Primary structure and function analysis of the Abrus precatorius agglutinin A chain by site-directed mutagenesis. Pro(199) Of amphiphilic alpha-helix H impairs protein synthesis inhibitory activity.

Abrus agglutinin (AAG), a low-toxicity protein from the plant Abrus precatorius, is less lethal than abrina (ABRa) in mice (LD(50) = 5 mg/kg versus 20 microg/kg of body weight). Nucleotide sequence analysis of a cDNA clone encoding full-length AAG showed an open reading frame with 1641 base pairs, corresponding to a 547-amino acid residue preproprotein containing a signal peptide and a linker region (two amino acid residues) between the AAG-A and AAG-B subunits. AAG had high homology to ABRa (77.8%). The 13 amino acid residues involved in catalytic function, which are highly conserved among abrins and ricins, were also conserved within AAG-A. The protein synthesis inhibitory activity of AAG-A (IC(50) = 3.5 nM) was weaker than that of ABRa-A (0.05 nM). Molecular modeling followed by site-directed mutagenesis showed that Pro(199) of AAG-A, located in amphiphilic helix H and corresponding to Asn(200) of ABRa-A, can induce bending of helix H. This bending would presumably affect the binding of AAG-A to its target sequence, GpApGpAp, in the tetraloop structure of the 28 S rRNA subunit and could be one of the major factors contributing to the relatively weak protein synthesis inhibitory activity and toxicity of AAG.