The effect of mutations surrounding and within the active site on the catalytic activity of ricin A chain.
Models for the binding of the sarcin-ricin loop (SRL) of 28S ribosomal RNA to ricin A chain (RTA) suggest that several surface exposed arginine residues surrounding the active site cleft make important interactions with the RNA substrate. The data presented in this study suggest differing roles for these arginyl residues. Substitution of Arg48 or Arg213 with Ala lowered the activity of RTA 10-fold. Furthermore, substitution of Arg213 with Asp lowered the activity of RTA 100-fold. The crystal structure of this RTA variant showed it to have an unaltered tertiary structure, suggesting that the positively charged state of Arg213 is crucial for activity. Substitution of Arg258 with Ala had no effect on activity, although substitution with Asp lowered activity 10-fold. Substitution of Arg134 prevented expression of folded protein, suggesting a structural role for this residue. Several models have been proposed for the binding of the SRL to the active site of RTA in which the principal difference lies in the conformation of the second 'G' in the target GAGA motif in the 28S rRNA substrate. In one model, the sidechain of Asn122 is proposed to make interactions with this G, whereas another model proposes interactions with Asp75 and Asn78. Site-directed mutagenesis of these residues of RTA favours the first of these models, as substitution of Asn78 with Ser yielded an RTA variant whose activity was essentially wild-type, whereas substitution of Asn122 reduced activity 37.5-fold. Substitution of Asp75 failed to yield significant folded protein, suggesting a structural role for this residue.
Marsden
Catherine J
CJ
Department of Biological Sciences, University of Warwick, Coventry, UK.
Fülöp
Vilmos
V
Day
Philip J
PJ
Lord
J Michael
JM
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012313:RNA / Q000378:metabolism; D012276:Ricin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D014961:X-Ray Diffraction
D012313:RNA; D012276:Ricin; D001120:Arginine