubstituted analogs (181 vs 452; 4.57 x 10(3) vs 3.35 x 10(5)), but could not be determined for 10-Cl-H(2)OCbl because hydrolysis of the phosphodiester is competitive with the establishment of the base-off equilibrium. Substitution of H by Cl at C10 causes the bands in the electronic spectrum of 10-Cl-XCbl complexes to move to lower energy, which is consistent with an increase in electron density in the corrin pi-conjugated system. This increased electron density is not due to greater electron donation from the axial ligand as bonds between these and the metal are either longer (not shorter) or unchanged, and it most probably arises from pi-donation to the corrin by Cl at C10. As the donor power of X increases (H(2)O < CN(-) < Me), the corrin ring becomes more flexible to deformation, and the number of bond lengths and bond angles that are significantly different in XCbl and 10-Cl-XCbl increases; importantly, the C10-Cl bond length, d(C10)(-)(Cl), increases as well. Thus, despite the fact that chlorine is an inductively electron withdrawing substituent, its resonance electron donation is the more important effect on electron distribution in the corrin ring. Mulliken charges obtained from semiempirical RHF-SCF MO calculations using the ZINDO/1 model on XCbl and their 10-Cl analogs at the crystal structure geometry are shown to correlate reasonably well with (13)C NMR shifts and may be used to determine the pattern of electron distribution in these complexes. Substitution by Cl at C10 causes an increase in charge density at Co when X = H(2)O and CN(-), while the charge density on the four equatorial N donors remains virtually unchanged, but a decrease when X = Me, while the charge density on the equatorial N donors also decreases. In response, d(Co)(-)(NB3) increases in the first two complexes but the equatorial bond lengths remain virtually unchanged, while d(Co)(-)(NB3) remains unchanged and the average d(Co)(-)(N(eq)) decreases in 10-Cl-MeCbl. Furthermore, the partial charge on chlorine increases as the donor power of X increases. The small decrease in the pK(a) of coordinated H(2)O in 10-Cl-H(2)OCbl(+) compared to H(2)OCbl(+) itself (7.65 vs 8.09) is due to a decreased charge density on oxygen in 10-Cl-OHCbl compared to OHCbl. The picture that emerges, therefore, is of competitive electron donation by X and Cl toward the corrin system. In 10-Cl-CNCbl, the decrease in the C&tbd1;N bond length as Co-C increases compared to CNCbl suggests that dpi-ppi bonding between cobalt and cyanide is important. (13)C and (15)N NMR observations on 10-Cl-(13)C(15)NCbl are consistent with these effects.

Journal Article↗
PubMed2001

Crystal structure of a DinB family error-prone DNA polymerase from Sulfolobus solfataricus.

A new group of error-prone DNA polymerases overcomes the blockage posed to normal DNA replication by damaged template bases, suggesting an active site with a loose, flexible pocket that accommodates aberrant DNA structures. We have determined a 2.8 A resolution crystal structure of the Sulfolobus solfataricus Dbh protein, a DNA translesion polymerase closely related to Escherichia coli DNA polymerase IV and human polymerase kappa. A high error rate is observed for the Dbh polymerase in a range of 10(-2)-10(-3) for all 12 base substitution mispairs. The crystal structure of Dbh reveals an overall architecture resembling other DNA polymerases but has unique features that are likely to contribute to error-prone synthesis, including -1 frameshifting mutations.

Amino Acid Sequence↗
PubMed2002

NMR solution structure of the isolated Apo Pin1 WW domain: comparison to the x-ray crystal structures of Pin1.

The NMR solution structure of the isolated Apo Pin1 WW domain (6-39) reveals that it adopts a twisted three-stranded antiparallel beta-sheet conformation, very similar to the structure exhibited by the crystal of this domain in the context of the two domain Pin1 protein. While the B factors in the apo x-ray crystal structure indicate that loop 1 and loop 2 are conformationally well defined, the solution NMR data suggest that loop 1 is quite flexible, at least in the absence of the ligand. The NMR chemical shift and nuclear Overhauser effect pattern exhibited by the 6-39 Pin1 WW domain has proven to be diagnostic for demonstrating that single site variants of this domain adopt a normally folded structure. Knowledge of this type is critical before embarking on time-consuming kinetic and thermodynamic studies required for a detailed understanding of beta-sheet folding.

Amino Acid Sequence↗
PubMed2000

Crystal structure analysis of recombinant human uteroglobin and molecular modeling of ligand binding.

