Highly specific anti-estradiol antibodies: structural characterisation and binding diversity.

Subtle modulation of antibody-binding properties by protein engineering often lies with an accurate structural and energetic description of how an antigen is recognised. Thus, with the intent to increase the affinity and add a bias in favour of natural estradiol compared with its chemically modified immunogen, we have determined the crystal structure of two anti-estradiol monoclonal antibodies, 10G6D6 and 17E12E5. Although generated against the same estradiol derivative, these antibodies share little sequence identity, which is reflected in dissimilar binding pockets and in different positioning of the steroid. In both antibodies the characteristic 17-hydroxyl group is buried deeply at the bottom of hydrophobic pockets and stabilised by hydrogen bonds. Apart from this similarity, the steroid is oriented differently in the respective binding pockets. The high specificity of both antibodies has been mapped out, and even closely related steroids show low cross-reactivity. The structural studies of the complex formed between 10G6D6 and 6-CMO-estradiol have identified contacts between the 6-CMO coupling linker and an arginine residue from the heavy chain CDR2 segment. This segment is now being targeted by random mutagenesis to select mutants with a preference for natural estradiol compared to the branched hapten.

Stability of a structural scaffold upon activity transfer: X-ray structure of a three fingers chimeric protein.

Fasciculin 2 and toxin alpha proteins belong to the same structural family of three-fingered snake toxins. They act on different targets, but in each case the binding region involves residues from loops I and II. The superimposition of the two structures suggests that these functional regions correspond to structurally distinct zones. Loop I, half of loop II and the C-terminal residue of fasciculin 2 were therefore transferred into the toxin alpha. The inhibition constant of the resulting chimera is only 15-fold lower than that of fasciculin 2, and as expected the potency of binding to the toxin alpha target has been lost. In order to understand the structure-function relationship between the chimera and its "parent" molecules, we solved its structure by X-ray crystallography. The protein crystallized in space group P3(1)21 with a=b=58.5 A, and c=62.3 A. The crystal structure was solved by molecular replacement and refined to 2.1 A resolution. The structure belongs to the three-fingered snake toxin family with a core of four disulphide bridges from which emerge the three loops I, II and III. Superimposition of the chimera on fasciculin 2 or toxin alpha revealed an overall fold intermediate between those of the two parent molecules. The regions corresponding to toxin alpha and to fasciculin 2 retained their respective geometries. In addition, the chimera protein displayed a structural behaviour similar to that of fasciculin 2, i.e. dimerization in the crystal structure of fasciculin 2, and the geometry of the region that binds to acetylcholinesterase. In conclusion, this structure shows that the chimera retains the general structural characteristics of three-fingered toxins, and the structural specificity of the transferred function.

High resolution x-ray analysis of two mutants of a curaremimetic snake toxin.

A previous mutational analysis of erabutoxin a (Ea), a curaremimetic toxin from sea snake venom, showed that the substitutions S8G and S8T caused, respectively, 176-fold and 780-fold affinity decreases for the nicotinic acetylcholine receptor (AchR). In view of the fact that the side-chain of Ser8 is buried in the wild-type toxin, we wondered whether these affinity changes reflect a direct binding contribution of S8 to the receptor and/or conformational changes that could have occurred in Ea as a result of the introduced mutations. To approach this question, we solved X-ray structures of the two mutants S8G and S8T at high resolution (0.18 nm and 0.17 nm, with R factors of 18.0% and 17.9%, respectively). The data show that none of the mutations significantly modified the toxin structure. Even within the site where the toxin binds to the receptor the backbone conformation remained unchanged. Therefore, the low affinities of the mutants S8T and S8G cannot be explained by a large conformational change of the toxin structure. Although we cannot exclude the possibility that undetectable structural changes have occurred in the toxin mutants, our data support the view that, although buried between loop I and II, S8 is part of the functional epitope of the toxin.

Molecular evolution of snake toxins: is the functional diversity of snake toxins associated with a mechanism of accelerated evolution?

