Structural and functional properties of the Kunitz-type and C-terminal domains of Amblyomin-X supporting its antitumor activity.

Amblyomin-X is a Kunitz-type FXa inhibitor identified through the transcriptome analysis of the salivary gland from Amblyomma sculptum tick. This protein consists of two domains of equivalent size, triggers apoptosis in different tumor cell lines, and promotes regression of tumor growth, and reduction of metastasis. To study the structural properties and functional roles of the N-terminal (N-ter) and C-terminal (C-ter) domains of Amblyomin-X, we synthesized them by solid-phase peptide synthesis, solved the X-Ray crystallographic structure of the N-ter domain, confirming its Kunitz-type signature, and studied their biological properties. We show here that the C-ter domain is responsible for the uptake of Amblyomin-X by tumor cells and highlight the ability of this domain to deliver intracellular cargo by the strong enhancement of the intracellular detection of molecules with low cellular-uptake efficiency (p15) after their coupling with the C-ter domain. In contrast, the N-ter Kunitz domain of Amblyomin-X is not capable of crossing through the cell membrane but is associated with tumor cell cytotoxicity when it is microinjected into the cells or fused to TAT cell-penetrating peptide. Additionally, we identify the minimum length C-terminal domain named F2C able to enter in the SK-MEL-28 cells and induces dynein chains gene expression modulation, a molecular motor that plays a role in the uptake and intracellular trafficking of Amblyomin-X.

Crystal structures of two viral peptides in complex with murine MHC class I H-2Kb.

The x-ray structures of a murine MHC class I molecule (H-2Kb) were determined in complex with two different viral peptides, derived from the vesicular stomatitis virus nucleoprotein (52-59), VSV-8, and the Sendai virus nucleoprotein (324-332), SEV-9. The H-2Kb complexes were refined at 2.3 A for VSV-8 and 2.5 A for SEV-9. The structure of H-2Kb exhibits a high degree of similarity with human HLA class I, although the individual domains can have slightly altered dispositions. Both peptides bind in extended conformations with most of their surfaces buried in the H-2Kb binding groove. The nonamer peptide maintains the same amino- and carboxyl-terminal interactions as the octamer primarily by the insertion of a bulge in the center of an otherwise beta conformation. Most of the specific interactions are between side-chain atoms of H-2Kb and main-chain atoms of peptide. This binding scheme accounts in large part for the enormous diversity of peptide sequences that bind with high affinity to class I molecules. Small but significant conformational changes in H-2Kb are associated with peptide binding, and these synergistic movements may be an integral part of the T cell receptor recognition process.

Routes to catalysis: structure of a catalytic antibody and comparison with its natural counterpart.

The three-dimensional structure of a catalytic antibody (1F7) with chorismate mutase activity has been determined to 3.0 A resolution as a complex with a transition state analog. The structural data suggest that the antibody stabilizes the same conformationally restricted pericyclic transition state as occurs in the uncatalyzed reaction. Overall shape and charge complementarity between the combining site and the transition state analog dictate preferential binding of the correct substrate enantiomer in a conformation appropriate for reaction. Comparison with the structure of a chorismate mutase enzyme indicates an overall similarity between the catalytic mechanism employed by the two proteins. Differences in the number of specific interactions available for restricting the rotational degrees of freedom in the transition state, and the lack of multiple electrostatic interactions that might stabilize charge separation in this highly polarized metastable species, are likely to account for the observed 10(4) times lower activity of the antibody relative to that of the natural enzymes that catalyze this reaction. The structure of the 1F7 Fab'-hapten complex provides confirmation that the properties of an antibody catalyst faithfully reflect the design of the transition state analog.

Crystal structure of mouse CD1: An MHC-like fold with a large hydrophobic binding groove.

CD1 represents a third lineage of antigen-presenting molecules that are distantly related to major histocompatibility complex (MHC) molecules in the immune system. The crystal structure of mouse CD1d1, corresponding to human CD1d, at 2.8 resolution shows that CD1 adopts an MHC fold that is more closely related to that of MHC class I than to that of MHC class II. The binding groove, although significantly narrower, is substantially larger because of increased depth and it has only two major pockets that are almost completely hydrophobic. The extreme hydrophobicity and shape of the binding site are consistent with observations that human CD1b and CD1c can present mycobacterial cell wall antigens, such as mycolic acid and lipoarabinomannans. However, mouse CD1d1 can present very hydrophobic peptides, but must do so in a very different way from MHC class Ia and class II molecules.

