1UQ4

RICIN A-CHAIN (RECOMBINANT) R213D MUTANT

Summary for 1UQ4

• Entry DOI: 10.2210/pdb1uq4/pdb
• Related: 1APG 1BR5 1BR6 1FMP 1IFS 1IFT 1IFU 1IL3 1IL4 1IL5 1IL9 1OBS 1OBT 1RTC 2AAI
• Descriptor: RICIN, SULFATE ION (3 entities in total)
• Functional Keywords: hydrolase, glycosidase, toxin, glycoprotein
• Biological source: RICINUS COMMUNIS (CASTOR BEAN)
• Total number of polymer chains: 1
• Total formula weight: 29600.15

Authors

Marsden, C.J.,Fulop, V. (deposition date: 2003-10-15, release date: 2004-01-02, Last modification date: 2023-12-13)

Primary citation

Marsden, C.J.,Fulop, V.,Day, P.,Lord, J.M.
The Effect of Mutations Surrounding and within the Active Site on the Catalytic Activity of Ricin a Chain
Eur.J.Biochem., 271:153-, 2004

PubMed Abstract: 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.

PubMed: 14686928
DOI: 10.1046/J.1432-1033.2003.03914.X

Experimental method

X-RAY DIFFRACTION (1.9 Å)