Recent studies revealed that animal toxins with unrelated biological functions often possess a similar architecture. To tentatively understand the evolutionary mechanisms that may govern this principle of functional prodigality associated with a structural economy, two complementary approaches were considered. One of them consisted of investigating the rates of mutations that occur in cDNAs and/or genes that encode a variety of toxins with the same fold. This approach was largely adopted with phospholipases A2 from Viperidae and to a lesser extent with three-fingered toxins from Elapidae and Hydrophiidae. Another approach consisted of investigating how a given fold can accommodate distinct functional topographies. Thus, a number of topologies by which three-fingered toxins exert distinct functions were investigated either by making chemical modifications and/or mutational analyses or by studying the three-dimensional structure of toxin-target complexes. This review shows that, although the two approaches are different, they commonly indicate that most if not all the surface of a snake toxin fold undergoes natural engineering, which may be associated with an accelerated rate of evolution. The biochemical process by which this phenomenon occurs remains unknown.

A particularly labile Asp-Pro bond in the green mamba muscarinic toxin MTX2. Effect of protein conformation on the rate of cleavage.

The single Asp53-Pro54 bond of the MTX2 toxin from the mamba snake Dendroaspis angusticeps is rapidly and efficiently cleaved in acidic solution (pH 1.5-2.5) at 45 degrees C. Unfolding of the toxin slows down the cleavage reaction by several times. Modelling studies indicate that the native toxin conformation can catalyse the Asp53-Pro54 bond cleavage. The implications of this study are: (i) cleavage of Asp-Pro bond for sequence determination may occur better in absence than in presence of denaturant, (ii) mild acid conditions, commonly used in NMR structure determinations, may irreversibly affect the structural integrity of Asp-Pro containing peptides and proteins.

Solution structure of a green mamba toxin that activates muscarinic acetylcholine receptors, as studied by nuclear magnetic resonance and molecular modeling.

The three-dimensional solution structure of the MTX2 toxin (65 amino acids and 4 disulfides) from the green mamba venom (Dendroaspis angusticeps), a toxin that activates the pharmacological M1 muscarinic acetylcholine receptors, has been determined by nuclear magnetic resonance and molecular modeling. Seventeen structures were calculated from 810 distance and 68 dihedral angle restraints using DIANA and X-PLOR. The average rms deviation between the 17 refined structures and the energy-minimized average structure is 0.95 A for the backbone atoms. The overall folding of MTX2 consists of three loops stabilized by the four disulfides and forming a two- and a three-stranded beta-sheet. This structure appears to be very similar to that of other snake toxins, such as neurotoxins, fasciculins, and cardiotoxins, that also possess the same three-finger fold. For instance, the RMSd for the backbone atoms between MTX2 and the curaremimetic toxin alpha (from Naja nigricollis), the acetylcholinesterase inhibitor fasciculin 1 (from Dendroaspis angusticeps), and the cardiotoxic toxin gamma (from Naja nigricollis) are 1.86, 1.87, and 2.04 A, respectively. Local differences are observed between this toxin and the other structurally related toxins. Some of these differences could be relevant for the functional specificity of MTX2. In particular, this toxin presents a large twist at the tip of loop II due to a bulge (V31, T32; N35) that accommodates an inserted amino acid in the loop. This spatial arrangement brings the side chain of K34 in the beta-turn of the loop to be aligned with the beta-sheet. Hypotheses about a possible functional role of this lysine are described. Other characteristics in the side-chain distribution that could be related to the MTX2 function are presented.

On the site by which alpha-dendrotoxin binds to voltage-dependent potassium channels: site-directed mutagenesis reveals that the lysine triplet 28-30 is not essential for binding.

We constructed a synthetic gene encoding the published amino acid sequence of DTx from Dendroaspis angusticeps, a ligand of voltage-dependent postassium channels that facilitates neurotransmitter release. We expressed it in Escherichia coli as a fusion protein secreted in the culture medium. The recombinant DTx was generated in vitro by chemical treatment and recovered as two isoforms. One of them (rDTx), like the venom toxin, has an N-terminal pyroglutamate whereas the other (rQDTx) has a free N-terminal glutamine. Chromatographic differences between rDTx and natural DTx led us to re-examine the amino acid sequence of natural DTx. In contrast to what was previously published, position 12 was an Asp and not Asn. Despite this difference, rDTx and DTx had similar toxicity in mice and binding affinity to synaptosomes, suggesting that residue 12 is not important for DTx function. Nor is the N-terminal residue implicated in DTx function since rDTx and rQDTx also had similar biological activities. We also synthesized and expressed a mutant of the DTx gene in which the lysine triplet 28-30 was changed into Ala-Ala-Gly. The two resulting recombinant isoforms exhibited only small decreases in biological activity, excluding the possibility that the positively charged lysine triplet 28-30 of DTx is directly involved in the toxin functional site.

