Macromolecular X-Ray Crystallography Research Papers (original) (raw)

The role of IgE in allergic disease mechanisms is performed principally through its interactions with two receptors, FcεRI on mast cells and basophils, and CD23 (FcεRII) on B cells. The former mediates allergic hypersensitivity, the... more

The role of IgE in allergic disease mechanisms is performed principally through its interactions with two receptors, FcεRI on mast cells and basophils, and CD23 (FcεRII) on B cells. The former mediates allergic hypersensitivity, the latter regulates IgE levels, and both receptors, also expressed on antigen-presenting cells, contribute to allergen uptake and presentation to the immune system. We have solved the crystal structure of the soluble lectin-like "head" domain of CD23 (derCD23) bound to a subfragment of IgE-Fc consisting of the dimer of Cε3 and Cε4 domains (Fcε3-4). One CD23 head binds to each heavy chain at the interface between the two domains, explaining the known 2:1 stoichiometry and suggesting mechanisms for cross-linking membrane-bound trimeric CD23 by IgE, or membrane IgE by soluble trimeric forms of CD23, both of which may contribute to the regulation of IgE synthesis by B cells. The two symmetrically located binding sites are distant from the single FcεRI binding site, which lies at the opposite ends of the Cε3 domains. Structural comparisons with both free IgE-Fc and its FcεRI complex reveal not only that the conformational changes in IgE-Fc required for CD23 binding are incompatible with FcεRI binding, but also that the converse is true. The two binding sites are allosterically linked. We demonstrate experimentally the reciprocal inhibition of CD23 and FcεRI binding in solution and suggest that the mutual exclusion of receptor binding allows IgE to function independently through its two receptors.

Two compounds, tenuifodione (1) and tenuifone (2), and 12 known compounds, izalpinin (3), alpinone (4), arborinone (5), irilin B (6), irisone A (7), irisone B (8), betavulgarin (9), b-sitosterol (10), 5,7-dihydroxy-2 0... more

Two compounds, tenuifodione (1) and tenuifone (2), and 12 known compounds, izalpinin (3), alpinone (4), arborinone (5), irilin B (6), irisone A (7), irisone B (8), betavulgarin (9), b-sitosterol (10), 5,7-dihydroxy-2 0 ,6-dimethoxyisoflavone (11), 2 0 ,5-dihdroxy-6,7-methylenedioxy flavanone , irisoid A (13) and ethyl-b-D-glucopyranoside were isolated from the whole plant of Iris tenuifolia Pall. All compounds, except 12, were isolated for the first time from this plant. Compounds 2, 3 and 11 have shown a considerable DPPH radical scavenging activity. Structures of these compounds were identified on the basis of spectroscopic techniques, including 2D NMR. Compounds 3, 5 and 7 were also subjected to single-crystal X-ray diffraction analysis and their structures were unambiguously deduced.

Flexibility of intrinsically disordered tau protein is important for performing its functions. It is believed that alteration of the flexibility is instrumental to the assembly of tau protein into paired helical filaments (PHF) in... more

Flexibility of intrinsically disordered tau protein is important for performing its functions. It is believed that alteration of the flexibility is instrumental to the assembly of tau protein into paired helical filaments (PHF) in tauopathies. Tau flexibility represents the main obstacle for structure determination of its conformation in physiology and/or pathology. We have alleviated this inherited difficulty by using specific monoclonal antibodies as tau protein surrogate binding partners. In this work we compare two ''antibody mold structures": (1) X-ray structure of the free form of the Alzheimer's disease PHF core-specific antibody MN423 and (2) previously solved structure of the complex of MN423 with the PHF core C-terminal tau peptide. We found that MN423 combining site is in both structures identical. As a consequence, recombinant tau assumes in the complex a fold determined by the antibody combining site. Obtained results show that MN423 functions as a molecular mold for the PHF core segment, and opens the way for structure determination of other PHF core segments providing that other conformation-specific antibodies are available. Data from in silico docking of tau peptide into antibody mold, obtained in this study, show that biochemical data and computational approaches provide results comparable to X-ray crystallography.

The synthesis and structure-activity relationships (SAR) of p38a MAP kinase inhibitors based on a pyrazolo-pyrimidine scaffold are described. These studies led to the identification of compound 2x as a potent and selective inhibitor of... more

The synthesis and structure-activity relationships (SAR) of p38a MAP kinase inhibitors based on a pyrazolo-pyrimidine scaffold are described. These studies led to the identification of compound 2x as a potent and selective inhibitor of p38a MAP kinase with excellent cellular potency toward the inhibition of TNFa production. Compound 2x was highly efficacious in vivo in inhibiting TNFa production in an acute murine model of TNFa production. X-ray co-crystallography of a pyrazolo-pyrimidine analog 2b bound to unphosphorylated p38a is also disclosed.

Calyxamines A (1) and B (2) are 2,2,4,6,6-pentasubstituted piperidine alkaloids possessing novel carbon skeletons isolated from the marine sponge Calyx podatypa collected in Puerto Rico. Their structures, after derivatization with... more

Calyxamines A (1) and B (2) are 2,2,4,6,6-pentasubstituted piperidine alkaloids possessing novel carbon skeletons isolated from the marine sponge Calyx podatypa collected in Puerto Rico. Their structures, after derivatization with trifluoroacetic acid, have been determined by a combination of X-ray and spectroscopic methods. A plausible biogenetic pathway to the calyxamines is suggested.

