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

Abstract

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.

The structure of dual-variable-domain immunoglobulin molecules alone and bound to antigen

“…15,[21][22][23][24][25][26][27] The majority of structures reported in the literature are either Fab or Fab' fragments.…”

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

“…Because of the large number of potential peptide complexes that could be crystallized, rather than applying the standard approach where footprint screens are set up for the free Fab and for each of the peptide complexes, the results obtained from the crystallization of the 4B7 IgG-peptide complex (Stura, Satterthwait, Calvo, Stefanko, Langeveld & Kaslow, 1994) were used to minimize the initial screening with the Fab-peptide complexes (see Reverse Screening, Stura, Satterthwait, Calvo, Kaslow & Wilson, 1994).…”

“…MAb 4B7 was isolated from ascites fluid by ammonium sulfate precipitation, purified, and typed as described by Stura, Satterthwait, Calvo, Stefanko, Langeveld & Kaslow (1994). Cleavage of this IgG2a (~; light chain) with either papain or pepsin failed to yield material sufficiently homogeneous for crystallization.…”

“…Modeling of constrained Pfs25 peptides was based on the solution structure of EGF-like domains derived from NMR studies (Cooke et al, 1987;Kline et al, 1990). The linear peptide recognized by MAb 4B7 corresponds to a predicted/3-hairpin in the third EGFlike domain of Pfs25 (Kaslow et al, 1988;Stura, Satterthwait, Calvo, Stefanko, Langeveld & Kaslow, 1994). Two prototypical cyclic peptides corresponding to predicted /3-hairpin conformations were synthesized by replacing main-chain hydrogen bonds with covalent hydrogen-bond mimics at the base of the loop (Fig.…”

“…The epitope recognized by MAb 4B7 has been defined as lying within the ILDTSNPVKT sequence contained in the third EGFlike domain of the Pfs25 molecule (Stura, Satterthwait, Calvo, Stefanko, Langeveld & Kaslow, 1994). Several cyclic peptides have been synthesized to mimic the predicted/3-hairpin of this EGF-like domain.…”