Uteroglobin, a steroid-inducible, cytokine-like, secreted protein with immunomodulatory properties, has been reported to bind progesterone, polychlorinated biphenyls (PCB), and retinol. Structural studies may delineate whether binding of ligands is a likely physiological function of human uteroglobin (hUG). We report a refined crystal structure of uncomplexed recombinant hUG (rhUG) at 2.5-A resolution and the results of our molecular modeling studies of ligand binding to the central hydrophobic cavity of rhUG. The crystal structure of rhUG is very similar to that of reported crystal structures of uteroglobins. Using molecular modeling techniques, the three ligands--PCB, progesterone, and retinol--were docked into the hydrophobic cavity of the dimer structure of rhUG. We undocked the progesterone ligand by pulling the ligand from the cavity into the solvent. From our modeling and undocking studies of progesterone, it is clear that these types of hydrophobic ligands could slip into the cavity between helix-3 and helix-3' of the dimer instead of between helix-1 and helix-4 of the monomer, as proposed earlier. Our results suggest that at least one of the physiological functions of UG is to bind to hydrophobic ligands, such as progesterone and retinol.

Animals↗
PubMed2000

Crystal structures of spin labeled T4 lysozyme mutants: implications for the interpretation of EPR spectra in terms of structure.

High resolution (1.43-1.8 A) crystal structures and the corresponding electron paramagnetic resonance (EPR) spectra were determined for T4 lysozyme derivatives with a disulfide-linked nitroxide side chain [-CH(2)-S-S-CH(2)-(3-[2,2,5,5-tetramethyl pyrroline-1-oxyl]) identical with R1] substituted at solvent-exposed helix surface sites (Lys65, Arg80, Arg119) or a tertiary contact site (Val75). In each case, electron density is clearly resolved for the disulfide group, revealing distinct rotamers of the side chain, defined by the dihedral angles X(1) and X(2). The electron density associated with the nitroxide ring in the different mutants is inversely correlated with its mobility determined from the EPR spectrum. Residue 80R1 assumes a single g(+)()g(+)() conformation (Chi(1) = 286, X(2) = 294). Residue 119R1 has two EPR spectral components, apparently corresponding to two rotamers, one similar to that for 80R1 and the other in a tg(-)() conformation (Chi(1) = 175, X(2) = 54). The latter state is apparently stabilized by interaction of the disulfide with a Gln at i + 4, a situation also observed at 65R1. R1 residues at helix surface site 65 and tertiary contact site 75 make intra- as well as intermolecular contacts in the crystal and serve to identify the kind of molecular interactions possible for the R1 side chain. A single conformation of the entire 75R1 side chain is stabilized by a variety of interactions with the nitroxide ring, including hydrophobic contacts and two unconventional C-H.O hydrogen bonds, one in which the nitroxide acts as a donor (with tyrosine) and the other in which it acts as an acceptor (with phenylalanine). The interactions revealed in these structures provide an important link between the dynamics of the R1 side chain, reflected in the EPR spectrum, and local protein structure. A library of such interactions will provide a basis for the quantitative interpretation of EPR spectra in terms of protein structure and dynamics.

Bacteriophage T4↗
PubMed2005

Crystal structure of novel NADP-dependent 3-hydroxyisobutyrate dehydrogenase from Thermus thermophilus HB8.

3-Hydroxyisobutyrate, a central metabolite in the valine catabolic pathway, is reversibly oxidized to methylmalonate semialdehyde by a specific dehydrogenase belonging to the 3-hydroxyacid dehydrogenase family. To gain insight into the function of this enzyme at the atomic level, we have determined the first crystal structures of the 3-hydroxyisobutyrate dehydrogenase from Thermus thermophilus HB8: holo enzyme and sulfate ion complex. The crystal structures reveal a unique tetrameric oligomerization and a bound cofactor NADP+. This bacterial enzyme may adopt a novel cofactor-dependence on NADP, whereas NAD is preferred in eukaryotic enzymes. The protomer folds into two distinct domains with open/closed interdomain conformations. The cofactor NADP+ with syn nicotinamide and the sulfate ion are bound to distinct sites located at the interdomain cleft of the protomer through an induced-fit domain closure upon cofactor binding. From the structural comparison with the crystal structure of 6-phosphogluconate dehydrogenase, another member of the 3-hydroxyacid dehydrogenase family, it is suggested that the observed sulfate ion and the substrate 3-hydroxyisobutyrate share the same binding pocket. The observed oligomeric state might be important for the catalytic function through forming the active site involving two adjacent subunits, which seems to be conserved in the 3-hydroxyacid dehydrogenases. A kinetic study confirms that this enzyme has strict substrate specificity for 3-hydroxyisobutyrate and serine, but it cannot distinguish the chirality of the substrates. Lys165 is likely the catalytic residue of the enzyme.

Alcohol Oxidoreductases↗
PubMed1998

Tetragonal Crystal Structure of Mung Bean Inhibitor-porcine Trypsin Complex.

