Abstract:

<p>The extreme potency of the plant toxin, ricin, is due to its enzymatic subunit, RTA, which inactivates mammalian ribosomes with near-perfect efficiency. Here we characterized, at the functional and structural levels, seven alpaca single-domain antibodies (VHs) previously reported to recognize epitopes in proximity to RTA&#39;s active site. Three of the VHs, V2A11, V8E6, and V2G10, were potent inhibitors of RTA in vitro and protected Vero cells from ricin when expressed as intracellular antibodies (&quot;intrabodies&quot;). Crystal structure analysis revealed that the complementarity-determining region 3 (CDR3) elements of V2A11 and V8E6 penetrate RTA&#39;s active site and interact with key catalytic residues. V2G10, by contrast, sits atop the enzymatic pocket and occludes substrate accessibility. The other four VHs also penetrated/occluded RTA&#39;s active site, but lacked sufficient binding affinities to outcompete RTA-ribosome interactions. Intracellular delivery of high-affinity, single-domain antibodies may offer a new avenue in the development of countermeasures against ricin toxin.toxin, antibody, structure, intracellular.</p>