Changes in structure and dynamics of the Fv fragment of a catalytic antibody upon binding of inhibitor

Binding of the product inhibitor p-nitrophenol to the monoclonal esterolytic antibody NPN43C9 has been investigated by performing NMR spectroscopy of the heterodimeric variable-domain fragment (Fv) of the antibody in the presence and absence of inhibitor. Structural information from changes in chemical shift upon binding has been related to the changes in local dynamics in the active site of the catalytic antibody using NMR relaxation measurements. Significant changes in the chemical shifts of the backbone resonances upon binding extend beyond the immediate vicinity of the antigen binding site into the interface between the two associated polypeptides that form the Fv heterodimer, a possible indication that the binding of ligand causes a change in the relative orientations of the component light (V(L)) and heavy (V(H)) chain polypeptides. Significant differences in backbone dynamics were observed between the free Fv and the complex with p-nitrophenol. A number of resonances, including almost all of the third hypervariable loop of the light chain (L3), were greatly broadened in the free form of the protein. Other residues in the antigen-binding site showed less broadening of resonances, but still required exchange terms (R(ex)) in the model-free dynamics analysis, consistent with motion on a slow timescale in the active site region of the free Fv. Binding of p-nitrophenol caused these resonances to sharpen, but some R(ex) terms are still required in the analysis of the backbone dynamics. We conclude that the slow timescale motions in the antigen-binding site are very different in the bound and free forms of the Fv, presumably due to the damping of large-amplitude motions by the bound inhibitor.     

Diagnostic chemical shift markers for loop conformation and substrate and cofactor binding in dihydrofolate reductase complexes

Heteronuclear NMR methods have been used to probe the conformation of four complexes of Escherichia coli dihydrofolate reductase (DHFR) in solution. (1)H(N), (15)N, and (13)C(alpha) resonance assignments have been made for the ternary complex with folate and oxidized NADP(+) cofactor and the ternary complex with folate and a reduced cofactor analog, 5,6-dihydroNADPH. The backbone chemical shifts have been compared with those of the binary complex of DHFR with the substrate analog folate and the binary complex with NADPH (the holoenzyme). Analysis of (1)H(N) and (15)N chemical shifts has led to the identification of marker resonances that report on the active site conformation of the enzyme. Other backbone amide resonances report on the presence of ligands in the pterin binding pocket and in the adenosine and nicotinamide-ribose binding sites of the NADPH cofactor. The chemical shift data indicate that the enzyme populates two dominant structural states in solution, with the active site loops in either the closed or occluded conformations defined by X-ray crystallography; there is no evidence that the open conformation observed in some X-ray structures of E. coli DHFR are populated in solution.     

The apomyoglobin folding pathway revisited: structural heterogeneity in the kinetic burst phase intermediate

Extensive analysis of accurate quench-flow hydrogen exchange results indicates that the burst phase kinetic intermediate in the folding of apomyoglobin (apoMb) from urea is structurally heterogeneous. The structural variability is associated with the partial folding of the E helix during the burst phase (<6.4ms) of the folding process. Analysis of the effects of exchange-out of amide proton labels during the labeling pulse ( approximately pH 10) of the quench-flow process indicates that three of the amide protons in the E helix are in fact largely protected in the burst phase of folding, while the remainder of the E helix has a substantial complement of amide protons that show biphasic kinetics, i.e. are protected partly during the burst phase and partly during the slow phase of folding. The locations of these amide protons can be used to map the sites of structural heterogeneity in the kinetic molten globule. These sites include, besides the E helix, the ends of the A and B helices and part of the C helix. Our results give significant support to the hypothesis that the kinetic molten globule intermediate of apoMb is native-like.     

Wolbachia infection associated with all-female broods in Hypolimnas bolina (Lepidoptera: Nymphalidae): evidence for horizontal transmission of a butterfly male killer

Inherited bacteria that kill male hosts during embryogenesis infect a wide range of insect species. In order to ascertain if there are patterns to host infection, with particular male killing bacteria specialising on particular taxa, we investigated the male killing trait in the butterfly Hypolimnas bolina. All-female broods were first reported in this species in the 1920s. Investigation of this system in the Fiji Islands revealed the causal agent of sex ratio distortion in H. bolina to be a male killing Wolbachia bacterium. This bacterium is identical in wsp and ftsZ sequence to a male killer in the butterfly Acraea encedon in Tanzania, suggesting it has moved between host species, yet retained its phenotype. The prevalence of the Wolbachia was calculated for three different island groups of Fiji, and found to vary significantly across the country. Antibiotics failed to cure either the male killing trait or the Wolbachia infection. The implications of these results are discussed.     