Crystal structure of the mung bean inhibitor-porcine trypsin (1:2) ternary complex in the tetragonal crystal with space group I422 was determined at 0.25nm resolution. 56 residues of the mung bean inhibitor were resolved among which one more residue Pro11 was determined than the previously reported structure in the trigonal crystal of the same complex. The structure of the inhibitor in the tetragonal crystal is similar to that in the trigonal crystal and the complexes in tetragonal crystal is also in packing disorder as in trigonal crystal, i.e., the complexes pack in two orientations Ta : MaMb : Tb and Tb : MbMa : Ta(Ta, Tb=trypsin, Ma, Mb=loop I and loop II of mung bean inhibitor respectively). But there are some differences in the two crystal forms. First, the inhibitor in the tetragonal crystal has no pseudo- beta-sheet structure which the trigonal crystal has. Second, its conformation is somewhat different from that in the trigonal crystal.Analysis showed that the linkage peptides between the two regid domains of the inhibitor were flexible, which also accounted for the formation of different crystal forms of this complex. Moreover, comparing mung bean inhibitor to other Bowman-Birk inhibitors showed that the two double-stranded antiparallel beta-sheets and the reactive binding loops were highly conservative.

Journal Article↗
PubMed1998

Copper(II) Complexes with Unusual Axial Phenolate Coordination as Structural Models for the Active Site in Galactose Oxidase: X-ray Crystal Structures and Spectral and Redox Properties of [Cu(bpnp)X] Complexes.

The crystal structures of [Cu(bpnp)(SCN)].NH(4)SCN (1), [Cu(bpnp)(CH(3)COO)].CH(3)OH.C(8)H(10) (2), and [Cu(bpnp)ClO(4)] (3) [Hbpnp = 2-(bis(pyrid-2-ylmethyl)aminomethyl)-4-nitrophenol] reveal a distorted square pyramidal geometry around Cu(II) with an unusual axial coordination of phenolate. The mononuclear complex [Cu(bpnp)(SCN)].NH(4)SCN crystallizes in the triclinic space group P&onemacr; with a = 10.796(2) Å, b = 10.804(2) Å, c = 12.559(2) Å, alpha = 71.38(1) degrees, beta = 72.68(1) degrees, gamma = 61.69(1) degrees, and Z = 2. The mononuclear acetate [Cu(bpnp)(CH(3)COO)].CH(3)OH.C(8)H(10) crystallizes in the triclinic space group P&onemacr; with a = 10.480(6) Å, b = 12.116(4) Å, c = 12.547(3) Å, alpha = 98.77(3) degrees, beta = 113.37(3) degrees, gamma = 100.78(3) degrees, and Z = 2. The binuclear perchlorate complex crystallizes in the monoclinic space group C2/c with a = 13.417(3) Å, b = 20.095(2) Å, c= 16.401(2) Å, alpha = 102.21(2) degrees, and Z = 8. The coordination plane in all these complexes is comprised of the tertiary amine and two pyridine nitrogens. The fourth equatorial position is occupied by SCN(-)/CH(3)COO(-) in the mononuclear complexes but by the coordinated phenolate ion from the adjacent molecule in the perchlorate complex, resulting in its dimerization. The unusual occupation of phenolate ion in the axial site is possibly due to the steric constraint at copper imposed by the 5,5,6-chelate ring sequence. The thiocyanate/acetate coordination geometry is reminiscent of the active site of the radical copper enzyme galactose oxidase (GOase) with an axial phenolate and equatorial SCN(-)/CH(3)COO(-) ligands. Further, the present complexes exhibit several spectral features also similar to this enzyme. The addition of chloride or thiocyanate or acetate ions dissociates the dimeric structure of the perchlorate complex to produce the corresponding monomeric derivatives. The study of the interaction of the acetate complex with N(3)(-) and CN(-) ions provide insight into the anion binding properties of the enzyme. The sensitivity of the acetate complex to protons suggests the facile dissociation of the axial phenolate which then acts as a base to bind to protons. The implication of this reaction to the GOase mechanism is discussed.

Journal Article↗
PubMed2006

Variation of loop sequence alters stability of cytolethal distending toxin (CDT): crystal structure of CDT from Actinobacillus actinomycetemcomitans.