Crystal structure of the principal neutralization site of HIV-1.

The crystal structure of a complex between a 24-amino acid peptide from the third variable (V3) loop of human immunodeficiency virus-type 1 (HIV-1) gp 120 and the Fab fragment of a broadly neutralizing antibody (59.1) was determined to 3 angstrom resolution. The tip of the V3 loop containing the Gly-Pro-Gly-Arg-Ala-Phe sequence adopts a double-turn conformation, which may be the basis of its conservation in many HIV-1 isolates. A complete map of the HIV-1 principal neutralizing determinant was constructed by stitching together structures of V3 loop peptides bound to 59.1 and to an isolate-specific (MN) neutralizing antibody (50.1). Structural conservation of the overlapping epitopes suggests that this biologically relevant conformation could be of use in the design of synthetic vaccines and drugs to inhibit HIV-1 entry and virus-related cellular fusion.

Crystallographic evidence for preformed dimers of erythropoietin receptor before ligand activation.

Erythropoietin receptor (EPOR) is thought to be activated by ligand-induced homodimerization. However, structures of agonist and antagonist peptide complexes of EPOR, as well as an EPO-EPOR complex, have shown that the actual dimer configuration is critical for the biological response and signal efficiency. The crystal structure of the extracellular domain of EPOR in its unliganded form at 2.4 angstrom resolution has revealed a dimer in which the individual membrane-spanning and intracellular domains would be too far apart to permit phosphorylation by JAK2. This unliganded EPOR dimer is formed from self-association of the same key binding site residues that interact with EPO-mimetic peptide and EPO ligands. This model for a preformed dimer on the cell surface provides insights into the organization, activation, and plasticity of recognition of hematopoietic cell surface receptors.

An antibody exo Diels-Alderase inhibitor complex at 1.95 angstrom resolution.

A highly specific Diels-Alder protein catalyst was made by manipulating the antibody repertoire of the immune system. The catalytic antibody 13G5 catalyzes a disfavored exo Diels-Alder transformation in a reaction for which there is no natural enzyme counterpart and that yields a single regioisomer in high enantiomeric excess. The crystal structure of the antibody Fab in complex with a ferrocenyl inhibitor containing the essential haptenic core that elicited 13G5 was determined at 1.95 angstrom resolution. Three key antibody residues appear to be responsible for the observed catalysis and product control. Tyrosine-L36 acts as a Lewis acid activating the dienophile for nucleophilic attack, and asparagine-L91 and aspartic acid-H50 form hydrogen bonds to the carboxylate side chain that substitutes for the carbamate diene substrate. This hydrogen-bonding scheme leads to rate acceleration and also pronounced stereoselectivity. Docking experiments with the four possible ortho transition states of the reaction explain the specific exo effect and suggest that the (3R,4R)-exo stereoisomer is the preferred product.

Functional mimicry of a protein hormone by a peptide agonist: the EPO receptor complex at 2.8 A.

The functional mimicry of a protein by an unrelated small molecule has been a formidable challenge. Now, however, the biological activity of a 166-residue hematopoietic growth hormone, erythropoietin (EPO), with its class 1 cytokine receptor has been mimicked by a 20-residue cyclic peptide unrelated in sequence to the natural ligand. The crystal structure at 2.8 A resolution of a complex of this agonist peptide with the extracellular domain of EPO receptor reveals that a peptide dimer induces an almost perfect twofold dimerization of the receptor. The dimer assembly differs from that of the human growth hormone (hGH) receptor complex and suggests that more than one mode of dimerization may be able to induce signal transduction and cell proliferation. The EPO receptor binding site, defined by peptide interaction, corresponds to the smaller functional epitope identified for hGH receptor. Similarly, the EPO mimetic peptide ligand can be considered as a minimal hormone, and suggests the design of nonpeptidic small molecule mimetics for EPO and other cytokines may indeed be achievable.

Immune versus natural selection: antibody aldolases with enzymic rates but broader scope.

