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PDBsum entry 2d3d
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RNA binding protein
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PDB id
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2d3d
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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 nmr and X-Ray structures of the saccharomyces cerevisiae vts1 sam domain define a surface for the recognition of RNA hairpins.
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Authors
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T.Aviv,
A.N.Amborski,
X.S.Zhao,
J.J.Kwan,
P.E.Johnson,
F.Sicheri,
L.W.Donaldson.
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Ref.
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J Mol Biol, 2006,
356,
274-279.
[DOI no: ]
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PubMed id
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Abstract
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The SAM domain of the Saccharomyces cerevisiae post-transcriptional regulator
Vts1 has a high affinity towards RNA hairpins containing a CUGGC pentaloop. We
present the 1.6 Angstroms X-ray crystal structure of the Vts1 SAM domain in its
unliganded state, and the NMR solution structure of this domain in its RNA-bound
state. Both structures reveal a canonical five helix SAM domain flanked by
additional secondary structural elements at the N and C termini. The two
structures are essentially identical, implying that no major structural
rearrangements occur upon RNA binding. Amide chemical shift changes map the
RNA-binding site to a shallow, basic patch at the junction of helix alpha5 and
the loop connecting helices alpha1 and alpha2.
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Figure 1.
Figure 1. The crystal structure of free Vts1 SAM and the
NMR structure of SRE-bound Vts1 SAM are essentially identical. A
gene fragment encompassing the SAM domain (residues 407-523) of
S. cerevisiae VTS1 was amplified by PCR methods and inserted
into a modified pGEX vector. The resulting
glutathione-S-transferase fusion protein was purified by
affinity chromatography from soluble extracts of E. coli
BL21(DE3) grown in a BioFlow 110 fermentor at 25 °C.
Isotopically labeled Vts1^407-526 was obtained from
fermentations in minimal M9 medium containing 1.0 g/l [U-98%]
[15N]ammonium chloride and 4.0 g/l [U-99%] [13C]glucose as the
sole sources of nitrogen and carbon, respectively. Vts1^407-523
was cleaved from the fusion protein with TEV protease. Proteins
were further purified and buffer-exchanged using S-100
gel-filtration chromatography. For crystallography, the 13 kDa
Vts1 fragment was digested with thermolysin to produce a minimal
10 kDa fragment, Vts1^436-523, that was purified further by
heparin chromatography. A 19 nt SRE RNA
(5'-GGAGGCUCUGGCAGCUUUC-3') was prepared in milligram quantities
for NMR spectroscopy by phage T7 RNA polymerase-driven, in vitro
transcription.17 The SRE RNA was purified by denaturing 20% PAGE
and electroeluted. Renaturation of the SRE RNA hairpin was
achieved by rapidly chilling a solution that was preheated to 94
°C. (a) A fluorescence polarization binding assay7 of the
thermolysin-resistant Vts1 SAM domain demonstrates high-affinity
binding for the SRE (5'-AGGCUCUGGCAGUCU-3') but not a mutant SRE
bearing substitutions at the first and third loop positions. (b)
A modest decrease in binding affinity is observed when either 5
mM EDTA or 10 mM Ca^2+ is present in the binding buffer. (c)
Superposition of the free Vts1 SAM domain crystal structure with
the bound NMR structure reveals no significant conformational
changes. The SRE binding site is indicated in red. Superposition
of the crystal structures of the Vts1 SAM domain with the (d)
EphA4 SAM domain and the (e) Smaug SAM domain. (f) Superposition
of the 20 lowest energy NMR structures of the RNA-bound Vts1 SAM
domain.
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Figure 2.
Figure 2. The RNA-binding surface on the Vts1 SAM domain.
(a) Weighted-averaged18 amide 1H and 15N chemical shift changes
upon SRE RNA binding where d[av](HN)=[dH2+(dN/5)2)/2]1/2. Bars
are colored according to the magnitude of the chemical shift
change (red, >0.3 ppm; yellow, >0.1 ppm). (b) Chemical shift
changes, sequence conservation, and electrostatic charge are
mapped onto the surface of the free X-ray structure of Vts1 SAM
domain. HSQC perturbations are colored according to (a). The
linewidth of Lys467 sharpens significantly upon RNA binding and
is colored orange. Sequence identity (in purple) and sequence
similarity (in pink) among Vts1 homologs coincides with the SRE
binding surface deduced from chemical shift perturbations. The
RNA-binding site demonstrates basic charge distribution (red;
negative potential; blue, positive potential). Molecular
structure representations and electrostatic calculations were
made with pyMOL (http://pymol.sourceforge.net).
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2006,
356,
274-279)
copyright 2006.
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Secondary reference #1
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Title
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The RNA-Binding sam domain of smaug defines a new family of post-Transcriptional regulators.
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Authors
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T.Aviv,
Z.Lin,
S.Lau,
L.M.Rendl,
F.Sicheri,
C.A.Smibert.
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Ref.
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Nat Struct Biol, 2003,
10,
614-621.
[DOI no: ]
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PubMed id
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Figure 1.
Figure 1. Homology modeling of Smg SAM domain. (a) Sequence
alignment of SAM domains of Smg homologs and selected proteins.
GenBank accession numbers are indicated on the right. Secondary
structure elements corresponding to the EphB2 SAM domain crystal
structure are indicated. Conserved hydrophobic residues, green;
acidic residues, red; basic residues, blue. Mutations in Smg or
Vts1 that perturbed SRE binding, red stars; benign mutation,
green diamond. Species abbreviations: dm, D. melanogaster; ag,
Anopheles gambiae; hs, Homo sapiens; mm, Mus musculus; ce, C.
elegans; ca, Candida albicans; sp, S. pombe; sc, S. cerevisiae;
dd, Dictyostelium discoideum. (b) Cladogram representing overall
sequence similarity and domain architecture of the Smg homologs.
See text for description of SAM, SSR1 and SSR2 domains. Zif,
CCHC zinc-finger domain (SMART43). (c,d) Ribbon and surface
representations of the Smg SAM domain, respectively. In c,
secondary structure elements encompassing RNA-binding surface
are pink and conserved side chains specific to Smg homologs are
in ball-and-stick representation. In d, regions corresponding to
conserved basic and hydrophobic residues specific to the Smg
homologs are blue and green, respectively.
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Figure 2.
Figure 2. Protein determinants of the Smg and Vts1 SAM domains
required for SRE binding. (a) Schematic of
fluorescein-labeled model SRE, based on the nos 3' UTR27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, of loop;
asterisks indicate expected Watson-Crick base pairing in the
stem. Bases required for Smg binding, bold. (b) Mutational and
SRE binding analysis of Smg(584 -763). (c) Mutational and SRE
binding analysis of full-length Vts1(1 -523). (d) Mutational and
SRE binding analysis of Vts1^SAM.
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The above figures are
reproduced from the cited reference
with permission from Macmillan Publishers Ltd
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