The 2.8A x-ray structure of the heterodimeric toxin ricin has recently been obtained. It is proposed to extend data to higher resolution, using cold temperatures if necessary, and to refine the structure in a crystallographic sense. Accurate atomic positions will provide a basis to assess forces and modes of inter-action between the A and B chains, and between the protein chains and substrates. Crystals and a derivative of the cloned ricin A chain (rRTA) nave been obtained and this structure will be solved to high resolution and compared with the A chain of the heterodimer. In particular the protein will be examined for conformational changes occurring when the A and B chains are separated. Forces stabilizes the heterodimer may be reordered to activate the A chain or to increase its propensity to bind to cell membranes. Analogs of the known rRNA substrate will be soaked into ricin, and rRTA crystals to assess the mode of enzyme action. A collaboration with Cetus Corporation will produce key site directed mutants of the protein which will be analyzed crystallographically. The structures will be compared to functional effects to dissect the molecular basis of cell surface binding, of rRNA attack, of heterodimer interactions and of A chain-membrane interactions. The x-ray structure of ricin B chain reveals the lectin is an ideal candidate for conversion to a hydrolytic enzyme via site directed mutagenesis. The appropriate clones are available to carry out these studies. This exercise should help to define rules which will aid in the general redesign of proteins for medical or commercial purposes. Crystals, and at least one isomorphours derivative of the single chain toxin PAP have been obtained. The crystal structure will be solved, and refined and a comparison made to the ricin A chain. Substrate analogs will be bound to the active site to assess common feature and subtle differences, with the evolutionarily related A chain. Collaborations have been initiated to begin crystallization efforts on two additional toxins, SLT from E. coli and BPSI from barley.