TIMP-3 (tissue inhibitor of metalloproteinases 3) is unique among the TIMP inhibitors, in that it effectively inhibits the TNF-α converting enzyme (TACE). In order to understand this selective capability of inhibition, we crystallized the... more

TIMP-3 (tissue inhibitor of metalloproteinases 3) is unique among the TIMP inhibitors, in that it effectively inhibits the TNF-α converting enzyme (TACE). In order to understand this selective capability of inhibition, we crystallized the complex formed by the catalytic domain of recombinant human TACE and the N-terminal domain of TIMP-3 (N-TIMP-3), and determined its molecular structure with X-ray data to 2.3 Å resolution. The structure reveals that TIMP-3 exhibits a fold similar to those of TIMP-1 and TIMP-2, and interacts through its functional binding edge, which consists of the N-terminal segment and other loops, with the active-site cleft of TACE in a manner similar to that of matrix metalloproteinases (MMPs). Therefore, the mechanism of TIMP-3 binding toward TACE is not fundamentally different from that previously elucidated for the MMPs. The Phe34 phenyl side chain situated at the tip of the relatively short sA-sB loop of TIMP-3 extends into a unique hydrophobic groove of the TACE surface, and two Leu residues in the adjacent sC-connector and sE-sF loops are tightly packed in the interface allowing favourable interactions, in agreement with predictions obtained by systematic mutations by Gillian Murphy's group. The combination of favourable functional epitopes together with a considerable flexibility renders TIMP-3 an efficient TACE inhibitor. This structure might provide means to design more efficient TIMP inhibitors of TACE.

PDB Reference: adipocyte lipid-binding protein, 2hnx, r2hnxsf.

The synthesis and SAR of a series of arylsulfonylpiperazine inhibitors of 11b-HSD1 are described. Optimization rapidly led to potent, selective, and orally bioavailable inhibitors demonstrating efficacy in a cynomolgus monkey ex vivo... more

The synthesis and SAR of a series of arylsulfonylpiperazine inhibitors of 11b-HSD1 are described. Optimization rapidly led to potent, selective, and orally bioavailable inhibitors demonstrating efficacy in a cynomolgus monkey ex vivo enzyme inhibition model.

Organovanadium compounds have been shown to be insulin sensitizers in vitro and in vivo. One potential biochemical mechanism for insulin sensitization by these compounds is that they inhibit protein tyrosine phosphatases (PTPs) that... more

Organovanadium compounds have been shown to be insulin sensitizers in vitro and in vivo. One potential biochemical mechanism for insulin sensitization by these compounds is that they inhibit protein tyrosine phosphatases (PTPs) that negatively regulate insulin receptor activation and signaling. In this study, bismaltolato oxovanadium (BMOV), a potent insulin sensitizer, was shown to be a reversible, competitive phosphatase inhibitor that inhibited phosphatase activity in cultured cells and enhanced insulin receptor activation in vivo. NMR and X-ray crystallographic studies of the interaction of BMOV with two different phosphatases, HCPTPA (human low molecular weight cytoplasmic protein tyrosine phosphatase) and PTP1B (protein tyrosine phosphatase 1B), demonstrated uncomplexed vanadium (VO ) in the active site. Taken together, these findings support phosphatase inhibition as a mechanism for insulin sensitization by BMOV 4 and other organovanadium compounds and strongly suggest that uncomplexed vanadium is the active component of these compounds. 

The binding of warfarin to the following human serum albumin (HSA) mutants was examined: K195M, K199M, F211V, W214L, R218M, R222M, H242V, and R257M. Warfarin bound to human serum albumin (HSA) exhibits an intrinsic fluorescence that is... more

The binding of warfarin to the following human serum albumin (HSA) mutants was examined: K195M, K199M, F211V, W214L, R218M, R222M, H242V, and R257M. Warfarin bound to human serum albumin (HSA) exhibits an intrinsic fluorescence that is approximately 10-...

3 containing the 1-aminocyclooctane-1carboxylic acid residue (Ac 8 c) were synthesized and conformationally characterized by x-ray diffraction studies in the crystal state. Peptides 1 and 2 were also studied by NMR in CDCl 3 solution.... more

3 containing the 1-aminocyclooctane-1carboxylic acid residue (Ac 8 c) were synthesized and conformationally characterized by x-ray diffraction studies in the crystal state. Peptides 1 and 2 were also studied by NMR in CDCl 3 solution. Peptide 1 adopts a purely 3 10-helical conformation in crystals, stabilized by three intramolecular 1 ← 4 hydrogen bonds. Peptide 2 in crystals is largely 3 10-helical with distortion in the backbone at the N-terminus by the insertion of a water molecule between Ac 8 c (2) CO and Ala (6) NH groups. Peptide 3 forms a C 10-ring structure, i.e. a type III (III) β-turn conformation stabilized by an intramolecular 1 ← 4 hydrogen bond. Five cyclooctane rings assume boat-chair conformations, whereas the sixth [Ac 8 c(8) in 2] is appreciably distorted, resembling a chiral intermediate in the pseudorotational pathway from the boat-chair to the twisted boat-chair conformation. Internal bond angles of the cyclooctane rings are appreciably distorted from the tetrahedral value, a characteristic feature of the cyclooctane ring. Peptide 1 crystallized in the space group P2 1 2 1 2 1 with a = 11.900(4)Å, b = 18.728(6)Å, c = 20.471(3)Å and Z = 4. The final R 1 and wR 2 values are 0.0753 and 0.2107, respectively, for 3901 observed reflections [F o ≥ 3σ (F o)]. Peptide 2 crystallized in space group P2 1 with a = 12.961(5)Å, b = 17.710(10)Å, c = 15.101(7)Å, β = 108.45(4)°and Z = 2. The final R 1 and wR 2 values are 0.0906 and 0.1832, respectively, for 2743 observed reflections [F o ≥ 3σ (F o)]. 1 H-NMR studies on both the peptides strongly suggest the persistence of 3 10-helical conformations in solution. Peptide 3 crystallized in the space group P2 1 /n, with a = 10.018(1)Å, b = 20.725(1)Å, c = 12.915(1)Å and Z = 4. The final R 1 and wR 2 values are 0.0411 and 0.1105, respectively, for 3634 observed reflections [F o ≥ 4σ (F o)].

C-protein is a major component of skeletal and cardiac muscle thick filaments. Mutations in the gene encoding cardiac C-protein [cardiac myosin binding protein-C (cMyBP-C)] are one of the principal causes of hypertrophic cardiomyopathy.... more

C-protein is a major component of skeletal and cardiac muscle thick filaments. Mutations in the gene encoding cardiac C-protein [cardiac myosin binding protein-C (cMyBP-C)] are one of the principal causes of hypertrophic cardiomyopathy. cMyBP-C is a string of globular domains including eight immunoglobulin-like and three fibronectin-like domains termed C0-C10. It binds to myosin and titin, and probably to actin, and may have both a structural and a regulatory role in muscle function. To help to understand the pathology of the known mutations, we have solved the structure of the immunoglobulin-like C1 domain of MyBP-C by X-ray crystallography to a resolution of 1.55 Å. Mutations associated with hypertrophic cardiomyopathy are clustered at one end towards the C-terminus, close to the important C1C2 linker, where they alter the structural integrity of this region and its interactions.

