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PDBsum entry 1aze
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Complex (adaptor protein/peptide)
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PDB id
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1aze
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Contents |
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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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Molecular and cellular analysis of grb2 sh3 domain mutants: interaction with sos and dynamin.
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Authors
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M.Vidal,
N.Goudreau,
F.Cornille,
D.Cussac,
E.Gincel,
C.Garbay.
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Ref.
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J Mol Biol, 1999,
290,
717-730.
[DOI no: ]
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PubMed id
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Abstract
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Quantitative analysis of Grb2/dynamin interaction through plasmon resonance
analysis (BIAcore) using Grb2 mutants showed that the high affinity measured
between Grb2 and dynamin is essentially mediated by the N-SH3 domain of Grb2. In
order to study the interactions between Grb2 and either dynamin or Sos in more
detail, Grb2 N-SH3 domains containing different mutations have been analysed.
Two mutations were located on the hydrophobic platform binding proline-rich
peptides (Y7V and P49L) and one (E40T) located in a region that we had
previously shown to be essential for Grb2/dynamin interactions. Through NMR
analysis, we have clearly demonstrated that the structure of the P49L mutant is
not folded, while the other E40T and Y7V mutants adopt folded structures that
are quite similar to that described for the reference domain. Nevertheless,
these point mutations were shown to alter the overall stability of these domains
by inducing an equilibrium between a folded and an unfolded form. The complex
formed between the peptide VPPPVPPRRR, derived from Sos, and the E40T mutant was
shown to have the same 3D structure as that described for the wild-type SH3
domain. However, the VPPPVPPRRR peptide adopts a slightly different orientation
when it is complexed with the Y7V mutant. Finally, the affinity of the
proline-rich peptide GPPPQVPSRPNR, derived from dynamin, for the Grb2 N-SH3
domain was too low to be analyzed by NMR. Thus, the interaction between either
Sos or dynamin and the SH3 mutants were tested on a cellular homogenate by means
of a far-Western blot analysis. In these conditions, the P49L mutant was shown
to be devoid of affinity for Sos as well as for dynamin. The Y7V SH3 mutant
displayed a decrease of affinity for both Sos and dynamin, while the E40T mutant
exhibited a decrease of affinity only for dynamin. These results support the
existence of two binding sites between dynamin and the Grb2 N-SH3 domain.
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Figure 5.
Figure 5. (a) The 3D structures of the N-SH3 domains of Grb2 complexed with the proline-rich peptide
VPPPVPPRRR. Left, C32S-Y7V Grb2 N-SH3 mutant; right, reference domain. The interacting side-chains from the
proline-rich peptide are in red, the peptide backbone is in yellow, the interacting side-chains from the SH3 domain
are in orange, and the backbone of the SH3 is in blue. The essential differences appear at the level of subsite S1,
which comprises amino acid residues Y7 and Y52 in the reference domain and interacts with Pro2 of the peptide.
When Y7 is mutated as V7, the proline-rich peptide interacts with V7 and Y52 side-chains by means of Pro3 ring
instead of Pro2. (b) Superposition of the 3D structures of the N-SH3 domains of Grb2 complexed with the proline-
rich peptide: in yellow, the reference domain and in red, the mutant domain. Only tyrosine 7 or valine 7 are shown.
It appears that the difference is mainly in the way the peptide binds the protein.
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Figure 6.
Figure 6. Quantification of the displacement by N-
terminal SH3 domain mutants of Sos and dynamin
bound to GST-Grb2-N-SH3-SH2 fusion protein on ER 22
cell homogenate. When a mutant possesses affinity for
either Sos or dynamin, the corresponding band intensity
decreases.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(1999,
290,
717-730)
copyright 1999.
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Secondary reference #1
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Title
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Nmr structure of the n-Terminal sh3 domain of grb2 and its complex with a proline-Rich peptide from sos.
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Authors
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N.Goudreau,
F.Cornille,
M.Duchesne,
F.Parker,
B.Tocqué,
C.Garbay,
B.P.Roques.
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Ref.
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Nat Struct Biol, 1994,
1,
898-907.
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PubMed id
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