The mechanism of an inhibitory antibody on TF-initiated blood coagulation revealed by the crystal structures of human tissue factor, Fab 5G9 and TF.G9 complex.

The tissue factor (TF)-initiated blood coagulation protease cascade can be greatly inhibited in vivo by a potent anti-human-TF monoclonal antibody, 5G9. This antibody binds the carboxyl module of the extracellular domain of TF with a nanomolar binding constant and inhibits the formation of the TF.VIIa.X ternary initiation complex. We have determined the crystal structures of the extra-cellular modules of human TF, Fab 5G9, and their complex (TF.5G9) to 2.4 A, 2. 5 A, and 3.0 A, respectively, and measured the apparent inhibition constants of 5G9 on a panel of TF mutants. In our unliganded TF structure, a 7 degrees change in the relative orientation between the D1 and D2 modules was observed when compared with other published TF structures. Comparison of the free and bound Fab 5G9 indicates that small segmental and side chain variation of the antibody complementarity determining regions occurred on complexation with TF. The antibody-antigen recognition involves 18 TF antigen residues and 19 Fab residues from six CDR with one of the largest buried surface areas seen to date. A combination of structural and mutagenesis data indicate that Tyr156, Lys169, Arg200, and Lys201 play the major role in the antibody recognition. The TF. 5G9 structure provides insights into the mechanism by which the antibody 5G9 inhibits formation of the TF.VIIa.X ternary complex.

Crystallization, sequence and preliminary crystallographic data for transmission-blocking anti-malaria Fab 4B7 with cyclic peptides from the Pfs25 protein of P. falciparum.

X-ray quality crystals of an Fab fragment from a transmission-blocking monoclonal antibody 4B7 (MAb 4B7) against a sexual stage protein Pfs25 of Plasmodium falciparum were grown as uncomplexed and peptide-complexed forms. Initially, the intact immunoglobulin was crystallized because cleavage with pepsin or papain did not produce a homogeneous product. Further proteolytic trials with elastase produced a suitable Fab fragment from which crystals have been obtained, both for the free Fab and in complex with cyclic peptides and in the presence of linear peptides. While linear peptides bind to MAb 4B7, cyclic peptides modeled on a predicted beta-hairpin loop of the third EGF-like domain of Pfs25 bind better and readily co-crystallize with the Fab. The genes for the variable domain of the Fab have been cloned, sequenced and the primary amino-acid sequence for the complete Fab deduced. This work explores the use of glycerol as an additive and the modification of the peptide sequence outside the epitope for improving in the crystallization. Data sets have been collected from crystals of several Fab-peptide complexes and from uncomplexed Fab to resolutions ranging from 2.4 to 3.3 A. The packing arrangements of several crystal forms have been determined by molecular replacement, and refinement of their three-dimensional structures is in progress. The three-dimensional structure of this Fab complexed with the various peptides will aid in an understanding of the mode by which this antibody recognizes and prevents transmission of the malaria parasite.

Crystallization of an intact monoclonal antibody (4B7) against Plasmodium falciparum malaria with peptides from the Pfs25 protein antigen.

Monoclonal antibody 4B7 is a neutralizing antibody that binds the protein Pfs25 in the sexual stages of the malaria parasite Plasmodium falciparum and completely blocks transmission of the parasite from human serum to the mosquito host. Here we report the identification of the epitope on Pfs25 recognized by 4B7 and the crystallization of the intact murine monoclonal antibody with peptides corresponding to that epitope. This study highlights the importance of ligands in the crystallization of proteins. In this case peptides have been used to modulate the solubility of the peptide-IgG complex and may have provided different or additional crystal contacts to create or enhance a crystalline reticulum. Multiple crystal forms characterize this crystallization and the various peptides, differing both in length and sequence, have been used to investigate how such changes affect nucleation and crystal growth.

Evidence that the 5' end of rabbit globin mRNA is hybridized with its 3' end.

1. A radiopolyadenylated rabbit globin mRNA was treated with different concentrations of ribonuclease V1 from cobra venom. 2. The enzymatic digests were chromatographed on an aminophenylboronate-agarose column, which specifically captured the cap structure i.e. m7G(5') ppp (5') NmP. 3. When the capture fragment was chromatographed on a Sephadex G-100 column, its size was smaller than the native molecule and also bore radioactivity, i.e. a poly(A) tail. 4. These results provide evidence that the 5' end (which encompasses the cap structure) of rabbit globin mRNA is hybridized and in close proximity to its 3' end. 5. We conclude that this conformation is required for messenger translation efficiency.

An inhibitor(s) of globin mRNA translation in rabbit serum.

1. A factor found in rabbit serum inhibits globin mRNA translation in vitro. 2. Inhibition of globin mRNA translation has been demonstrated in a cell-free rabbit reticulocyte lysate. 3. The inactivation of globin mRNA translation is not attributed to either serum albumin or ribonuclease activities. 4. Dialyzing the inhibitor for 24 hr at 4 degrees C does not result in the diminution of the inhibiting activity. However, the activity of the inhibitor is destroyed by heating to 70-80 degrees C for 5 min or by treatment with trypsin for 2 hr. 5. Ion exchange chromatography points to the inhibitor being a neutral protein, whereas, polyacrylamide gel electrophoresis reveals one major band with mol. wt 43 kDa. 6. The activity of the inhibiting material 3-fold greater in anemic serum than in normal serum. 7. These studies suggest that rabbit serum contains a protein inhibitor that may play a physiological role in regulating protein synthesis in red cells.