PDBsum entry 1svk

Hydrolase, signaling protein

PDB id: 1svk

Contents

Protein chain

313 a.a. *

Ligands

ALF-GDP

Metals

_MG

Waters ×336

* Residue conservation analysis

PDB id:

1svk

Name:

Hydrolase, signaling protein

Title:

Structure of the k180p mutant of gi alpha subunit bound to alf4 and gdp

Structure:

Guanine nucleotide-binding protein g(i), alpha-1 subunit. Chain: a. Synonym: adenylate cyclase-inhibiting g alpha protein. Engineered: yes. Mutation: yes

Source:

Rattus norvegicus. Norway rat. Organism_taxid: 10116. Gene: gnai1, gnai-1. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.00Å R-factor: 0.197 R-free: 0.239

Authors:

C.J.Thomas,X.Du,P.Li,Y.Wang,E.M.Ross,S.R.Sprang

Key ref:

C.J.Thomas et al. (2004). Uncoupling conformational change from GTP hydrolysis in a heterotrimeric G protein alpha-subunit. Proc Natl Acad Sci U S A, 101, 7560-7565. PubMed id: 15128951 DOI: 10.1073/pnas.0304091101

Date:

29-Mar-04 Release date: 01-Jun-04

Protein chain

P10824  (GNAI1_RAT) -  Guanine nucleotide-binding protein G(i) subunit alpha-1 from Rattus norvegicus

Seq: 354 a.a.

Struc: 313 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.6.1.46 - Transferred entry: 3.6.5.1.
• Reaction: GTP + H₂O = GDP + phosphate

DOI no: 10.1073/pnas.0304091101

Proc Natl Acad Sci U S A 101:7560-7565 (2004)

PubMed id: 15128951

Uncoupling conformational change from GTP hydrolysis in a heterotrimeric G protein alpha-subunit.

C.J.Thomas, X.Du, P.Li, Y.Wang, E.M.Ross, S.R.Sprang.

ABSTRACT

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.

Selected figure(s)

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.

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).

Figures were selected by an automated process.