Keywords: HIV-1 protease inhibitors; structure-based drug design; pyrrolidine-based inhibitors; crystallography; deviating binding modes Structure-based drug design is an integral part of industrial and academic drug discovery projects.... more

Keywords: HIV-1 protease inhibitors; structure-based drug design; pyrrolidine-based inhibitors; crystallography; deviating binding modes Structure-based drug design is an integral part of industrial and academic drug discovery projects. Initial lead structures are, in general, optimized in terms of affinity using iterative cycles comprising synthesis, biological evaluation, computational methods, and structural analysis. X-ray crystallography commonly suggests the existence of a single well-defined state, termed binding mode, which is generally assumed to be consistent in a series of similar ligands and therefore used for the following optimization process. During the further development of symmetrically disubstituted 3,4-amino-pyrrolidines as human immunodeficiency virus type 1 protease inhibitors, we discovered that, by modification of the P1/P1′ moieties of our lead structure, the activity of the inhibitors towards the active-site mutation Ile84Val was altered, however, not being explainable with the initial underlying structure-activity relationship. The cocrystallization of the most potent derivative in complex with the human immunodeficiency virus type 1 protease surprisingly led to two different crystal forms (P2 1 2 1 2 1 and P6 1 22). Structural analysis revealed two completely different binding modes; the interaction of the pyrrolidine nitrogen atom with the catalytic aspartates remains as the only similarity. The study presented clearly demonstrates that structural biology has to escort the entire lead optimization process not to fail by an initially observed binding orientation.

We aimed to compare the apical sealing ability of two experimental retrograde root-filling cements and mineral trioxide aggregate using a fluid filtration method. X-ray diffraction (XRD) and X-ray fluorescence spectrometry (XRF) were used... more

We aimed to compare the apical sealing ability of two experimental retrograde root-filling cements and mineral trioxide aggregate using a fluid filtration method. X-ray diffraction (XRD) and X-ray fluorescence spectrometry (XRF) were used to evaluate structural and qualitative characteristics. Thirty single-rooted extracted teeth were treated, root-end prepared, and obturated using MTA and two experimental cements. Fluid filtration was measured during a 5-minute period after 4, 24, and 48 hours and 1, 2, and 12 weeks. The results were statistically compared by using a two-way analysis of variance (p Ͻ 0.05). The marginal adaptation was evaluated by using a SEM replica technique. XRD analyses showed similar patterns. XRF showed lower amounts of SiO 2 and FeO 3 in the experimental cements. All cements showed a reduced fluid flow rate over time. No significant differences were found between the cements. The SEM replica indicated a good marginal adaptation to dentinal walls. Both experimental cements showed suitable properties as retrograde rootfilling materials. (J Endod 2007;33:742-745)

A series of four-coordinate square-planar nickel(II) complexes of o-phenylenebis(N -methyloxamidate) (L 1 ) and related o-phenylene(N -methyloxamidate)oxamate (L 2 ) and o-phenylenebis(oxamate) (L 3 ) tetradentate ligands have been... more

A series of four-coordinate square-planar nickel(II) complexes of o-phenylenebis(N -methyloxamidate) (L 1 ) and related o-phenylene(N -methyloxamidate)oxamate (L 2 ) and o-phenylenebis(oxamate) (L 3 ) tetradentate ligands have been synthesized and characterized structurally, spectroscopically and electrochemically. The parent nickel(II)-L 1 complex presents an intense MLCT band in the UV region (k max = 357 nm) and a distinctive 1 s → 4p CT satellite in the Ni K-edge XANES spectrum (E = 8339.2 eV). These features together with the short Ni-N(amidate) bond lengths (1.85-1.93 Å ) as revealed by the analysis of the Ni K-edge EXAFS spectrum and confirmed by single-crystal X-ray diffraction are typical of square-planar low spin (S = 0) Ni II ions. The dianionic nickel(II) complexes, [Ni II L i ] 2− (i = 1-3), experience two redox processes in acetonitrile at 25 • C. The first redox process, at moderately low potentials (E 1 = 0.12-0.52 V vs. SCE), is a reversible one-electron metal-centered oxidation to the corresponding monoanionic nickel(III) complexes, [Ni III L i ] − . The second redox process, at relatively high potentials (E 2 = 0.86-1.04 V vs. SCE), is a quasireversible to irreversible one-electron oxidation largely centered on the o-benzenediamidate fragment of the non-innocent ligand, yielding the corresponding neutral nickel(III) complexes with a o-benzosemiquinonediimine p-cation radical ligand, [Ni III (L i ) • + ]. The singly and doubly oxidized species of the parent nickel(II)-L 1 complex have been prepared by chemical oxidation and characterized spectroscopically in acetonitrile at −40 • C. The stable singly oxidized nickel(III)-L 1 species presents an intense LMCT band in the NIR region (k max = 910 nm) and a rhombic X-band EPR spectrum (g 1 = 2.193, g 2 = 2.080 and g 3 = 2.006) characteristic of square-planar low spin (S = 1/2) Ni III ions. The unstable double oxidized nickel(III)-L 1 p-cation radical species exhibits a rather intense visible band (k max = 645 nm) that is tentatively assigned as a MLCT transition from the Ni III -benzosemiquinone type ground state to the Ni IV excited state. 2 5 1 6 D a l t o n T r a n s . , 2 0 0 5 , 2 5 1 6 -2 5 2 6 T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5

Here we report the first crystal structure of the SH3 domain of the cellular Src tyrosine kinase (c-Src-SH3) domain on its own. In the crystal two molecules of c-Src-SH3 exchange their -RT loops generating an intertwined dimer, in which... more

Here we report the first crystal structure of the SH3 domain of the cellular Src tyrosine kinase (c-Src-SH3) domain on its own. In the crystal two molecules of c-Src-SH3 exchange their -RT loops generating an intertwined dimer, in which the two SH3 units, preserving the binding site configuration, are oriented to allow simultaneous binding of two ligand molecules. The dimerization of c-Src-SH3 is induced, both in the crystal and in solution, by the binding of a PEG molecule at the dimer interface, indicating that this type of conformations are energetically close to the native structure. These results have important implications respect to in vivo oligomerization and amyloid aggregation.