Structural and mechanistic studies show that when the selection criteria of the immune system are changed, catalytic antibodies that have the efficiency of natural enzymes evolve, but the catalytic antibodies are much more accepting of a wide range of substrates. The catalytic antibodies were prepared by reactive immunization, a process whereby the selection criteria of the immune system are changed from simple binding to chemical reactivity. This process yielded aldolase catalytic antibodies that approximated the rate acceleration of the natural enzyme used in glycolysis. Unlike the natural enzyme, however, the antibody aldolases catalyzed a variety of aldol reactions and decarboxylations. The crystal structure of one of these antibodies identified the reactive lysine residue that was selected in the immunization process. This lysine is deeply buried in a hydrophobic pocket at the base of the binding site, thereby accounting for its perturbed pKa.

Dual conformations for the HIV-1 gp120 V3 loop in complexes with different neutralizing fabs.

BACKGROUND: The third hypervariable (V3) loop of HIV-1 gp120 has been termed the principal neutralizing determinant (PND) of the virus and is involved in many aspects of virus infectivity. The V3 loop is required for viral entry into the cell via membrane fusion and is believed to interact with cell surface chemokine receptors on T cells and macrophages. Sequence changes in V3 can affect chemokine receptor usage, and can, therefore, modulate which types of cells are infected. Antibodies raised against peptides with V3 sequences can neutralize laboratory-adapted strains of the virus and inhibit syncytia formation. Fab fragments of these neutralizing antibodies in complex with V3 loop peptides have been studied by X-ray crystallography to determine the conformation of the V3 loop. RESULTS: We have determined three crystal structures of Fab 58.2, a broadly neutralizing antibody, in complex with one linear and two cyclic peptides the amino acid sequence of which comes from the MN isolate of the gp120 V3 loop. Although the peptide conformations are very similar for the linear and cyclic forms, they differ from that seen for the identical peptide bound to a different broadly neutralizing antibody, Fab 59.1, and for a similar peptide bound to the MN-specific Fab 50.1. The conformational difference in the peptide is localized around residues Gly-Pro-Gly-Arg, which are highly conserved in different HIV-1 isolates and are predicted to adopt a type II beta turn. CONCLUSIONS: The V3 loop can adopt at least two different conformations for the highly conserved Gly-Pro-Gly-Arg sequence at the tip of the loop. Thus, the HIV-1 V3 loop has some inherent conformational flexibility that may relate to its biological function.

Complex between Peptostreptococcus magnus protein L and a human antibody reveals structural convergence in the interaction modes of Fab binding proteins.

BACKGROUND: Peptostreptococcus magnus protein L (PpL) is a multidomain, bacterial surface protein whose presence correlates with virulence. It consists of up to five homologous immunoglobulin binding domains that interact with the variable (VL) regions of kappa light chains found on two thirds of mammalian antibodies. RESULTS: We refined the crystal structure of the complex between a human antibody Fab fragment (2A2) and a single PpL domain (61 residues) to 2.7 A. The asymmetric unit contains two Fab molecules sandwiching a single PpL domain, which contacts similar VL framework regions of two light chains via independent interfaces. The residues contacted on VL are remote from the hypervariable loops. One PpL-Vkappa interface agrees with previous biochemical data, while the second is novel. Site-directed mutagenesis and analytical-centrifugation studies suggest that the two PpL binding sites have markedly different affinities for VL. The PpL residues in both interactions are well conserved among different Peptostreptococcus magnus strains. The Fab contact positions identified in the complex explain the high specificity of PpL for antibodies with kappa rather than lambda chains. CONCLUSIONS: The PpL-Fab complex shows the first interaction of a bacterial virulence factor with a Fab light chain outside the conventional combining site. Structural comparison with two other bacterial proteins interacting with the Fab heavy chain shows that PpL, structurally homologous to streptococcal SpG domains, shares with the latter a similar binding mode. These two bacterial surface proteins interact with their respective immunoglobulin regions through a similar beta zipper interaction.

Crystallization and preliminary crystallographic data for an antiprogesterone monoclonal antibody Fab' and steroid-Fab' complexes.

