Structural Complementation of a Monomeric Ribosomal-Inactivating Protein

Abstract

Exotoxin A (ETA) from the Gram-negative bacterium Pseudomonas aeruginosa is a ribosomal-inactivating protein (RIP) that acts by ADP-ribosylation of the post-translationally modified histidine, diphthamide, in elongation factor 2. Apoptotic cell death typically ensues upon intoxication. Distinct from toxins with multiple subunits, ETA is a single-chain protein with three functional domains. The catalytic domain (domain III, or PE3) of pseudomonal exotoxin A is a 213-residue monomer with extensive sequence and structural homology with the A chain of diphtheria toxin and the more recently-characterized cholix toxin from Vibrio cholerae. We are interested in developing split fragments of PE3 capable of structural complementation. Based on B-factors in the crystal structure of PE3, we have identified several disordered loops as candidates for severance, none of which are spatially close to the active site. We have produced and characterized fusion constructs each consisting of a split fragment and one subunit of a heterospecific, antiparallel coiled-coil. We report one such pair that remains inactive individually but undergoes complementation to yield a functionally active RIP in vitro and we are characterizing its biophysical properties. To our knowledge, this pair of split PE3 fragments represents the first example structural complementation of a monomeric protein toxin and may have significant biotechnological and therapeutic applications. Our ultimate goal is to engineer a cellular toxin with multiple specificities to be achieved by conditional co-expression inside a target cell.