Cytolethal distending toxin (CDT) secreted by Actinobacillus actinomycetemcomitans induces cell cycle arrest of cultured cells in the G2 phase. The crystal structure of the natural form of A. actinomycetemcomitans DCT (aCDT) has been determined at 2.4 A resolution. aCDT is a heterotrimer consisting of the three subunits, aCdtA, aCdtB, and aCdtC. Two crystallographically independent aCDTs form a dimer through interactions of the aCdtB subunits. The primary structure of aCDT has 94.3% identity with that of Haemophilus ducreyi CDT (hCDT), and the structure of aCDT is quite similar to that of hCDT reconstituted from the three subunits determined recently. However, the molecular packings in the crystal structures of aCDT and hCDT are quite different. A careful analysis of molecular packing suggests that variation of the amino acid residues in a nonconserved loop (181TSSPSSPERRGY192 of aCdtB and 181NSSSSPPERRVY192 of hCdtB) is responsible for the different oligomerization of very similar CDTs. The loop of aCdtB has a conformation to form a dimer, while the loop conformation of hCdtB prevents hCDT from forming a dimer. Although dimerization of aCDT does not affect toxic activity, it changes the stability of protein. aCDT rapidly aggregates and loses toxic activity in the absence of sucrose in a buffered solution, while hCDT is stable and retains toxic activity. Another analysis of crystal structures of aCDT and hCDT suggests that the receptor contact area is in the deep groove between CdtA and CdtC, and the characteristic "aromatic patch" on CdtA.

Aggregatibacter actinomycetemcomitans↗
PubMed2004

Module-based assembly of copper(II) chloranilate compounds: syntheses, crystal structures, and magnetic properties of [[Cu(2)(CA)(terpy)(2)][Cu(CA)(2)]](n)() and [[Cu(2)(CA)(terpy)(2)(dmso)(2)][Cu(CA)(2)(dmso)(2)](EtOH)](n)(H(2)CA = chloranilic acid, terpy = 2,2':6',2' '-terpyridine, dmso = dimethyl sulfoxide).

Two new copper(II) compounds of chloranilate and 2,2':6',2' '-terpyridine have been synthesized, and the structures have been solved by the single-crystal X-ray diffraction method. The crystal structure of [[Cu(2)(CA)(terpy)(2)][Cu(CA)(2)]](n)(1), where H(2)CA = chloranilic acid and terpy = 2,2':6',2' '-terpyridine, consists of two modules, the dimer unit [Cu(2)(CA)(terpy)(2)](2+) and the anionic mononuclear unit [Cu(CA)(2)](2)(-), forming an alternated chain. The chain is stabilized by semicoordinating and additional but efficient secondary bonding interactions. The crystal structure of [[Cu(2)(CA)(terpy)(2)(dmso)(2)][Cu(CA)(2)(dmso)(2)](EtOH)](n)(2), where dmso = dimethyl sulfoxide, consists of solvent molecules and two discrete modules, the dimer unit [Cu(2)(CA)(terpy)(2)(dmso)(2)](2+) and the anionic mononuclear unit [Cu(CA)(2)(dmso)(2)](2)(-). The dimer units form a layer by secondary bonding interactions, and the monomer units and ethanol molecules are introduced between the layers. The magnetic properties of 1 and 2 have been investigated in the temperature range 2.0-300 K. A weak ferromagnetic interaction was observed in 1, J(a) = 2.36 cm(-)(1) and zJ(b) = -0.68 cm(-)(1) while no exchange coupling was observed in 2.

Journal Article↗
PubMed1998

Crystal structure of porcine cathepsin H determined at 2.1 A resolution: location of the mini-chain C-terminal carboxyl group defines cathepsin H aminopeptidase function.

BACKGROUND: Cathepsin H is a lysosomal cysteine protease, involved in intracellular protein degradation. It is the only known mono-aminopeptidase in the papain-like family and is reported to be involved in tumor metastasis. The cathepsin H structure was determined in order to investigate the structural basis for its aminopeptidase activity and thus to provide the basis for structure-based design of synthetic inhibitors. RESULTS: The crystal structure of native porcine cathepsin H was determined at 2.1 A resolution. The structure has the typical papain-family fold. The so-called mini-chain, the octapeptide EPQNCSAT, is attached via a disulfide bond to the body of the enzyme and bound in a narrowed active-site cleft, in the substrate-binding direction. The mini-chain fills the region that in related enzymes comprises the non-primed substrate-binding sites from S2 backwards. CONCLUSIONS: The crystal structure of cathepsin H reveals that the mini-chain has a definitive role in substrate recognition and that carbohydrate residues attached to the body of the enzyme are involved in positioning the mini-chain in the active-site cleft. Modeling of a substrate into the active-site cleft suggests that the negatively charged carboxyl group of the C terminus of the mini-chain acts as an anchor for the positively charged N-terminal amino group of a substrate. The observed displacements of the residues within the active-site cleft from their equivalent positions in the papain-like endopeptidases suggest that they form the structural basis for the positioning of both the mini-chain and the substrate, resulting in exopeptidase activity.

Amino Acid Sequence↗