PDB file 1GIA

This structure of the a subunit of an inhibitory G Protein, with bound GTPgS, was solved by D. E. Coleman et al., in 1994.

G_a,b,g

PDB file 1GP2

This file depicts a rat heterotrimeric inhibitory G Protein, with bound GDP. This structure was solved by M. A. Wall et al., in 1996.

Recommended display options:

Display as cartoon, and color chain, to distinguish the subunits a (chain a), b (chain b), g (chain g). To see the GDP, select hetero-ligand, display as spacefill, and color CPK.

The WD-repeat in the b subunit forms a sevenfold b propeller.
Select protein-sheet and specify a different color, to emphasize the b structure.
Locate where the ends of the b protein overlap, with both ends of the protein segment contributing to one of the propeller blades.
Question: Why might this overlap of N and C -terminal ends of the protein have evolved?

Examine the domain of subunit a that makes contact with subunit b at the face of the b-propeller.
Select 200-215a and color magenta.
This is the "switch II" domain of subunit a, that changes position when GTP replaces GDP, resulting in dissociation of G_a from G_bg.

With your teammate at an adjacent computer, compare this display of a-GDP complexed to bg, and the one of a-GTP (GTP analog). To facilitate this comparison, to make the b & g subunits on this display disappear, use the menu to select chain b then select hide selected; then select chain g and hide selected. Rotate so that both images have the same orientation in relation to the guanine ring and first two phosphates of GDP/GTP.
Compare the position of switch II (should be colored magenta) with GDP and the GTP analog.

The analog GTPgS was used in place of the normal ligand GTP.

Recommended display options:

Team up with someone at an adjacent computer. Together explore this structure of G_a-GTP.
Keep the display on one computer while together you display G_abg-GDP on the other computer.
Compare the position of switch II in the two cases.

Color chain, and display as cartoon.
Select hetero-ligand, display as spacefill, and color CPK. Note how GTPgS sits in a deep cleft in the protein. Identify the yellow S substituting for an oxygen in the terminal phosphate of GTP.
Questions:
Why is GTPgS used in place of GTP during protein crystallization?
Mg⁺⁺ is required for activation of G_a. What atoms of the protein and the GTP analog coordinate Mg⁺⁺?

Select protein-sheet and then select-change color to, and specify a color, to highlight the 6-stranded b-sheet adjacent to the nucleotide binding site.

Enter into the command line select 40-47, and then use the mouse menu to specify a color.
Explore how this " P-loop " is adjacent to the terminal phosphates of GTP.

Select 178-184, and color CPK.
This is Switch I, one of the regions that changes position when GTP substitutes for GDP.
Change display to ball & stick.
Look for the invariant threonine that interacts with Mg.

To visualize Switch II, select 200-215 and display as sticks.
Look for the conserved glutamine residue that helps to position the attacking water molecule at the active site for GTP hydrolysis.

Now change the display for residues 200-215 (Switch II) to cartoon and color magenta (Use this exact color so that you will be able to compare the position of Switch II in G_abg-GDP.)
The a-helix in this domain ends in a region that interacts with Adenylate Cyclase. The helix also interacts with the inhibitory b subunit when G_a has bound GDP. The change in conformation of switch II with GDP/GTP exchange may contribute to altered affinity of G_a for G_b and for Adenyate Cyclase.

Поиск по сайту: