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PDBsum entry 1obc
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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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Structural and mechanistic basis of pre- And posttransfer editing by leucyl-Trna synthetase.
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Authors
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T.L.Lincecum,
M.Tukalo,
A.Yaremchuk,
R.S.Mursinna,
A.M.Williams,
B.S.Sproat,
W.Van den eynde,
A.Link,
S.Van calenbergh,
M.Grøtli,
S.A.Martinis,
S.Cusack.
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Ref.
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Mol Cell, 2003,
11,
951-963.
[DOI no: ]
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PubMed id
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Abstract
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The aminoacyl-tRNA synthetases link tRNAs with their cognate amino acid. In some
cases, their fidelity relies on hydrolytic editing that destroys incorrectly
activated amino acids or mischarged tRNAs. We present structures of leucyl-tRNA
synthetase complexed with analogs of the distinct pre- and posttransfer editing
substrates. The editing active site binds the two different substrates using a
single amino acid discriminatory pocket while preserving the same mode of
adenine recognition. This suggests a similar mechanism of hydrolysis for both
editing substrates that depends on a key, completely conserved aspartic acid,
which interacts with the alpha-amino group of the noncognate amino acid and
positions both substrates for hydrolysis. Our results demonstrate the economy by
which a single active site accommodates two distinct substrates in a
proofreading process critical to the fidelity of protein synthesis.
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Figure 1.
Figure 1. Editing Reactions, Editing Substrates, and
Sequence Conservation in the Editing Domain(A) LeuRS
aminoacylation and editing reactions. Editing reactions are
indicated by the dashed arrows. Although tRNA has been shown to
be a cofactor for pretransfer editing by IleRS (Baldwin and
Berg, 1966), its role in LeuRS editing is unknown.(B) Diagrams
of the analogs used in this work of LeuRS pre- and posttransfer
editing substrates for the case of noncognate norvaline (Nva).
Left: posttransfer substrate analog,
2′-(L-norvalyl)amino-2′-deoxyadenosine (Nva2AA), mimicking
the 3′ end of the aminoacyl-2′-ester Nva-tRNA^Leu. Right:
pretransfer substrate analog,
5′-O-[N-(L-norvalyl)sulphamoyl]adenosine (NvaAMS), a sulfamoyl
analog of norvalyl-adenylate. In each case, the labile ester
linkages were replaced by a nonhydrolyzable amino linkage to
permit structural studies.(C) Alignment of conserved regions
within the editing (CP1) domain of selected LeuRS (L), ValRS
(V), and IleRS (I) enzymes. The “threonine-rich region”
contains two highly conserved threonines (arrowed) discussed in
the text. In the second region, separated by a bracket, a
conserved glycine-rich loop is followed by a completely
conserved aspartic acid (arrowed) that was mutated to alanine.
Abbreviations: Sc, S. cerevisiae; Ce, Caenorhabditis elegans;
Hs, Homo sapiens; Nc, Neurospora crassa; Ec, E. coli; Tt,
Thermus thermophilus; Bs, Bacillus subtilis; Gs, Geobacillus
stearothermophilus; Sa, Staphylococcus aureus; cyt, cytoplasmic;
mit, mitochondrial.
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Figure 2.
Figure 2. Electron Density of the Editing Substrates(A)
Simulated omit map (Brunger et al., 1998) for the pretransfer
substrate analog (NvaAMS) in the editing (top) and synthetic
(bottom) active site. Resolution is 2.2 Å. In both
molecules, the ribose is in the C2′ endo conformation.(B)
Location of the NvaAMS in the synthetic and editing active sites
of LeuRSTT.(C) Unbiased difference map (2.0 Å resolution)
for the posttransfer editing substrate analog (Nva2AA) in the
editing site. The ribose is in the C3′ endo conformation.(D)
Competitive inhibition of E. coli LeuRS editing of Ile-tRNA^Leu
by Nva2AA. Editing of Ile-tRNA^Leu by wild-type E. coli LeuRS in
the absence of inhibitor exhibited a K[M] of 0.2 μM.
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The above figures are
reprinted
by permission from Cell Press:
Mol Cell
(2003,
11,
951-963)
copyright 2003.
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Secondary reference #1
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Title
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The 2 a crystal structure of leucyl-Trna synthetase and its complex with a leucyl-Adenylate analogue.
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Authors
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S.Cusack,
A.Yaremchuk,
M.Tukalo.
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Ref.
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EMBO J, 2000,
19,
2351-2361.
[DOI no: ]
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PubMed id
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Figure 4.
Figure 4 (A) The LeuAMS binding site showing the major
interacting residues. Hydrogen bonds are shown as dashed green
lines and a tightly bound water as a green sphere. The
catalytically essential class 1 motifs H^49IGH and M^638SKS are
shown in cyan and red, respectively. The side chain of Tyr43 is
omitted for clarity, but is visible in (C). (B) The
conformational changes associated with LeuAMS binding. The view
is the same as in (A). The pink ribbon diagram, pink side chains
and pink labels correspond to the apo-structure (mercury
derivative) and the grey ribbon and yellow side chains belong to
the LeuAMS-bound structure. Upon binding of the adenosine
moiety, the HIGH and MSKS loops towards the active centre,
Gln574 and Glu540 move to bind the ribose tightly, and helices
H18 and H3 refold to permit packing of the ZN-1 domain close to
the active site [see the text and (C)]. A sulfate ion (not
shown) is bound to His49 and His52 in the apo-structure, but not
in the LeuAMS-bound structure. (C) Proximity of Arg178 to the
active center in the LeuAMS complex. Colouring as in (B) with
water molecules as green spheres and the Zn-1 atom as a red
sphere. One of the zinc ligands (His179) and the adenosine
moiety of the LeuAMS are omitted for clarity. The positions of
Leu544 and Leu84 sterically prevent the packing of the ZN-1
domain close to the active site in the apo-structure.
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Figure 5.
Figure 5 Final electron density at 2.0 Å resolution for
LeuAMS in the active site of LeuRSTT contoured at 2  .
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The above figures are
reproduced from the cited reference
which is an Open Access publication published by Macmillan Publishers Ltd
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Secondary reference #2
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Title
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Crystallization and preliminary crystallographic analysis of thermus thermophilus leucyl-Trna synthetase and its complexes with leucine and a non-Hydrolysable leucyl-Adenylate analogue.
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Authors
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A.Yaremchuk,
S.Cusack,
O.Gudzera,
M.Grøtli,
M.Tukalo.
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Ref.
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Acta Crystallogr D Biol Crystallogr, 2000,
56,
667-669.
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PubMed id
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