Interactive Protein Tutorial
A case study of the Ras small GTP-binding protein
This tutorial allows you to explore some of the protein structural features of the cellular form of Harvey Ras (c-H-ras proto-oncogene). Many of the observations can be extended to other members of the ras superfamily of GTP-binding proteins including other forms of ras and more distantly related sub familis like Rho/rac, Rab, Ran, Arf/Arl etc. In addition many of the sturtcural features described here can also be found amongst the alpha subunits of heterotrimeric G-proteins.
We will begin the tutorial by considering the inactive GDP-bound form of the protein.
If at any point you are having trouble following the direction of the amino acid chain through the protein structure change the view to "rockets" (right mouse click, Style > Structures > Rockets).
Ras is a small compact globular protein
Here we can modify the view of the protein changing from ball and stick to space fill modes. The Ras protein is seen to be a compact particle with no obvious domains. At only 21KDa mass this is only to be expected.The ras fold and localisation of the guanine nucleotide
Changing to a cartoon rendered of secondary structure we can see that ras is composed of a mixture of alpha helices and beta strands. The beta strands are organised into a twisted beta sheet with some parallel and anti-parallel components. The alpha helices are packed on both sides of the beta sheet. As is often the case with ligand-binding proteins whose tertiary structure (fold) is arranged in this way the ligand - here GDP shown in space fill in red - is bound between the loops found at one end of this sheet.Magnesium ion
This button adds the magnesium ion here shown in yellow in space fill mode. Most guanine and adenine nucleotides are found in cells as complexes with magnesium ions - this includes ATP and here GDP. Here the magnesium ion fills its six-fold co-ordination sphere with bonds to oxygens found in the phosphate groups of GDP and a serine residue in the protein and four "structural" water molecules.The guanine nucleotide binding pocket
Here the GDP molecule is made transparent and the surface mapped with dots so that you can see how tightly surrounded the ligand is by protein structural elements. These will now be revealed sequentially.The G1 or P loop
G1 region is also known as the diphosphate binding loop or P loop as it binds and correctly positions the alpha and beta phosphates of the nucleotides correctly. Importantly this loop contains the G12 amino acid which is the site of a common activating G12V mutation. You should be familiar with this from your lecture on structure-function relationships in ras GAPs. Because G1 has a key role in binding guanine nucleotides some elements of its sequence are said to be characteristic of this function and may be used as molecular "fingerprints" to identify G-proteins related to ras.The G2 region or Switch 1
G2 connects sub-sites that bind magnesium and the gamma phosphate of GTP if it is present (see below). This region will therefore make characteristic movements as the ras protein switches between GDP and GTP forms. G2 contains an important threonine residue absolutely conserved in Ras-like and G alpha-like proteins, when GTP is bound this residue will coordinate to the magnesium ion. T to N mutations here lead to forms of the G protein that cannot be activated and which are very useful experimentally as suppressors of signalling when over-expressed in cells. Note that the G1 loop from the previous view has been coloured blue to help you maintain your orientation.The G3 region or Switch 2
Like G1 this G3 region also contain consensus amino acids that "fingerprint" or characterise ATP or GTP binding sites in many proteins. For example G3 contains amino acid Q61 which is the site of a common Q61L activating mutation. The catalytic role of this amino acid should also be familiar to you from your lecture on ras GAP.The G4 region
This region is important in recognising the non-phosphate parts of the guanine, for example the NKXD motif which interacts with the ribose ring. This motif is found in almost all guanine and many adenine nucleotide binding pockets. Use your mouse to hover over the space filled residues and identify the sequence in question - what is the X residue in c-H-ras? Hint: it contains a sulphur atom.The G5 region
The G5 region or loop is also important in recognising the guanine nucleotide base.The "business end" of ras
This button now reveals all five regions G1 to G5 and shows that together they constitute the majority of the ras protein that interacts with the guanine nucleotide - here GDP is still shown in dots for transparency. As you can see the pocket is tightly packed around the MgGDP complex except for G3/Switch 2 which appears quite distant.The guanine nucleotide binding pocket
The reminder of the ras protein is largely structural scaffolding for the G1 to G5 se components and here they are subtracted so that you can concentrate on this component. The GDP ligand molecule is then revealed in space fill. Can you identify the guanine base and the phosphate groups and their interactions? Nitrogens in the base are prominent in deep blue and phosphate oxygens in seen in bright red.Simplifying the G1 to G5 loops
Here the view of the molecular model is simplified for clarity.Adding back the rest of ras
A complete structural view now allows you see the guanine nucleotide binding pocket in its "structural context".We will now begin to focus our attention on the structural rearrangements that ras undergoes when it switches from its inactive GDP-bound form to its active GTP-bound configuration. Obviously those components of the guanine nucleotide binding pocket that recognise the parts of GDP and GDP which are exactly the same in each molecule i.e the base and ribose ring and the alpha and beta phosphates, are of little importance. Our discussion will focus on the G2/Switch 1 and G3 Switch 2 regions.
A closer look at Switch 1
Here you can see that the G2/Switch 1 region is in close contact with phosphate groups and magnesium ion and also occupies space near where the gamma phosphate of GTP must fit when ras is activated.A closer look at Switch 2
Here you can see that the G3/Switch 2 region is distant from the phosphate groups but not the magnesium ion. Again importantly it also occupies space where the gamma phosphate of GTP must fit when ras is activated.Switch 1 and 2 together
It is obvious Switch 1 and Switch 2 when viewed together will have to make significant changes to accommodate GTP rather than GDP.Now you can turn to GTP-bound active ras
GDP-ras has now been replaced with a model of the active form of the protein.The GTP binding site.
Again a tight fitting nucleotide-binding pocket is formed from the G1 to G5 regions seen in GDP ras. Here an analogue of GTP has been used in which the oxygen bridge between the beta and gamma phosphates has been replaced by an imino (NH) group to make a nucleotide that cannot be broken down to GDP by hydrolysis. The nitrogen atom is visible as a bright blue sphere between the beta and gamma phosphates.Simplifying the G1 to G5 loops
This view should be largely familiar to you from your earlier exploration of the GDP-bound form of ras. As you will notice the changes in the ras structure have not been very dramatic but thee subtle readjustments constitute the "signal output", the switch from "off" to "on" for this crucial signalling protein. When you run this tutorial you should look for the difference between the two forms in several representations and renderings.Identifying Switch 1
Here Switch 1 is again revealed in transparent dots.Identifying Switch 2
Switch 2 is now shown and appears to have taken a more compact form as amino acids in the loop make adjustments to accommodate and bind the gamma phosphate group.Identifying Switch 1 and 2 together
Beside making room for the extra highly charged bulk of the gamma phosphate the changes in these two switch regions are due in large part to a change in the coordination behaviour of the magnesium ion when the gamma phosphate is in place. As with the GDP form the magnesium retains coordination to two water molecules, one hydroxyl group provided by a serine in the P-loop (G1) and one to the beta phosphate. However, two coordinate bonds to structural water are lost and replaced with one bond to a hydroxyl group provided by the essential threonine in Switch I and one to the gamma phosphate of GTP.You should now repeat this tutorial to see if you can identify the changes in the switch regions between the two forms. Use mouse hover to identify and label specific residues and use the right mouse button menu to change your viewing options.