Curli are functional amyloid fibres that constitute the major protein component of the extracellular matrix in pellicle biofilms formed by Bacteroidetes and Proteobacteria (predominantly of the α and γ classes). They provide a fitness... more

Curli are functional amyloid fibres that constitute the major protein component of the extracellular matrix in pellicle biofilms formed by Bacteroidetes and Proteobacteria (predominantly of the α and γ classes). They provide a fitness advantage in pathogenic strains and induce a strong pro-inflammatory response during bacteraemia. Curli formation requires a dedicated protein secretion machinery comprising the outer membrane lipoprotein CsgG and two soluble accessory proteins, CsgE and CsgF. Here we report the X-ray structure of Escherichia coli CsgG in a non-lipidated, soluble form as well as in its native membrane-extracted conformation. CsgG forms an oligomeric transport complex composed of nine anticodon-binding-domain-like units that give rise to a 36-stranded β-barrel that traverses the bilayer and is connected to a cage-like vestibule in the periplasm. The transmembrane and periplasmic domains are separated by a 0.9-nm channel constriction composed of three stacked concentric ...

A new natural pentacyclic compound, named kingianin A, was isolated as a racemic mixture from the barks of Endiandra kingiana (Lauraceae). Its structure was elucidated by comprehensive analysis of NMR spectroscopic data, X-ray... more

A new natural pentacyclic compound, named kingianin A, was isolated as a racemic mixture from the barks of Endiandra kingiana (Lauraceae). Its structure was elucidated by comprehensive analysis of NMR spectroscopic data, X-ray crystallography, and ECD calculations. The pentacyclic skeleton may be formed by a Diels-Alder reaction between two monomers having a bicyclo[4.2.0]octadiene backbone formed by a stereospecific electrocyclization of a linear compound of polyketide origin.

A series of novel N-aryl substituted thieno[2,3-d]pyrimidin-4(3H)-ones were designed and synthesized as potential inhibitors of chorismate mutase. Synthesis of this class of compounds was carried out by using Cu-mediated C-N bond forming... more

A series of novel N-aryl substituted thieno[2,3-d]pyrimidin-4(3H)-ones were designed and synthesized as potential inhibitors of chorismate mutase. Synthesis of this class of compounds was carried out by using Cu-mediated C-N bond forming reaction between thieno[2,3-d]pyrimidin-4(3H)-ones and aryl boronic acids. The reaction can be performed in an open flask as the conversion was found to be not sensitive to the presence of air or atmospheric moisture. A range of compounds were prepared by using this method and single crystal X-ray diffraction study was performed using a representative compound. In vitro pharmacological data of some of the compounds synthesized along with dose response studies using active molecules are presented. In silico interactions of these molecules with chorismate mutase are also presented.

Five new meroterpenoids, purpurogenolides A-E (1-5), and four known metabolites (6-9) were isolated from the solid substrate fermentation cultures of the fungus Penicillium purpurogenum MHz 111. The structures of the new meroterpenoids... more

Five new meroterpenoids, purpurogenolides A-E (1-5), and four known metabolites (6-9) were isolated from the solid substrate fermentation cultures of the fungus Penicillium purpurogenum MHz 111. The structures of the new meroterpenoids were elucidated by analysis of spectroscopic and spectrometric data (1D and 2D NMR, IR, and HRESIMS). The absolute configurations of 1 and 5 were determined by single-crystal X-ray crystallographic analysis, and those of 2-4 were elucidated on the basis of experimental and calculated electronic circular dichroism spectra. Compounds 2-4 and 6 showed inhibition of nitric oxide production in lipopolysaccharide-activated BV-2 microglial cells with IC50 values of 0.8-30.0 μM.

Human monoclonal antibody (mAb) 447-52D neutralizes a broad spectrum of HIV-1 isolates, whereas murine mAb 0.5 , raised against gp120 of the X4 isolate HIV-1 IIIB , neutralizes this strain specifically. Two distinct gp120 V3 peptides, V3... more