Crystals of an Fab' from an antiprogesterone monoclonal antibody (IgG1) have been grown from 1.5 M-ammonium sulfate, pH 5.5 to 8.5. The single crystals are hexagonal rods, space group P6222 (or P6422), with cell dimensions a = b = 135.2 A, and c = 124.2 A. Cocrystals of the Fab' with progesterone, pregnanedione and other related steroids have been grown. The complex crystals have different morphology but are isomorphous with the native crystals. A hydroxy-progesterone derivative obtained by substituting an iodo-benzoyl group at the 11 alpha-hydroxyl position looks promising as a suitable heavy-atom candidate in addition to other potential conventional heavy-atom derivatives. All crystals diffract to at least 2.8 A resolution and are suitable for high-resolution X-ray diffraction studies.

Structure of Azotobacter vinelandii 7Fe ferredoxin at 1.35 A resolution and determination of the [Fe-S] bonds with 0.01 A accuracy.

The crystal structure of Azotobacter vinelandii ferredoxin I (FdI) at 100 K has been refined at 1.35 A resolution by full matrix block diagonal least-squares methods with anisotropic temperature factors for all non-hydrogen atoms and with hydrogen atoms included in the model. Fe-S bonds within the [3Fe-4S]+ and [4Fe-4S]2+ clusters of the protein are determined with an accuracy of at least 0.01 A. Analysis of metric parameters reveals greater variation in bonds and angles within the [3Fe-4S]+ cluster than in the [4Fe-4S]2+ cluster, whereas the opposite is true regarding the cysteine Sgamma atoms ligating to the two [Fe-S] cores. The [3Fe-4S]+ core is asymmetrically distorted by the protein matrix but relatively uniformly ligated by its three Cys ligands; in contrast the tetrahedral [4Fe-4S]2+ core is relatively symmetric but non-uniformily ligated by its four Cys ligands, three of which occur in a conserved CysxxCysxxCys residue motif. Comparison of the [3Fe-4S]+ clusters in FdI and Desulfovibrio gigas ferredoxin II, refined at 1.7 A resolution, indicates that within the limit of accuracy of the two refinements the cuboidal core is differently distorted in the two proteins. Comparison of the [3Fe-4S]+ core in FdI with the structure of a reduced [Fe3S4]o synthetic analog indicates that the protein-bound cluster displays distortions not intrinsic to the core itself. Nevertheless, both [3Fe-4S]+ and [Fe3S4]o cores have metric features consistent with expected trends due to net charge on Fe and valency of S, and both exhibit a splayed configuration with respect to their three mu2S atoms in the absence of a fourth Fe. Comparison of the [4Fe-4S]2+ cluster in FdI with the structures of [Fe4S4]2+ synthetic analogs shows that the protein bound and synthetic cubanes are very similar in geometric parameters, including the presence of tetragonal distortion in the FdI cluster common to this oxidation state.

Crystallization and preliminary crystallographic data for Rab guanine nucleotide dissociation inhibitor (RabGDI) from bovine brain.

X-ray quality crystals of Rab guanine nucleotide dissociation inhibitor (RabGDI) from bovine brain expressed in Escherichia coli have been obtained from 1.73 M ammonium sulfate. The crystals are prismatic long rods and belong to the monoclinic space group P21 with approximate cell dimensions a = 91.9 A, b = 43.5 A, c = 63.2 A, beta = 104.5 degrees and one molecule per asymmetric unit. The crystals are stable in the X-ray beam and diffract to at least 2.3 A. Reverse screening, streak seeding and macroseeding methods were used to obtain and improve the crystals.

Three-dimensional structure of an anti-steroid Fab' and progesterone-Fab' complex.

The monoclonal anti-progesterone antibody DB3 binds progesterone with nanomolar affinity (Ka approximately 10(9) M-1), suggesting high specificity. However, DB3 also cross-reacts with similar affinity with a subgroup of structurally distinct, progesterone-like steroids. Crystals of the unliganded Fab' and various steroid-Fab' complexes are isomorphous and belong to the hexagonal space group, P6(4)22, with unit cell dimensions of a = b = 135 A, c = 124 A. Structures of free and progesterone-bound Fab' have been determined by X-ray crystallography at 2.7 A resolution using molecular replacement techniques. Progesterone is bound in a hydrophobic pocket formed mainly by the interaction of three complementarity determining regions L1, H2 and H3. The orientation of the ligand in the binding site was aided by both crystallographic and biochemical analyses of substituted steroids. The indole side-chain of TrpH100 of the DB3 has two different conformations, inter-converting "open" and "closed" forms of the antibody combining site. The TrpH100 indole thus appears to be acting as an antibody-derived surrogate ligand for its own hydrophobic binding pocket. These structures provide the first atomic view of how a steroid interacts with a protein and offer a structural explanation for the restriction of the anti-progesterone response to the VGAM3.8 family of VH genes.