X-ray structure at 1.55 A of toxin gamma, a cardiotoxin from Naja nigricollis venom. Crystal packing reveals a model for insertion into membranes.

The crystal structure of toxin gamma from Naja nigricollis has been solved and refined to 1.55 A resolution. The final R-factor, computed with all X-ray data available, is 17.9%. The three-dimensional structure is characterized by a core formed by two beta-sheets organized in three extended loops. It is similar to that of cardiotoxin V4II from Naja mossambica mossambica, with the exception of the hydrophobic loop I. The flexibility and variability of the loops contrast sharply with the rigidity of the molecular core and its high degree of structural conservation among the cardiotoxin family. The most flexible loop II adopts different conformations in the three monomers forming the crystal asymmetric unit. These monomers form a trimer around an approximate 3-fold axis, with conserved hydrophobic side-chains on the outside and hydrophilic residues in the central channel or involved in interactions with the other molecules. The trimer thus resembles a membrane protein with a central channel that could allow the passage of small ions. It is proposed as a model for the insertion of cardiotoxin into a membrane.

Three-dimensional crystal structure of recombinant erabutoxin a at 2.0 A resolution.

Recombinant erabutoxin a (Ea(r)) has been crystallized by vapour diffusion in hanging drops. The crystals belong to space group P2(1)2(1)2(1) with cell dimensions a = 55.8 A, b = 53.4 A, c = 40.8 A. Diffraction data have been recorded on a FAST detector up to 2.0 A. The atomic crystal structure of Ea(r) has been determined by initial refinement of the structure of the isotoxin erabutoxin b (Eb) the crystals of which were grown under identical conditions. The R-factor was 23% at 2.0 A resolution. The secondary and tertiary structures of Ea(r) are shown to be identical with that of wild-type Eb, within the experimental error.

A toxin that recognizes muscarinic acetylcholine receptors. Preparation and characterization of crystals suitable for structural analysis.

Muscarinic toxin 2 from Dendroaspis angusticeps has been crystallized by vapour diffusion, in sodium acetate using sodium thiocyanate as a precipitant. Trigonal crystals (space group P3(1)21 or P3(2)21) have been obtained. The unit cell parameters are a = b = 64.2 A and c = 37.1 A. The presence of one molecule per asymmetric unit is estimated.

Structure determination of a dimeric form of erabutoxin-b, crystallized from a thiocyanate solution.

Erabutoxin-b, M(r) = 6861.1, a single 62 amino-acid chain folded by four disulfide bridges, was crystallized in a new orthorhombic form by using thiocyanate as crystallizing agent. The space group is P2(1)2(1)2(1) with a = 53.36 (4), b = 40.89 (4), c = 55.71 (5) A, V = 121533.1 A and Z = 8. X-ray diffraction data were recorded at the LURE synchrotron facility (lambda = 1.405 A). The structure was solved by molecular replacement and shows a dimeric association through an anti-parallel beta-sheet around the twofold non-crystallographic axis. The two independent molecules, one SCN- ion and 97 associated water molecules were refined by molecular dynamics and annealing techniques to R = 19.6% (10,913 Fobs, resolution 5-1.7 A). The thiocyanate ion is located at the interface of the dimer and close to the non-crystallographic twofold axis.

Preliminary X-ray analysis of crystals of fasciculin 1, a potent acetylcholinesterase inhibitor from green mamba venom.