Hepatitis B virus core antigen: enhancement of its production in Escherichia coli,and interaction of the core particles with the viral surface antigen

The core antigen (HBcAg) of hepatitis B Virus (HBV) can be expressed in Escherichia coil where it assembles into icosahedral particles containing 240 or 180 subunits. Analysis of the two kinds of particles by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that a substantial proportion of their subunits were smaller than the full-length HBcAg monomer and of variable size, but all had the same N-terminal sequence showing that the smaller species were heterogeneous in their arginine-rich C-terminal regions. Around 50% of these arginine residues are encoded by the triplet AGA which is rare in E. coli. Supplementation of the level of AGA tRNA in the cell by transformation with plasmids expressing the T4 AGA tRNA gene significantly enhanced the yield of HBcAg. Fusion phage carrying a ligand specific for HBcAg showed no significant difference in the affinity for the two sizes of HBcAg particles, but in similar reactions in solution HBV surface antigen exhibited differential affinities for the same two HBcAg preparations.     

Fc-dependent depletion of activated T cells occurs through CD40L-specific antibody rather than costimulation blockade

Although the underlying mechanisms are not well understood, it is generally believed that antigen recognition by T cells in the absence of costimulation may alter the immune response, leading to anergy or tolerance. Further support for this concept comes from animal models of autoimmunity and transplantation, where treatments based on costimulation blockade, in particular CD40 ligand (CD40L)-specific antibodies, have been highly effective. We investigated the mechanisms of action of an antibody to CD40L and provide evidence that its effects are dependent on the constant (Fc) region. Prolongation of graft survival is dependent on both complement- and Fc receptor-mediated mechanisms in a major histocompatibility complex (MHC)-mismatched skin transplant model. These data suggest that antibodies to CD40L act through selective depletion of activated T cells, rather than exerting immune modulation by costimulation blockade as currently postulated. This finding opens new avenues for treatment of immune disorders based on selective targeting of activated T cells.     

NMR solution structure of the inserted domain of human leukocyte function associated antigen-1

The interaction between the leukocyte function-associated antigen-1 (LFA-1) and the intercellular adhesion molecule is thought to be mediated primarily via the inserted domain (I-domain) in the alpha-subunit. The activation of LFA-1 is an early step in triggering the adhesion of leukocytes to target cells decorated with intercellular adhesion molecules. There is some disagreement in the literature over the respective roles of conformational changes in the I-domain and of divalent cations (Mg(2+), Mn(2+)) in the activation of LFA-1 for intercellular adhesion molecule binding. X-ray crystallographic structures of the I-domains of LFA-1 and Mac-1 in the presence and absence of cations show structural differences in the C-terminal alpha-helix; this change was proposed to represent the active and inactive conformations of the I-domain. However, more recent X-ray results have called this proposal into question. The solution structure of the Mg(2+) complex of the I-domain of LFA-1 has been determined by NMR methods, using a model-based approach to nuclear Overhauser enhancement spectroscopy peak assignment. The protein adopts the same structure in solution as that of the published I-domain X-ray structures, but the C-terminal region, where the X-ray structures are most different from each other, is different again in the solution structures. The secondary structure of this helix is well formed, but NMR relaxation data indicate that there is considerable flexibility present, probably consisting of breathing or segmental motion of the helix. The conformational diversity seen in the various X-ray structures could be explained as a result of the inherent flexibility of this C-terminal region and as a result of crystal contacts. Our NMR data are consistent with a model where the C-terminal helix has the potential flexibility to take up alternative conformations, for example, in the presence and absence of the intercellular adhesion molecule ligand. The role of divalent cations appears from our results not to be as a direct mediator of a conformational change that alters affinity for the ligand. Rather, the presence of the cation appears to be involved in some other way in ligand binding, perhaps by acting as a bridge to the ligand and by modulation of the charge of the binding surface.     

Dynamics of the metallo-beta-lactamase from Bacteroides fragilis in the presence and absence of a tight-binding inhibitor

A significant determinant for the broad substrate specificity of the metallo-beta-lactamases from Bacteroides fragilis and other similar organisms is the presence of a plastic substrate binding site that is nevertheless capable of tight substrate binding in the Michaelis complex. To achieve these two competing ends, the molecule apparently employs a flexible flap that closes over the active site in the presence of substrate. These characteristics imply that dynamic changes are an important component of the mechanism of action of these enzymes. The backbone and tryptophan side chain dynamics of the metallo-beta-lactamase from B. fragilis have been examined using (15)N NMR relaxation measurements. Two states of the protein were examined, in the presence and absence of a tight-binding inhibitor. Relaxation measurements were analyzed by the model-free method. Overall, the metallo-beta-lactamase molecule is rigid and shows little flexibility except in loops. The flexibility of the loop that covers the active site is not unusually great as compared to the other loops of the protein. Local motion on a picosecond time scale was found to be very similar throughout the protein in the presence and absence of the inhibitor, but a significant difference was observed in the motions on a nanosecond time scale (tau(e)). Large-amplitude motions with a time constant of about 1.3 ns were observed for the flexible flap region (residues 45-55) in the absence of the inhibitor. These motions were completely damped out in the presence of the inhibitor. In addition, the motion of a tryptophan side chain at the tip of the beta-hairpin of the flap shows a very significant difference in motion on the ps time scale. These results indicate that the motions of the polypeptide chain in the flap region can be invoked to explain both the wide substrate specificity (the free form has considerable amplitude of motion in this region) and the catalytic efficiency of the metallo-beta-lactamase (the motions are damped out when the inhibitor and by implication a substrate binds in the active site).