Human monoclonal antibody (mAb) 447-52D neutralizes a broad spectrum of HIV-1 isolates, whereas murine mAb 0.5 , raised against gp120 of the X4 isolate HIV-1 IIIB , neutralizes this strain specifically. Two distinct gp120 V3 peptides, V3 MN and V3 IIIB , adopt alternative-hairpin conformations when bound to 447-52D and 0.5 , respectively, suggesting that the alternative conformations of this loop play a key role in determining the coreceptor specificity of HIV-1. To test this hypothesis and to better understand the molecular basis underlying an antibody's breadth of neutralization, the solution structure of the V3 IIIB peptide bound to 447-52D was determined by NMR. V3 IIIB and V3 MN peptides bound to 447-52D exhibited the same N-terminal strand conformation, while the V3 IIIB peptide revealed alternative N-terminal conformations when bound to 447-52D and 0.5. Comparison of the three known V3 structures leads to a model in which a 180°change in the orientation of the side chains and the resulting oneresidue shift in hydrogen bonding patterns in the N-terminal strand of the-hairpins markedly alter the topology of the surface that interacts with antibodies and that can potentially interact with the HIV-1 coreceptors. Predominant interactions of 447-52D with three conserved residues of the N-terminal side of the V3 loop, K312, I314, and I316, can account for its broad cross reactivity, whereas the predominant interactions of 0.5 with variable residues underlie its strain specificity. Human immunodeficiency virus type 1 (HIV-1) 1 utilizes two membrane-bound molecules to gain entry into cells: CD4 and one of several types of chemokine receptors. Most HIV strains can utilize either the receptor for CC chemokines, CCR5, or the receptor for CXC chemokines, CXCR4, and are thus termed R5-and X4-tropic viruses, respectively. A minority of HIV strains that use both of these receptors are termed dual-tropic (1). The third hypervariable region of envelope glycoprotein gp120 (V3 loop, residues 303-340) is directly involved in the binding of gp120 to the chemokine receptors (2, 3). Analysis of the HIV-1 genome demonstrated that the V3 loop contains major determinants responsible for the phenotype of the virus and its cell tropism and that the V3 sequence determines whether the virus binds to CCR5 or CXCR4 (4). Results of alanine-scanning mutagenesis led to the conclusion that Lys305, Ile307, Arg313, and Phe315 are involved in CCR5 utilization (5). [These residues are numbered Lys312, Ile314, Arg322, and Phe324 according to the numbering system used in this paper (6).] A single mutation in the V3 loop, D329R, transforms an R5 virus into an X4 virus (7). Exchange of the V3 loop of the IIIB strain (an X4 virus) with the V3 loop of an R5 virus creates an R5-like virus with infectivity that is inhibited by chemokines specific for CCR5 (8). Many HIV-1-neutralizing antibodies in infected individuals or in immunized animals are directed against the V3 loop, which was designated, accordingly, the "principal neutralizing determinant" (PND) of HIV-1 (9). HIV-neutralizing antibodies against V3 are thought to prevent the binding of gp120 to either CCR5 or CXCR4, thus abolishing viral fusion with its target cell (3, 10). HIV-1 has developed a number of highly effective mechanisms for evading the immune system and especially for escaping neutralization by anti-V3 antibodies (11, 12). Study of chimeric gp120s from R5 and X4 strains and sequence analyses revealed that neutral-† This study was supported by the NIH Grants GM 53329 (J.A.), AI36085 (S.Z.-P.), and HL 59725 (S.Z.-P.) and research funding from the U.S. Department of Veterans Affairs (S.Z.-P.). J.A. is the Dr. Joseph and Ruth Owades Professor of Chemistry. ‡ The coordinates have been deposited in the Protein Data Bank as entries 1U6U (average structure) and 1U6V (ensemble of low-energy structures).

The regulation of the trp repressor system of Escherichia coli is frequently modeled by a single equilibrium, that between the aporepressor (TR) and the corepressor, L-tryptophan (Trp), at their intracellular concentrations. The actual... more

The regulation of the trp repressor system of Escherichia coli is frequently modeled by a single equilibrium, that between the aporepressor (TR) and the corepressor, L-tryptophan (Trp), at their intracellular concentrations. The actual mechanism, which is much more complex and more finely tuned, involves multiple equilibria: TR and Trp association, TR oligomerization, specific and nonspecific binding of various states of TR to DNA, and interactions between these various species and ions. TR in isolation exists primarily as a homodimer, but the state of oligomerization increases as the TR concentration goes up and/or the salt concentration goes down, leading to species with lower affinity for DNA. We have used multinuclear, multidimensional NMR spectroscopy to investigate structural changes that accompany the oligomerization of TR. For these investigations, the superrepressor mutant EK18 (TR with Glu 18 replaced by Lys) was chosen because it exhibits less severe oligomerization at higher protein concentration than other known variants; this made it possible to study the dimer to tetramer oligomerization step by NMR. The NMR results suggest that the interaction between TR dimers is structurally linked to folding of the DNA binding domain and that it likely involves direct contacts between the C-terminal residues of the C-helix of one dimer with the next dimer. This implies that oligomerization can compete with DNA binding and thus serves as a factor in the fine-tuning of gene expression.

The crystal structure of recombinant ferritin from Helicobacter pylori has been determined in its apo, low-iron-bound, intermediate, and high-ironbound states. Similar to other members of the ferritin family, the bacterial ferritin... more

The crystal structure of recombinant ferritin from Helicobacter pylori has been determined in its apo, low-iron-bound, intermediate, and high-ironbound states. Similar to other members of the ferritin family, the bacterial ferritin assembles as a spherical protein shell of 24 subunits, each of which folds into a four-α-helix bundle. Significant conformational changes were observed at the BC loop and the entrance of the 4-fold symmetry channel in the intermediate and high-iron-bound states, whereas no change was found in the apo and low-iron-bound states. The imidazole rings of His149 at the channel entrance undergo conformational changes that bear resemblance to heme configuration and are directly coupled to axial translocation of Fe ions through the 4-fold channel. Our results provide the first structural evidence of the translocation of Fe ions through the 4-fold channel in prokaryotes and the transition from a protein-dominated process to a mineral-surfacedominated process during biomineralization.

Transaldolase (Tal) is involved in the central carbon metabolism, i.e. the non-oxidative pentose phosphate pathway, and is therefore a ubiquitous enzyme. However, Tals show a low degree in sequence identity and vary in length within the... more

Transaldolase (Tal) is involved in the central carbon metabolism, i.e. the non-oxidative pentose phosphate pathway, and is therefore a ubiquitous enzyme. However, Tals show a low degree in sequence identity and vary in length within the enzyme family which previously led to the definition of five subfamilies. We wondered how this variation is conserved in structure and function. To answer this question we characterised and compared the Tals from Bacillus subtilis, Corynebacterium glutamicum and Escherichia coli, each belonging to a different subfamily, with respect to their biochemical properties and structures. The overall structure of the Tal domain, a (b ⁄ a) 8 -barrel fold, is well conserved between the different subfamilies but the enzymes show different degrees of oligomerisation (monomer, dimer and decamer). The substrate specificity of the three enzymes investigated is quite similar which is reflected in the conservation of the active site, the phosphate binding site as well as the position of a catalytically important water molecule. All decameric enzymes characterised so far appear to be heat stable no matter whether they originate from a mesophilic or thermophilic organism. Hence, the thermostability might be due to the structural properties, i.e. tight packing, of these enzymes.

