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PDBsum entry 1yfs
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References listed in PDB file
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Key reference
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Title
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Breaking sieve for steric exclusion of a noncognate amino acid from active site of a tRNA synthetase.
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Authors
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M.A.Swairjo,
P.R.Schimmel.
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Ref.
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Proc Natl Acad Sci U S A, 2005,
102,
988-993.
[DOI no: ]
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PubMed id
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Abstract
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The genetic code is fixed in aminoacylation reactions catalyzed by
aminoacyl-tRNA synthetases. Amino acid discrimination occurs at two sites: one
for amino acid activation and aminoacylation and one for editing misactivated
amino acids. Although the active site sieves out bulkier amino acids,
misactivation occurs with substrates whose side chains are smaller than the
cognate one. Paradoxically, although alanyl-tRNA synthetase activates glycine as
well as alanine, the sterically larger (than alanine) serine is also
misactivated. Here, we report crystal structures of an active fragment of
Aquifex aeolicus alanyl-tRNA synthetase complexed, separately, with Mg2+-ATP,
alanine, glycine, and serine. Ala and Gly are bound in similar orientations in a
side-chain-accommodating pocket, where alpha-amino and carboxyl groups are
stabilized by salt bridges, and the carboxyl by an H-bond from the side chain
NH2 of Asn-194. In contrast, whereas the same two salt bridges stabilize bound
Ser, H-bonding of the highly conserved (among class II tRNA synthetases) Asn-194
side chain NH2 to the Ser OH, instead of to the carboxyl, forces pocket
expansion. Significantly, in the Mg2+-ATP complex, Asn-194 coordinates a
Mg2+-alpha-phosphate bridge. Thus, the sieve for Ser exclusion is broken because
of selective pressure to retain Asn-194 for Mg2+-ATP and Ala binding.
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Figure 4.
Fig. 4. ATP binding by AlaRS. (A) Simulated annealed omit
F[o] - F[c] electron density map (resolution, 2.15 Å;
contour, 2.8  ) for the active site
region of AlaRS[453]/Mg2+-ATP complex, superimposed on the
refined model. The model for ATP, magnesium ions, and
surrounding atoms within a sphere of 3.2 Å was omitted
from map calculation. (B) Similar view showing active site
residues involved in ATP or magnesium binding. Model colors are
as in Fig. 1. Bound magnesium ions and water molecules are shown
as gray and red spheres, respectively. For clarity, some water
molecules and interactions with the ribose are not shown. (C)
Schematic of the interactions between enzyme, ATP, and
magnesium. Residues from motifs 2 and 3 are shown in orange and
cyan, respectively. Residues in black belong to strands in the
central  -sheet of the
active-site domain. Side chain conservation patterns among AlaRS
sequences from 80 organisms are shown in brackets (percentage
occurrence shown only for side chains present in >4% of the
sequences). Side chains without adjacent bracketed numbers are
invariant.
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Figure 5.
Fig. 5. ATP-induced conformational changes in AlaRS[453]
active site. Cyan ribbon and side chains, apo AlaRS[453]; yellow
ribbon and colored side chains, complex with Mg2+-ATP.
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Secondary reference #1
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Title
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Alanyl-Trna synthetase crystal structure and design for acceptor-Stem recognition.
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Authors
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M.A.Swairjo,
F.J.Otero,
X.L.Yang,
M.A.Lovato,
R.J.Skene,
D.E.Mcree,
L.Ribas de pouplana,
P.Schimmel.
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Ref.
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Mol Cell, 2004,
13,
829-841.
[DOI no: ]
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PubMed id
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Figure 2.
Figure 2. Aminoacylation of RNA Substrates by
Aa-AlaRS453(A) The sequences of A. aeolicus tRNA^Ala and
synthetic RNA substrates tested for aminoacylation by
Aa-AlaRS453. Base pairs in the acceptor-stem region of
Ec-minihelix^Ala that differ from the A. aeolicus sequence are
labeled with asterisks.(B) Aminoacylation of wild-type
Aa-minihelix^Ala (closed circles) and G3:C70 Aa-minihelix^Ala
(open circles) by Aa-AlaRS453 at 37°C (pH 7.5). Inset: the
same experiment repeated at 55°C. Aminoacylation of
Ec-minihelix^Ala was done at 37°C only (closed triangles).
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Figure 6.
Figure 6. Docking Model of tRNA onto Aa-AlaRS453(A) Overall
view with the protein shown as a C[α] trace representation with
the van der Waals surface superposed on top and the three
domains colored as in Figure 3. The tRNA is shown in gray. Both
the complex (top) and the separated enzyme and tRNA (bottom) are
shown. Functionally important sites on protein and
tRNA––identified in previous mutagenesis and biochemical
studies––are shown in CPK representation in purple and blue,
respectively.(B) Potential C domain interaction with G3:U70 from
the major groove side of the acceptor stem. The five helices of
the domain and the 3:70 and 2:71 base pairs are labeled. Side
chains on α14 are shown as sticks. The short loop in
Aa-AlaRS453 which marks the site of the D. melanogaster
mitochondrial AlaRS-specific insertion is colored in magenta.
The dotted line hypothetically indicates the longer loop and
predicted helical pair formed by the insertion in D.
melanogaster mitochondrial AlaRS. The arrow indicates the
direction of the potential C domain shift, from 3:70 to 2:71
position up the acceptor stem.
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The above figures are
reproduced from the cited reference
with permission from Cell Press
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