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PDBsum entry 1uq5
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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The effect of mutations surrounding and within the active site on the catalytic activity of ricin a chain.
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Authors
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C.J.Marsden,
V.Fülöp,
P.J.Day,
J.M.Lord.
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Ref.
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Eur J Biochem, 2004,
271,
153-162.
[DOI no: ]
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PubMed id
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Abstract
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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.
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Figure 1.
Fig. 1. Models of hexanucleotide binding in the active
site of RTA(based upon [10]). The structures are shown as stereo
images with the C[1]G[2]A[3]G[4]A[5]G[6] (where A[3] is the
target for depurination by RTA) in red. C[1] of the
hexanucleotide is at the bottom left and the target adenine is
at the top of each model. The sidechains of RTA are shown in
blue. (A) Model 1 has the tetraloop bound in the active site of
RTA with G[4] stacked upon the G[2]-A[5] pair and able to make
interactions with Asn122. (B) Model 2 is a variation of model 1
with the tetraloop bound in a conformation where G[4] stacks
with Tyr80 and makes interactions with Asp75 and Asn78. Drawn
with MOLSCRIPT[34,35].
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Figure 4.
Fig. 4. Electron density of RTA R213D in the vicinity of
residue 213. The backbone and sidechains of the R213D
substitution are shown as stereo images in thick ball and stick
and the position of the Arg213 side-chain of the wild-type
enzyme is overlayed and shown in thin ball and stick. The SIGMAA
[33] weighted 2mF[o]-  F[c] electron
density using phases from the final model is contoured at 1  level, where
represents the
rms electron density for the unit cell. Contours more than 1.4
Å from any of the displayed atoms have been removed for
clarity. Drawn with MOLSCRIPT[34,35].
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The above figures are
reprinted
by permission from the Federation of European Biochemical Societies:
Eur J Biochem
(2004,
271,
153-162)
copyright 2004.
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Secondary reference #1
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Title
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X-Ray structure of recombinant ricin a-Chain at 1.8 a resolution.
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Authors
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S.A.Weston,
A.D.Tucker,
D.R.Thatcher,
D.J.Derbyshire,
R.A.Pauptit.
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Ref.
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J Mol Biol, 1994,
244,
410-422.
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PubMed id
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