Fasciculin 1 from Dendroaspis angusticeps has been crystallized by vapour diffusion, in sodium acetate using sodium thiocyanate as precipitant. Tetragonal crystals (space group P4(1)2(1)2 or P4(3)2(1)2) diffract to 1.8 A resolution. The unit cell parameters are a = 40.4 A and c = 81.1 A. We estimated the presence of one molecule in the asymmetric unit.

Pregnancies resulting in infants with acquired immunodeficiency syndrome or AIDS-related complex.

Thirty-four children have been cared for at SUNY-Health Science Center at Brooklyn with diagnoses of either acquired immunodeficiency syndrome (AIDS) or AIDS-related complex. Reported here are descriptions of pregnancies resulting in these children. Few of the mothers (four of 32) were symptomatic; however, low birth weight (11 of 34), preterm birth (11 of 34), and premature rupture of membranes (ten of 32) were common. Of 33 patients whose mode of delivery was known, cesarean section was used in ten cases, including one elective repeat. Among mothers who had children before their affected offspring, the average birth weight of the older children significantly exceeded that of the affected (3241 +/- 508 versus 2712 +/- 722 g, P less than .05). The affected child's age at onset of symptoms did not correlate with birth weight, mode of delivery, or status of membranes at the onset of labor.

Pregnancies resulting in infants with acquired immunodeficiency syndrome or AIDS-related complex: follow-up of mothers, children, and subsequently born siblings.

Although several hundred cases of acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC) in infants have been reported, there is little information available concerning the follow-up of mothers and these children or subsequently born children. Thirty-four children with perinatally acquired AIDS and ARC (19 AIDS; 15 ARC) have been followed at the Downstate Medical Center. Although no mother had AIDS or ARC during her pregnancy, after an average follow-up (+/- SD) of 27.8 +/- 21.6 months, five had AIDS and ten had ARC. For 22 of the mothers, T4/T8 ratios were obtained; 15 of these were less than 1, and five were between 1 and 1.5. Among 11 subsequently born siblings for whom HTLV-III antibody status was known, four were positive; of these, two had ARC and one had AIDS. We conclude that the diagnosis of AIDS or ARC in a child indicates a risk for the development of illness in the mother and subsequently born siblings.

Pediatric acquired immunodeficiency syndrome: demonstration of B lymphocyte defects in vitro.

Acquired immunodeficiency syndrome (AIDS) in childhood is characterized by recurrent bacterial infections, a feature common in antibody deficiency disorders. The present study was aimed at investigating B lymphocyte function in 15 children aged 6 months to 6 years with AIDS or AIDS-related complex (ARC). Spontaneous secretion of immunoglobulins by freshly isolated peripheral blood B cells and the generation of immunoglobulin and antibody-secreting cells in lymphocyte cultures after polyclonal and antigenic stimulation were quantified in hemolytic plaque assays. Despite excessive spontaneous immunoglobulin secretion, responses elicited by B cells after in vitro stimulation were depressed in these children. Responses to T-dependent as well as to T-independent stimuli were affected. Studies of immunoregulatory T cells and intrinsic B cell function suggested that deficient precursor B cells and abnormal immunoregulation contributed to the defects in B cell differentiation. These findings indicate that B lymphocyte dysfunction is an integral feature of HTLV III infection in children who clinically present as either AIDS or AIDS-related complex.

Low circulating thymulin-like activity in children with AIDS and AIDS-related complex.

Thymic secretory function was assessed by determining levels of circulating thymulin-like activity in plasma of 21 pediatric patients infected with the HTLV-III/LAV retrovirus. All the patients had serum antibodies against p41 antigens of HTLV-III on Western blot analyses. In accordance with the latest definition established by the Centers for Disease Control, 14 patients had the acquired immunodeficiency syndrome (AIDS) and the remaining 7 were classified as having AIDS-related complex. Their ages ranged from 1 to 7 years, with 10 being less than 1 year of age. Circulating thymulin activity, normally highest in healthy children under 15 years of age, was undetectable in 11 patients and below normal range for age in the remaining. OKT4/OKT8 ratios of T-cell subsets in peripheral blood were below normal in the majority of patients. Our findings suggest that thymic epithelial injury may be an early event in HTLV-III/LAV-related disease and may precede the development of clinical and/or immunologic aberrations.