Crystallization of murine major histocompatibility complex class I H-2Kb with single peptides.

X-ray quality crystals of a soluble murine class I H-2Kb molecule complexed with three different peptide antigens were grown in several forms by streak seeding and macroseeding methods. Co-crystals with VSV-8 (RGYVYGQL), OVA-8 (SIINFEKL) and SEV-9 (FAPGNYPAL) peptides were grown either from NaH2PO4/HPO4 or from polyethylene glycol 4000 within the pH range 5.0 to 7.5, with the use of 4-methyl-2-pentane diol (MPD) as an additive. The VSV-8 crystals grew in space groups P1, with cell dimensions a = 63.1 A, b = 69.1 A, c = 72.0 A, alpha = 89.9 degrees, beta = 77.1 degrees, gamma = 123.3 degrees and P2(1)2(1)2, with a = 138.1 A, b = 88.6 A, c = 45.7 A, and diffract to 2.9 and 2.3 A, respectively. Crystals of the SEV-9 complex grew from similar crystallization conditions to those of the orthorhombic VSV-8 complex with similar cell parameters and diffract to at least 2.5 A resolution. Crystals of the OVA-8 complex were obtained from either phosphate (space group C2, a = 118.7 A, b = 61.6 A, c = 85.3 A, beta = 108.4 degrees) or polyethylene glycol (space group P1, a = 64.5 A, b = 71.0 A, c = 66.3 A, alpha = 89.7 degrees, beta = 95.7 degrees, gamma = 123.3 degrees) and diffract to 3 A resolution. The crystallization procedures used here significantly increased the rate and production of X-ray quality crystals.

Towards structure-based drug design: crystal structure of a multisubstrate adduct complex of glycinamide ribonucleotide transformylase at 1.96 A resolution.

An inhibitor complex structure of glycinamide ribonucleotide transformylase (GAR-Tfase; EC 2.1.2.2) from Escherichia coli has been determined with a multisubstrate adduct BW1476U89 to an R-value of 19.1% at 1.96 A resolution. The structure was determined by a combination of molecular and single isomorphous replacement using data from two different monoclinic crystal lattices and collecting data from crystals soaked in 20% (w/v) methyl-pentanediol as cryoprotectant for shock-freezing at -150 degrees C. The multisubstrate adduct is bound in an extended crevice at the interface between the two functional domains of the enzyme. This inhibitor is positioned in the binding site by three sets of tight interactions with its phosphate, glutamate and pyrimidone ring moieties, while its interventing linker atoms are more flexible and adopt two distinct sets of conformations. The highly conserved Arg103, His108 and Gln170 residues that are key in ligand binding and catalysis (His108), have compensatory conformational variation that gives some clues as to their role in substrate specificity and in the formyl transfer. The molecular design of 1476U89 as a multisubstrate adduct inhibitor (Ki approximately 100 pM at pH 8.5), is confirmed as it closely mimics the shape, molecular interaction and combined binding constants of the natural 10-formyltetrahydrofolate (10-CHO-H4F; Km approximately 77.4 microM at pH 8.5) and glycinamide-ribonucleotide (GAR; Km approximately 8.1 microM at pH 8.5) substrates. The stereochemistry of this ligand complex suggests that His108 may act as an electrophile stabilizing the oxyanion of the tetrahedral intermediate that is formed as a result of the direct attack on the 10-CHO-H4F by the amino group of GAR. Structural comparison of the folate binding modes among GAR-Tfase, dihydrofolate reductase and thymidylate synthase reveals that folate derivates bound to GAR-Tfase differentially adopt the trans conformation for the dihedral angle between atoms C-6 and C-9 providing a handle for targeting specific folate-dependent enzymes. The structural information derived from two different discrete conformations of the ligand in the binding site also suggests several leads for the de novo design of inhibitors of GAR-Tfase that may develop into useful chemotherapeutic agents.

Structural analysis of antibody specificity. Detailed comparison of five Fab'-steroid complexes.

