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PDBsum entry 3onw
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Signaling protein
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PDB id
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3onw
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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 determinants of affinity enhancement between goloco motifs and g-Protein alpha subunit mutants.
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Authors
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D.E.Bosch,
A.J.Kimple,
D.W.Sammond,
R.E.Muller,
M.J.Miley,
M.Machius,
B.Kuhlman,
F.S.Willard,
D.P.Siderovski.
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Ref.
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J Biol Chem, 2011,
286,
3351-3358.
[DOI no: ]
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PubMed id
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Abstract
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GoLoco motif proteins bind to the inhibitory G(i) subclass of G-protein α
subunits and slow the release of bound GDP; this interaction is considered
critical to asymmetric cell division and neuro-epithelium and epithelial
progenitor differentiation. To provide protein tools for interrogating the
precise cellular role(s) of GoLoco motif/Gα(i) complexes, we have employed
structure-based protein design strategies to predict gain-of-function mutations
that increase GoLoco motif binding affinity. Here, we describe fluorescence
polarization and isothermal titration calorimetry measurements showing three
predicted Gα(i1) point mutations, E116L, Q147L, and E245L; each increases
affinity for multiple GoLoco motifs. A component of this affinity enhancement
results from a decreased rate of dissociation between the Gα mutants and GoLoco
motifs. For Gα(i1)(Q147L), affinity enhancement was seen to be driven by
favorable changes in binding enthalpy, despite reduced contributions from
binding entropy. The crystal structure of Gα(i1)(Q147L) bound to the RGS14
GoLoco motif revealed disorder among three peptide residues surrounding a well
defined Leu-147 side chain. Monte Carlo simulations of the peptide in this
region showed a sampling of multiple backbone conformations in contrast to the
wild-type complex. We conclude that mutation of Glu-147 to leucine creates a
hydrophobic surface favorably buried upon GoLoco peptide binding, yet the
hydrophobic Leu-147 also promotes flexibility among residues 511-513 of the
RGS14 GoLoco peptide.
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Secondary reference #1
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Title
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Structure-Based protocol for identifying mutations that enhance protein-Protein binding affinities.
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Authors
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D.W.Sammond,
Z.M.Eletr,
C.Purbeck,
R.J.Kimple,
D.P.Siderovski,
B.Kuhlman.
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Ref.
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J Mol Biol, 2007,
371,
1392-1404.
[DOI no: ]
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PubMed id
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Figure 2.
Figure 2. Binding curves for select affinity increasing
mutations compared to wild-type for (a) Gα[i1]:GoLoco and (b)
E6AP:UbcH7.
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The above figure is
reproduced from the cited reference
with permission from Elsevier
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Secondary reference #2
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Title
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Structural determinants for goloco-Induced inhibition of nucleotide release by galpha subunits.
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Authors
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R.J.Kimple,
M.E.Kimple,
L.Betts,
J.Sondek,
D.P.Siderovski.
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Ref.
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Nature, 2002,
416,
878-881.
[DOI no: ]
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PubMed id
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Figure 1.
Figure 1: alpha- [i1]
[glyph.gif] GDP
in complex with the RGS14 GoLoco region. a, Ribbon drawing of
R14GL peptide (red) in contact with the Ras-like (green) and
all-helical (yellow) domains of G  [i1].
Also shown are the three switch regions of G [i1]
(blue), GDP (magenta) and Mg2+ (orange). b, Molecular surface of
R14GL (red) and G   (cyan)
contacts on G [i1]
GDP,
denoting shared switch II residue contacts (magenta).
Highlighted are G [i1]
residues that contact the R14GL peptide and are different within
G [o].
c, Space fill model of [i1]
GDP
in its R14GL-bound conformation (switch regions in blue,
[148]alpha B - [149]alpha C loop in yellow) and G [150]beta [1]
[151]gamma [2]-bound conformation (in cyan).
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Figure 2.
Figure 2: Role of the GoLoco motif Asp-Gln-Arg triad in GDI
activity. a, Stereo view of the remodelled nucleotide binding
pocket. The G [i1]
Arg 178 side chain (transparent yellow) is re-oriented (green)
to form a salt bridge with Glu 43 of the G [i1]
subunit, thus allowing Arg r516 of the GoLoco motif (red) to
approach and interact with the -
and -phosphate
oxygens (and bridging oxygen) of GDP. b, GST -RGS14 GoLoco
fusion proteins (GST -RGS14[496 -531]) bearing alanine (R516A)
or leucine (R516L) substitutions show decreased GDI activity
relative to the wild-type GST -RGS14[496 -531] (WT), as measured
both by BODIPY -GTP S
binding (left) and by AlF[4]^--induced increase of intrinsic
tryptophan fluorescence (right).
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The above figures are
reproduced from the cited reference
with permission from Macmillan Publishers Ltd
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