The steroid hormone receptors are characterized by binding to relatively rigid, inflexible endogenous steroid ligands. Other members of the nuclear receptor superfamily bind to conformationally flexible lipids and show a corresponding... more

The steroid hormone receptors are characterized by binding to relatively rigid, inflexible endogenous steroid ligands. Other members of the nuclear receptor superfamily bind to conformationally flexible lipids and show a corresponding degree of elasticity in the ligand-binding pocket. Here, we report the X-ray crystal structure of the oestrogen receptor a (ERa) bound to an oestradiol derivative with a prosthetic group, ortho-trifluoromethlyphenylvinyl, which binds in a novel extended pocket in the ligand-binding domain. Unlike ER antagonists with bulky side groups, this derivative is enclosed in the ligand-binding pocket, and acts as a potent agonist. This work shows that steroid hormone receptors can interact with a wider array of pharmacophores than previously thought through structural plasticity in the ligand-binding pocket.

Thiazolidinedione (TZD) compounds targeting the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) demonstrate unique benefits for the treatment of insulin resistance and type II diabetes. TZDs include... more

Thiazolidinedione (TZD) compounds targeting the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) demonstrate unique benefits for the treatment of insulin resistance and type II diabetes. TZDs include rosiglitazone, pioglitazone and rivoglitazone, with the latter being the most potent. The TZDs are only marginally selective for the therapeutic target PPARγ as they also activate PPARα and PPARδ homologues to varying degrees, causing off-target effects. While crystal structures for TZD compounds in complex with PPARγ are available, minimal structural information is available for TZDs bound to PPARα and PPARδ. This paucity of structural information has hampered the determination of precise structural mechanisms involved in TZD
selectivity between PPARs. To help address these questions molecular dynamic simulations were performed of rosiglitazone, pioglitazone and rivoglitazone in complex with PPARα, PPARδ, and PPARγ in order to better understand the mechanisms of PPAR selectivity. The simulations revealed that TZD interactions with residues Tyr314 and Phe318 of PPARα and residues Phe291 and Thr253 of PPARδ as well as the omega loop, are key determinants of TZD receptor selectivity. Notably, in this study, we solve the first X-ray crystal structure of rivoglitazone bound to any PPAR. Rivoglitazone forms a unique hydrogen bond network with the residues of the PPARγ co-activator binding surface (known as AF2) and makes more extensive contacts with helix 3 and the β- sheet as compared to model TZD compounds such as rosiglitazone.

GDP-4-keto-6-deoxy-D-mannose epimerase/reductase is a bifunctional enzyme responsible for the last step in the biosynthesis of GDP-L-fucose, the substrate of fucosyl transferases. Several cell-surface antigens, including the leukocyte... more

GDP-4-keto-6-deoxy-D-mannose epimerase/reductase is a bifunctional enzyme responsible for the last step in the biosynthesis of GDP-L-fucose, the substrate of fucosyl transferases. Several cell-surface antigens, including the leukocyte Lewis system and cell-surface antigens in pathogenic bacteria, depend on the availability of GDP-L-fucose for their expression. Therefore, the enzyme is a potential target for therapy in pathological states depending on selectin-mediated cell-to-cell interactions. Previous crystallographic investigations have shown that GDP-4-keto-6-deoxy-Dmannose epimerase/reductase belongs to the short-chain dehydrogenase/reductase protein homology family. The enzyme active-site region is at the interface of an N-terminal NADPH-binding domain and a C-terminal domain, held to bind the substrate. The design, expression and functional characterization of seven site-speci®c mutant forms of GDP-4-keto-6-deoxy-D-mannose epimerase/reductase are reported here. In parallel, the crystal structures of the native holoenzyme and of three mutants (Ser107Ala, Tyr136Glu and Lys140Arg) have been investigated and re®ned at 1.45-1.60 A Ê resolution, based on synchrotron data (R-factors range between 12.6 % and 13.9 %). The re®ned protein models show that besides the active-site residues Ser107, Tyr136 and Lys140, whose mutations impair the overall enzymatic activity and may affect the coenzyme binding mode, side-chains capable of proton exchange, located around the expected substrate (GDP-4-keto-6-deoxy-D-mannose) binding pocket, are selectively required during the epimerization and reduction steps. Among these, Cys109 and His179 may play a primary role in proton exchange between the enzyme and the epimerization catalytic intermediates. Finally, the additional role of mutated active-site residues involved in substrate recognition and in enzyme stability has been analyzed.

PLAA (ortholog of yeast Doa1/Ufd3, also know as human PLAP or phospholipase A2-activating protein) has been implicated in a variety of disparate biological processes that involve the ubiquitin system. It is linked to the maintenance of... more

PLAA (ortholog of yeast Doa1/Ufd3, also know as human PLAP or phospholipase A2-activating protein) has been implicated in a variety of disparate biological processes that involve the ubiquitin system. It is linked to the maintenance of ubiquitin levels, but the mechanism by which it accomplishes this is unclear. The C-terminal PUL (PLAP, Ufd3p, and Lub1p) domain of PLAA binds p97, an AAA ATPase, which among other functions helps transfer ubiquitinated proteins to the proteasome for degradation. In yeast, loss of Doa1 is suppressed by altering p97/Cdc48 function indicating that physical interaction between PLAA and p97 is functionally important. Although the overall regions of interaction between these proteins are known, the structural basis has been unavailable. We solved the high resolution crystal structure of the p97-PLAA complex showing that the PUL domain forms a 6-mer Armadillo-containing domain. Its N-terminal extension folds back onto the inner curvature forming a deep ridge that is positively charged with residues that are phylogenetically conserved. The C terminus of p97 binds in this ridge, where the side chain of p97-Tyr 805 , implicated in phosphorylation-dependent regulation, is buried. Expressed in doa1⌬ null cells, point mutants of the yeast ortholog Doa1 that disrupt this interaction display slightly reduced ubiquitin levels, but unlike doa1⌬ null cells, showed only some of the growth phenotypes. These data suggest that the p97-PLAA interaction is important for a subset of PLAA-dependent biological processes and provides a framework to better understand the role of these complex molecules in the ubiquitin system.

† E.P. and R.A. contributed equally to this work.