Structures of the Fab' fragment of the anti-progesterone antibody DB3 in complex with five cross-reactive steroids (aetiocholanolone, 5 beta-androstane-3,17-dione, 5 alpha-pregnane-20-one-3 beta-ol-hemisuccinate, progesterone-11 alpha-ol-hemisuccinate and progesterone) have been determined by X-ray crystallography to a maximum resolution of 2.7 A. These different steroids compete with progesterone binding with affinities in the nanomolar range despite substantial differences in their three-dimensional structures. Comparison of the unliganded DB3 Fab' and these five steroid-Fab' complexes reveals that all the steroid ligands bind to an "open" conformation of the Fab' as defined by the orientation of the indole side-chain of TrpH100, whereas in the unliganded or "closed" form the binding site is occluded by TrpH100. Small but significant conformational changes take place in the antibody to maximize the physical and chemical complementarity with each ligand. The various cross-reactive ligands are accommodated in the binding site in two distinct orientations. We term these binding modes syn and anti, as they are defined by the orientation of the steroid beta face relative to TrpH50. In all cases, the steroid D ring is inserted into a hydrophobic cavity formed mainly by TrpH50, TyrH97, TrpH100 and PheH100b; a hydrogen bond interaction with AsnH35 to the keto group at position C17 or C20 orients the steroid in the pocket. The AsnH35 hydrogen bond and the interaction with TrpH50 account for the restricted heavy chain response to immunization with progesterone-like steroids derivatized at the 11 alpha position. Cross-reactivity of the antibody with different steroids is explained by alternative binding pockets for the A ring, which generates different ligand orientations in the binding site. This study suggests which factors are most likely to contribute to the observed antibody specificity, such as linker position and the paucity of functional groups on the immunogenic hapten.

Crystal structure of glycinamide ribonucleotide transformylase from Escherichia coli at 3.0 A resolution. A target enzyme for chemotherapy.

The atomic structure of glycinamide ribonucleotide transformylase, an essential enzyme in purine biosynthesis, has been determined at 3.0 A resolution. The last three C-terminal residues and a sequence stretch of 18 residues (residues 113 to 130) are not visible in the electron density map. The enzyme forms a dimer in the crystal structure. Each monomer is divided into two domains, which are connected by a central mainly parallel seven-stranded beta-sheet. The N-terminal domain contains a Rossmann type mononucleotide fold with a phosphate ion bound to the C-terminal end of the first beta-strand. A long narrow cleft stretches from the phosphate to a conserved aspartic acid, Asp144, which has been suggested as an active-site residue. The cleft is lined by a cluster of residues, which are conserved between bacterial, yeast, avian and human enzymes, and likely represents the binding pocket and active site of the enzyme. GAR Tfase binds a reduced folate cofactor and glycinamide ribonucleotide for the catalysis of one of the initial steps in purine biosynthesis. Folate analogs and multi-substrate inhibitors of the enzyme have antineoplastic effects and the structure determination of the unliganded enzyme and enzyme-inhibitor complexes will aid the development of anti-cancer drugs.

Comparison of AMP and NADH binding to glycogen phosphorylase b.

The binding sites for the allosteric activator, AMP, to glycogen phosphorylase b are described in detail utilizing the more precise knowledge of the native structure obtained from crystallographic restrained least-squares refinement than has hitherto been available. Localized conformational changes are seen at the allosteric effector site that include shifts of between 1 and 2 A for residues Tyr75 and Arg309 and very small shifts for the region of residues 42 to 44 from the symmetry-related subunit. Kinetic studies demonstrate that NADH inhibits the AMP activation of glycogen phosphorylase b. Crystallographic binding studies at 3.5 A resolution show that NADH binds to the same sites on the enzyme as AMP, i.e. the allosteric effector site N, which is close to the subunit-subunit interface, and the nucleoside inhibitor site I, which is some 12 A from the catalytic site. The conformations of NADH at the two sites are different but both conformations are "folded" so that the nicotinamide ring is close (approx. 6 A) to the adenine ring. These conformations are compared with those suggested from solution studies and with the extended conformations observed in the single crystal structure of NAD+ and for NAD bound to dehydrogenases. Possible mechanisms for NADH inhibition of phosphorylase activation are discussed.