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PDBsum entry 1svk
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Hydrolase, signaling protein
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PDB id
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1svk
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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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Uncoupling conformational change from gtp hydrolysis in a heterotrimeric g protein alpha-Subunit.
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Authors
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C.J.Thomas,
X.Du,
P.Li,
Y.Wang,
E.M.Ross,
S.R.Sprang.
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Ref.
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Proc Natl Acad Sci U S A, 2004,
101,
7560-7565.
[DOI no: ]
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PubMed id
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Abstract
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Heterotrimeric G protein alpha (G alpha) subunits possess intrinsic GTPase
activity that leads to functional deactivation with a rate constant of
approximately 2 min(-1) at 30 degrees C. GTP hydrolysis causes conformational
changes in three regions of G alpha, including Switch I and Switch II. Mutation
of G202-->A in Switch II of G alpha(i1) accelerates the rates of both GTP
hydrolysis and conformational change, which is measured by the loss of
fluorescence from Trp-211 in Switch II. Mutation of K180-->P in Switch I
increases the rate of conformational change but decreases the GTPase rate, which
causes transient but substantial accumulation of a low-fluorescence G
alpha(i1).GTP species. Isothermal titration calorimetric analysis of the binding
of (G202A)G alpha(i1) and (K180P)G alpha(i1) to the GTPase-activating protein
RGS4 indicates that the G202A mutation stabilizes the pretransition state-like
conformation of G alpha(i1) that is mimicked by the complex of G alpha(i1) with
GDP and magnesium fluoroaluminate, whereas the K180P mutation destabilizes this
state. The crystal structures of (K180P)G alpha(i1) bound to a slowly
hydrolyzable GTP analog, and the GDP.magnesium fluoroaluminate complex provide
evidence that the Mg(2+) binding site is destabilized and that Switch I is
torsionally restrained by the K180P mutation. The data are consistent with a
catalytic mechanism for G alpha in which major conformational transitions in
Switch I and Switch II are obligate events that precede the bond-breaking step
in GTP hydrolysis. In (K180P)G alpha(i1), the two events are decoupled
kinetically, whereas in the native protein they are concerted.
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Figure 2.
Fig. 2. Catalytic sites of G  [i1]·
GDP·Mg2+·AlF[4]^- complexes. Atoms are rendered as
follows: carbon, gold; nitrogen, cyan; oxygen, red; fluorine,
yellow; aluminum, gray; and phosphorus, green. Mg2+ is shown as
a blue sphere; and  phosphate oxygen atoms
are shown. Metal-coordination interactions are indicated by gray
dashed lines, and hydrogen bonds are indicated by red dashed
lines. (A) Wild-type G [i1]. (B) For (K180P)G
[i1], major (a,
occupancy,  0.25) and minor (b,
occupancy, 0.75) conformations of
Ser-47 are shown.
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Figure 3.
Fig. 3. Difference-distance analysis of wild type and
(K180P)G [i1] in complexes with
GppNHp·Mg2+ and GDP·Mg2+·AlF[4.] Changes in
contacts between C in residues 165-207 in
G [i1] (rows) and
residues 35-76 and 140-226 in (K180P)G [i1] (columns) for the
GNP-bound complexes (Left), and the AlF-bound complexes (Center)
are shown. In Right, the elements from the AlF matrix are
subtracted from the corresponding elements in the GNP matrix.
Values are  -weighted and
color-coded according to direction and magnitude (red, negative;
blue, positive). Contour values range from ± to 0.
Matrix elements corresponding to residue pairs separated by >10
Å were set at 0. The dark line represents self-vectors (i
= j).
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