Major structural differences between pokeweed antiviral protein and ricin A-chain do not account for their differing ribosome specificity.
Pokeweed antiviral protein (PAP) and the A-chain of ricin (RTA) are two members of a family of ribosome-inactivating proteins (RIPS) that are characterised by their ability to catalytically depurinate eukaryotic ribosomes, a modification that makes the ribosomes incapable of protein synthesis. In contrast to RTA, PAP can also inactivate prokaryotic ribosomes. In order to investigate the reason for this differing ribosome specificity, a series of PAP/RTA hybrid proteins was prepared to test for their ability to depurinate prokaryotic and eukaryotic ribosomes. Information from the X-ray structures of RTA and PAP was used to design gross polypeptide switches and specific peptide insertions. Initial gross polypeptide swaps created hybrids that had altered ribosome inactivation properties. Preliminary results suggest that the carboxy-terminus of the RIPs (PAP 219-262) does not contribute to ribosome recognition, whereas polypeptide swaps in the amino-terminal half of the proteins did affect ribosome inactivation. Structural examination identified three loop regions that were different in both structure and composition within the amino-terminal region. Directed substitution of RTA sequences into PAP at these sites, however, had little effect on the ribosome inactivation characteristics of the mutant PAPs, suggesting that the loops were not crucial for prokaryotic ribosome recognition. On the basis of these results we have identified regions of RIP primary sequence that may be important in ribosome recognition. The implications of this work are discussed.
Chaddock
J A
JA
Department of Biological Sciences, University of Warwick, Coventry, UK.
Monzingo
A F
AF
Robertus
J D
JD
Lord
J M
JM
Roberts
L M
LM
D000998:Antiviral Agents / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016254:Mutagenesis, Insertional; D009699:N-Glycosyl Hydrolases; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes / Q000378:metabolism*; D012276:Ricin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D000998:Antiviral Agents; D009838:Oligodeoxyribonucleotides; D010940:Plant Proteins; D011994:Recombinant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D012276:Ricin; D009699:N-Glycosyl Hydrolases; C024275:pokeweed antiviral protein