The crystal structure of native chicken fibrinogen (320 kDa) complexed with two synthetic peptides has been determined at a resolution of 2.7 Å. The structure provides the first atomic-resolution view of the polypeptide chain arrangement... more

The crystal structure of native chicken fibrinogen (320 kDa) complexed with two synthetic peptides has been determined at a resolution of 2.7 Å. The structure provides the first atomic-resolution view of the polypeptide chain arrangement in the central domain where the two halves of the molecule are joined, as well as of a putative thrombin-binding site. The amino-terminal segments of the R and chains, including fibrinopeptides A and B, are not visible in electron density maps, however, and must be highly disordered. The RC domain is also very disordered. A residue by residue analysis of the coiled coils with regard to temperature factor shows a strong correlation between mobility and plasmin attack sites. It is concluded that structural flexibility is an inherent feature of fibrinogen that plays a key role in both its conversion to fibrin and its subsequent destruction by plasmin.

Background: Feedback inhibition of biosynthetic threonine deaminase (TD) from Escherichia coli provided one of the earliest examples of protein-based metabolic regulation. Isoleucine, the pathway end-product, and valine, the product of a... more

Background: Feedback inhibition of biosynthetic threonine deaminase (TD) from Escherichia coli provided one of the earliest examples of protein-based metabolic regulation. Isoleucine, the pathway end-product, and valine, the product of a parallel pathway, serve as allosteric inhibitor and activator, respectively. This enzyme is thus a useful model system for studying the structural basis of allosteric control mechanisms.

PDB Reference: Bacillus amyloliquefaciens -amylase, 3bh4.

Predicting protein-protein interfaces from a three-dimensional structure is a key task of computational structural proteomics. In contrast to geometrically distinct small molecule binding sites, protein-protein interface are notoriously... more

Predicting protein-protein interfaces from a three-dimensional structure is a key task of computational structural proteomics. In contrast to geometrically distinct small molecule binding sites, protein-protein interface are notoriously difficult to predict. We generated a large nonredundant data set of 1494 true protein-protein interfaces using biological symmetry annotation where necessary. The data set was carefully analyzed and a Support Vector Machine was trained on a combination of a new robust evolutionary conservation signal with the local surface properties to predict protein-protein interfaces. Fivefold cross validation verifies the high sensitivity and selectivity of the model. As much as 97% of the predicted patches had an overlap with the true interface patch while only 22% of the surface residues were included in an average predicted patch. The model allowed the identification of potential new interfaces and the correction of mislabeled oligomeric states. Proteins 2005;60:353-366.

Mammalian genomes encode seven catalytic proteasome subunits, namely, β1c, β2c, β5c (assembled into constitutive 20S proteasome core particles), β1i, β2i, β5i (incorporated into immunoproteasomes), and the thymoproteasome-specific subunit... more

Mammalian genomes encode seven catalytic proteasome subunits, namely, β1c, β2c, β5c (assembled into constitutive 20S proteasome core particles), β1i, β2i, β5i (incorporated into immunoproteasomes), and the thymoproteasome-specific subunit β5t. Extensive research in the past decades has yielded numerous potent proteasome inhibitors including compounds currently used in the clinic to treat multiple myeloma and mantle cell lymphoma. Proteasome inhibitors that selectively target combinations of β1c/β1i, β2c/β2i, or β5c/β5i are available, yet ligands truly selective for a single proteasome activity are scarce. In this work we report the development of cell-permeable β1i and β5i selective inhibitors that outperform existing leads in terms of selectivity and/or potency. These compounds are the result of a rational design strategy using known inhibitors as starting points and introducing structural features according to the X-ray structures of the murine constitutive and immunoproteasome 20S core particles.

The exploitation of catalytic promiscuity and the application of de novo design have recently opened the access to novel, non-natural enzymatic activities. Here we describe a structural bioinformatic method for predicting catalytic... more

The exploitation of catalytic promiscuity and the application of de novo design have recently opened the access to novel, non-natural enzymatic activities. Here we describe a structural bioinformatic method for predicting catalytic activities of enzymes based on three-dimensional constellations of functional groups in active sites ('catalophores'). As a proof-ofconcept we identify two enzymes with predicted promiscuous ene-reductase activity (reduction of activated C-C double bonds) and compare them with known ene-reductases, that is, members of the Old Yellow Enzyme family. Despite completely different amino acid sequences, overall structures and protein folds, high-resolution crystal structures reveal equivalent binding modes of typical Old Yellow Enzyme substrates and ligands. Biochemical and biocatalytic data show that the two enzymes indeed possess ene-reductase activity and reveal an inverted stereopreference compared with Old Yellow Enzymes for some substrates. This method could thus be a tool for the identification of viable starting points for the development and engineering of novel biocatalysts.

Objective-To design a new class of selective neuronal nitric oxide synthase (nNOS) inhibitors and demonstrate that administration in a rabbit model for cerebral palsy (CP) prevents hypoxiaischemia induced deaths and reduces the number of... more

Objective-To design a new class of selective neuronal nitric oxide synthase (nNOS) inhibitors and demonstrate that administration in a rabbit model for cerebral palsy (CP) prevents hypoxiaischemia induced deaths and reduces the number of newborn kits exhibiting signs of CP.

The CD1 family is a large cluster of non-polymorphic, major histocompatibility complex (MHC) class-I-like molecules that bind distinct lipid-based antigens that are recognized by T cells. The most studied group of T cells that interact... more

The CD1 family is a large cluster of non-polymorphic, major histocompatibility complex (MHC) class-I-like molecules that bind distinct lipid-based antigens that are recognized by T cells. The most studied group of T cells that interact with lipid antigens are natural killer T (NKT) cells, which characteristically express a semi-invariant T-cell receptor (NKT TCR) that specifically recognizes the CD1 family member, CD1d. NKT-cell-mediated recognition of the CD1d-antigen complex has been implicated in microbial immunity, tumour immunity, autoimmunity and allergy. Here we describe the structure of a human NKT TCR in complex with CD1d bound to the potent NKT-cell agonist a-galactosylceramide, the archetypal CD1d-restricted glycolipid. In contrast to T-cell receptor-peptide-antigen-MHC complexes, the NKT TCR docked parallel to, and at the extreme end of the CD1d-binding cleft, which enables a lock-and-key type interaction with the lipid antigen. The structure provides a basis for the interaction between the highly conserved NKT TCR a-chain and the CD1d-antigen complex that is typified in innate immunity, and also indicates how variability of the NKT TCR b-chain can impact on recognition of other CD1d-antigen complexes. These findings provide direct insight into how a T-cell receptor recognizes a lipid-antigen-presenting molecule of the immune system.

