Interactive Protein Tutorial

A case study of the Src tyrosine kinase.

This tutorial allows you to explore some of the protein structural features of the Src tyrosine kinase (pp60 cSrc) and by implication therefore also the other members of this family. It also provides the opportunity for you unify the insights gained from the other tutorials on kinase domains, SH2 domains and SH3 domains. You should remember from your lectures that Src family kinases are composed of three principal domains; SH1 - Src Homology 1 - which is a kinase domain, SH2 - Src Homology 2 - a phosphotyrosine binding domain and SH3 - Src Homology 3 - which binds type 2 polyproline helices.

For independent study the original PDB entry is 1FMK.

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).

Src has a "clumpy" looking structure and does not appear compact

As you can see from the ball and stick representation the Src kinase is a complex structure that in some orientations appears somewhat similar to a flat, square box. With a little more observation it is also apparent that the protein appears to be built from four discrete clumps of "structure" - one in each quadrant of the flat, square box. When you are satisfied that you can visualise this use this button to change the rendering to cartoon form. You might then like to orientate the Src protein in a fashion similar to that shown in your lecture slides. If you stop the rotation you should see that these three clumps are the kinase small lobe (mostly beta structure), the kinase large lobe (mostly alpha structure, with a central 4 helix bundle), an SH2 domain and an SH3 domain lying close to a centrally-located, extended (white) chain with a couple of prominent twists in it.

Viewing the Src kinase domain

Use this button to hide the SH2 and SH3 domains. You should now see that the "centrally-located, extended (white) chain with a couple of prominent twists in it" is the long linker region familiar from the tutorial on the kinase domains. However in Src it is not a prominent alpha helix but a structure that in many ways resembles a type 2 polyproline helix even though its sequence does not conform to the expected consensus for these structures (it has only one proline). You can verify this by hovering your mouse over the structure and waiting for residue labels to appear. In addition you can also see the long C-terminal tail of Src protruding from the large lobe of the kinase domain.

The C-terminal inhibitory phosphorylation site

Src is ordinarily found in its inactive, auto-inhibited form - this is the structure we are exploring here. Src family tyrosine kinases are inactive because their C-terminal tails are phosphorylated at a tyrosine residue by Csk for example. Until this inhibition is released other activating events like orientation of the catalytic site and activation loop (auto)phosphorylation cannot follow. This button reveals the location of this C-terminal phosphorylation site and displays the phosphotyrosine residue in space-fill format. This button has also changed the display of the linker region running along the back of the kinase domain. You can re-orientate the molecule so that you can look along this linker structure from one end and see the tightly-wound triangular cross-section of the helix structure that it adopts.

The SH2 domain interacts with the phosphorylated tail

Here you can reveal the SH2 domain and see that it is bound to the Src C-terminal tyrosine phosphorylation site. As expected the phosphotyrosine residue occupies the pY pocket. You may remember that the consensus sequence for optimal binding to this SH2 domain is pYEEI. However in the C-terminal peptide the sequence is pYQPG - there are no acid residues for favourable interactions at pY+1 and +2 nor an aliphatic hydrophobic residue to occupy the pY+3 pocket. (You can verify this sequence by letting your mouse rest over the residues of the tail - a label will appear after a few moments.) As a result this interaction obstructs the SH2 domain binding site but with only low affinity - the interaction is unstable with a high off-rate (short "half-life"). Because the interaction between the SH2 domain and tail is intra-molecular there is a high on-rate which is why the C-terminal tail is an effective competitive inhibitor of the binding of other phospho-peptides to the SH2 domain - the local concentration of the low affinity inhibitor is effectively very high!

The SH3 domain binds to the back of the kinase domain

You can now hide the SH2 domain and reveal the SH3 domain. It should be obvious that the SH3 domain is interacting with the tightly wound linker helix which lies along the back of the kinase domain. The interaction between these two components is similar to that described in detail in the SH3 domain tutorial. However as with the SH2 domain the interaction is not very strong because sequence of the linker, and therefore the geometry adopted, is not optimal. Again this leads to a situation where a fast off-rate is balanced by a fast on-rate arising from the intra-molecular nature of the association. What is not shown yet is the consequences of the interaction of the SH3 domain with the linker. Steric clashes are created between the SH3 domain and the kinase domain that inhibit the catalytic activity. These include an induced twist between the two lobes of the kinase domain and dislocation of the "PSTAIRE-like" or "C-like " helices familiar from your lectures on CDKs and PKA.

The SH2 and SH3 domains pack against each other and stabilise their orientations

Use this button to reveal the SH2 domain once more. You should now be able to see how the SH2 and SH3 domains pack against one another when Src is organised in this compact and auto-inhibited form. If you trace the peptide chain between the SH3 and SH2 domains it should be apparent that this is relatively short and provides little conformational flexibility. As a result when either the SH2 or SH3 domains has engaged its intra-molecular binding site the other domain is positioned appropriately to interact with its own target. As a consequence the weak interactions of the two domains are converted into on much stronger combined interaction â€“ the effect is near multiplicative on the combined dissociation rate, rather than additive, much like the avidity effect seen in IgG binding to antigens.

The tight packing of domains against each other enforces inhibition

Lastly you can now show the structure of Src in space fill mode. This will reveal that the Src protein is indeed a compact structure with each domain packed tightly against the next. You should now be able to appreciate how the steric clashes between the SH3 and kinase domains occur. In addition the close positioning of SH3 and SH2 domains against each other is clearly visible.

As a final exercise change the representation of Src so that you can view both the polypeptide chain and the molecular surface. Use you right mouse button (Select > All; Style > Structures > Backbone; Surfaces > Molecular Surface). Does this give you a better appreciation of the compact nature of the inactive Src kinase?