Interactive Protein Tutorial
Sequence and structure of domains that bind phosphotyrosine-containing peptides.
This tutorial considers two protein modules that can bind phosphotyrosine. Although we will concentrate on the SH2 (src homology 2) domain we will also examine the PTB (phosphotyrosine binding domain) as both are important modules in signal transduction. You should note the differences in the architecture of these two modules.
We will consider an SH2 domain first using the Lck- SH2 domain (PDB = 1LCJ) as an example. Remember that Lck is a non-receptor tyrosine kinase of the Src superfamily and that it has a great deal in common with similar domains from other members of this family. However, as described in your lecture notes the function of the different SH2 domains, described in terms of their selectivity and affinity for different ligands, is a direct function of substitutions of particular amino acids at key sites in the SH2 domain structure.
The SH2 domain:
With this button you can reveal the secondary structural elements within the wireframe model. Note that this domain is built from a central anti-parallel beta sheet flanked on either side by two alpha helices. The gaps between either side of the beta sheet and an alpha helix represents two distinct binding pockets. The tops and bottoms of these pockets are described by loops in the structure. Because there are two pockets, each capable of recognising one distinct feature in the complimentary ligand, the binding of some peptide ligands is said to fit a "plug and socket" model with the SH2 domain representing the socket. For the rest of this tutorial it will be useful first time around to adopt a standard orientation. Stop the rotation (you can restart it later as you like) and by taking note of the position of the smallest beta strand position the molecule until it look something like this.The phosphopeptide ligand
We can add a peptide ligand to demonstrate this. Notice that the ligand is bound in an extended conformation and that it lies orthogonal to the central beta sheet.The phosphotyrosine residue
All high affinity peptide ligands for SH2 domains contain phosphotyrosine residues. The pTyr residue in the ligand can be revealed with this button, the amino acid side chain is spacefilled and coloured grey whilst the phosphate group is "cpk" coloured. You can see clearly that this group fits into one pocket on the side of the central beta sheet. The top of the pocket is marked by a loop between beta 1 and beta 2. Make sure that you can identify the strands, helices and loops in the secondary structure. For your convenience the N-terminal has been coloured blue. Take careful note of the secondary structure. In this representation of this LCK-SH2 domain, there is a marked difference from that found in many papers and some text books describing SH2 domains. We see only four beta strands but descriptions often refer to five. This is because the beta-D strand has been "welded" seamlessly onto the end of the beta-C strand making one long beta-C strand. In a conventional representation the longest strand, beta-C, running from G58 to L69 would be broken somewhere around K65 to R67 to give a small beta-D strand that would then lie anti-parallel to the final small beta strand G73 to Y75.Acid side chains
You will remember from your lecture notes that the Lck-SH2 domain is said to bind ligands of type Group IA. In other words, it strongly selects the consensus "pY-psi-psi-phi" where psi is an acidic and phi an aliphatic hydrophobic amino acid. Here we can reveal the two acidic residues at Y+1 andY+2 and colour them red. Note that because the peptide is in an extended conformation that the side chains "point" in opposite directions and run parallel to the edge of the central beta sheet.Lastly, we can add the Y+3 aliphatic hydrophobic amino acid, in this case isoleucine. The consensus sequence of the peptide ligand is therefore pYEEI, the classical Src-SH2 binding peptide. Note that this residue is almost completely buried in a pocket formed by the central beta sheet, the β-helix and two loops, one proceeding the helix and one following it and running out to the C-terminal.
The pY+3 residue
Since all peptides that bind strongly to SH2 domains will contain a phosphotyrosine residue, the pTyr binding pocket contains little information on peptide selectivity besides an affinity for pTyr. However, it will contain key basic residues, like arginine, to co-ordinate the negatively charged phosphate group. These may be mutated to produce loss-of-function mutants e.g. β-B5, in PLC-γ1 SH2 domains. This is because the edge of the beta sheet forming part of the pY+3 binding pocket contains most of the "information" that codes for selectivity for pYEEI over other ligands, for example pY-X-psi-X of group III ligands. We shall now look at how this selectivity is achieved by the rest of the SH2 domain including, the pY+3-binding pocket, in more detail. It is a good exercise to cross-reference these features to the primary and secondary structure of Fyn-SH2 domain.Binding the Y+1 residue
Firstly, we will examine key residues on the edge of the beta-sheet. Using this button you can reveal lysine 179 and see that the positively charged side chain is in close contact with the negatively charged pY+1 glutamate of he phosphopeptide ligand.βD5
Use this button to add tyrosine 181, this is the beta-D5 residue that enforces a strong selectivity for peptides of Group I peptide ligands. You will remember from your lecture notes that substitution of this residue can switch the selectivity of the SH2 domain to Group III ligands. Although it is not shown here the residue between K179 and Y181 is histidine 180. You will remember that the geometry of extended conformations (beta strands) means that this residue "points" in the opposite direction from its neighbours. Consequently the positively charged H180 sits next to the phosphate group of the peptide pTyr residue and so helps to form the cationic pocket that localises and selects this part of the ligand.Binding the Y+2 residue
This button will show you R184 which makes an electrostatic bond to the pY+2 glutamate of the ligand. Taken together with K179 you should now appreciate why peptides with glutamate at pY+1 and +2 are strongly selected by SH2 domains of this class. We can now start to build the predominantly hydrophobic pocket that selects isoleucine at pY+3.Insert I193
Insert S194
which is not hydrophobic, but which packs it's non-polar methylene group against the side chain of the ligand isoleucine.Insert Y209
Insert leucine 216
to complete the pocket.You can now display the ligand pY+3 isoleucine residue as a transparent green surface. This will help you to see that this residue sits in a tight pocket built from four hydrophobic amino acids and the non-polar part of a fifth residue.
We can now examine the PTB domain of Shc. Display the structure of the domain with this button. You will notice immediately that PTB domains have an entirely different architecture from SH2 domains and are the same fold as PH domains.
Here you can add the phosphopeptide ligand in ball and stick representation. Note that this ligand binds at a different site from that which would be occupied by inositol phosphates or lipids if this was a PH domain. You will also notice that that the peptide is bound in an extended conformation parallel to the large helix that packs against one "open end" of the central beta barrel. Note also that the peptide makes a sharp turn near one end of this helix.
If you add the phosphotyrosine residue (shown in cpk colours). Here a stable analogue of a phosphate residue, a thiophosphate group, has been incorporated, hence the large sulphur atom is visible instead of an oxygen. Note that this "phosphotyrosine" is near the sharp turn in the ligand peptide and packs against the side of the beta-barrel and between loops. These loops contain residues that select for the phosphorylated forms of suitable peptide ligands.
MENUS
These displays were created by providing JMol with a series of scripts (lists of commands). Many of these commands can be applied directly. Press the button opposite to reset to ball and stick mode, then place the cursor over the display and press the right mouse button.
Rendering Options: (if Jmol isn't working, try JSmol!)