The structure of the bacteriophage phi X174 was examined in a 2.7 A resolution map and refined, using 6.0 A to 3.0 A resolution data with F > or = 5 sigma (F). The final R-factor was 20.9% and the root-mean-square deviation from... more

The structure of the bacteriophage phi X174 was examined in a 2.7 A resolution map and refined, using 6.0 A to 3.0 A resolution data with F > or = 5 sigma (F). The final R-factor was 20.9% and the root-mean-square deviation from idealized bond lengths was 0.021 A. The Hendrickson-Konnert refinement was restrained by the phases derived from the molecular replacement icosahedral averaging procedure. The mature phage capsid consists of 60 copies of the F protein with 426 amino acids, the G protein with 175 amino acids and the J protein with 37 amino acids, as well as 12 copies of the H protein with 328 amino acids. The entire polypeptide chain of the F and G protein, all but the first N-terminal residue of the J protein, and 178 solvent molecules were included in the refinement calculations. The secondary structural features of the F, G and J proteins and their interactions with each other are described. The majority of the protein-protein interactions are between the icosahedral 5-fold related interfaces of the F and of the G proteins. These pentameric units of the F and G proteins form the 9S and 6S assembly intermediates, respectively. The J protein lacks any secondary structure and acts as a linking arm between the icosahedral 5-fold related F proteins. Water molecules were introduced only after phase extension to 2.7 A resolution had been completed. The F protein is associated with lower "thermal" parameters and exhibits greater water order in its environment than the G and J proteins. The largest thermal parameters occur in residues on the viral surface. The solvent contributes to the interactions between the proteins. There is an interface of solvent molecules between the F and the G pentamers which stabilizes the pentameric G protein spikes in a crater centered at each of the icosahedral 5-fold vertices of the F protein capsid. Sequence alignments of the F, G and J amino acid sequences for the homologous bacteriophages G4, alpha 3, phi K and phi X174 with respect to the phi X174 structure demonstrated the conservation of functionally important residues on the viral surface.

The pathogenic bacterium Staphylococcus aureus counteracts the host immune defense by excretion of the 85 residue staphylococcal complement inhibitor (SCIN). SCIN inhibits the central complement convertases; thereby, it reduces... more

The pathogenic bacterium Staphylococcus aureus counteracts the host immune defense by excretion of the 85 residue staphylococcal complement inhibitor (SCIN). SCIN inhibits the central complement convertases; thereby, it reduces phagocytosis following opsonization and efficiently blocks all downstream effector functions. In this study, we present the crystal structure of SCIN at 1.8 Å resolution and the identification of its active site. Functional characterization of structure based chimeric proteins, consisting of SCIN and the structurally but nonfunctional homologue open reading frame-D, indicate an 18-residue segment (Leu-31-Gly-48) crucial for SCIN activity. In all complement activation pathways, chimeras lacking these SCIN residues completely fail to inhibit production of the potent mediator of inflammation C5a. Inhibition of alternative pathway-mediated opsonization (C3b deposition) and formation of the lytic membrane attack complex (C5b-9 deposition) are strongly reduced for these chimeras as well. For inhibition of the classical/lectin pathway-mediated C3b and C5b-9 deposition, the same residues are critical although additional sites are involved. These chimeras also display reduced capacity to stabilize the C3 convertases of both the alternative and the classical/lectin pathway indicating the stabilizing effect is pivotal for the complement inhibitory activity of SCIN. Because SCIN specifically and efficiently inhibits complement, it has a high potential in anti-inflammatory therapy. Our data are a first step toward the development of a second generation molecule suitable for such therapeutic complement intervention.

A new series of adamantane-isothiourea hybrid derivatives, namely 4-arylmethyl (Z)-N'-(adamantan-1-yl)-morpholine-4-carbothioimidates 7a-e and 4-arylmethyl (Z)-N'-(adamantan-1-yl)-4-phenylpiperazine-1-carbothioimidates 8a-e were... more

A new series of adamantane-isothiourea hybrid derivatives, namely 4-arylmethyl (Z)-N'-(adamantan-1-yl)-morpholine-4-carbothioimidates 7a-e and 4-arylmethyl (Z)-N'-(adamantan-1-yl)-4-phenylpiperazine-1-carbothioimidates 8a-e were prepared via the reaction of N-(adamantan-1-yl)morpholine-4-carbothioamide 5 and N-(adamantan-1-yl)-4-phenylpiperazine-1-carbothioamide 6 with benzyl or substituted benzyl bromides, in acetone, in the presence of anhydrous potassium carbonate. The structures of the synthesized compounds were confirmed by ¹H-NMR, (13)C-NMR, electrospray ionization mass spectral (ESI-MS) data, and X-ray crystallographic data. The in vitro antimicrobial activity of the new compounds was determined against certain standard strains of pathogenic bacteria and the yeast-like pathogenic fungus Candida albicans. Compounds 7b, 7d and 7e displayed potent broad-spectrum antibacterial activity, while compounds 7a, 7c, 8b, 8d and 8e were active against the tested Gram-positive bac...

Fragment-based ligand screening is now established as an emerging paradigm for drug discovery. Here we examine the recent literature looking at how structural biology has been used in a variety of successful fragment-screening... more

Fragment-based ligand screening is now established as an emerging paradigm for drug discovery. Here we examine the recent literature looking at how structural biology has been used in a variety of successful fragment-screening applications. We argue that the determination of experimental binding modes has proved to be one of the mainstays of successful fragment-based approaches and that this reflects the difficulty in optimising a fragment to a lead molecule in the absence of structural information. We focus on antimicrobial research where fragment-based drug discovery allows control of the physical properties of the emerging lead molecule.