Signal Transduction Education
Signal Module at U-Bordeaux - Cell Transformation - Biocomputing Practical

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Signal transduction at the molecular level
The first event in EGF-mediated signaling is dimerization of its growth factor receptors.
This is followed by an inter-phosphorylation, resulting in tyrosine phosphorylated residues in the COOH-terminal region of the intracellular tyrosine kinase domain. One of these phosphotyrosines is recognized by the SH2-domain of the adaptor protein Grb2. Grb2 is linked, via its SH3 domain to the proline rich-region of the GTP exchange protein Sos (a Ras-GEF). The consequence of receptor tyrosine phosphorylation is the recruitment of Sos to the plasma membrane. Sos interacts with the membrane bound Ras and opens the nucleotide binding pocket causing the release of GDP. Due to an excess of GTP over GDP, GTP will now occupy the nucleotide binding pocket of Ras and impose a change in protein folding. This, in turn, allows Ras to interact with down stream effectors like the protein kinase Raf or the lipid kinase PI 3-kinase. The extracellular signal has been brought across the membrane and will induce a cellular response..

In this practical we will give you an introduction to the use of Web-based databases and programmes that enable you to identify the different components of the above depicted signal transduction cascade. It is a practical about “protein domains” and how these are involved in the formation of signal transduction cascades. These domains, you will discover, are omnipresent in a great variety of proteins involved in signal transduction.

1. If this material has your interest, we suggest you to read chapter 1, 2, 3, 4 and 13 of “Protein Structure” by Branden & Tooze (Garland Publishing ISBN 0-8153-2305-0 (paperback edition)
2. This practical is linked to Chapter 4 (pp71-73, pp85-95), Chap 11 and Chap 18 of the course book “Signal Transduction”

You will have to construct a slide show report of this practical. Make sure the wonderful protein-domain images that you will be creating during this practical are carefully stored (as GIF, JPG or BMP files).

-- The adaptor protein Grb2 --

Your search starts at the Protein Data Bank operated by the Research Collaboratory for Structural Bioinformatics.

To find information about Grb2, just type its name (keyword) and press search. How many structures do you find? How would you select the right one?

Select 1TZE, we now start to explore the GRB2-SH2 domain of this protein.

Click on “download files”, then click on top item “PDB file”, finally click on “Enregistrer” save the file as “Grb2-SH2-pTyr peptide 1TZE”

You next open “PyMoll” in which you open your newly saved “Grb2-SH2-pTyr peptide1TZE” file.

Start exploring the protein by identifying the number of chains. Click on “Display”, then on “Sequence”. You go back to “Display” and you then click on “Sequence Mode” and select “Chains”. You will find E I “ appearing on the black screen. The E represents the SH2 domain, the I represents the short tyrosine containing peptide (inhibitor) and the “ reprents water molecules. Bring the cursor to “B” and click on it. Then go to “select” and click on “S” and select cartoon (action 3). Then go back to “select”, click on “H” and select “lines”. You know have the structure in cartoon form. You now select chain “ I “ but you show it in “Sticks” rather than “Cartoon”. Go back to “Display”, then “Sequence Mode” and select “Residue Codes”. You get all the amino acids that are represented on the screen. Find the short peptide and select PTR (phosphotyrosine) and N (Asparagine). Click on “C” (colour), select “by element” and take the option with “CHNOS”. This will allow you to see the two important amino acids and to detect the phosphotyrosine (P in orange).

Finally, select the OOOOO’s (after you have deselect the PTR and N!) and click on “H”, select “Everything”.

When you turn the image around, which protein folds do you discern?

You can move the image around with three mouse buttons, left is “ turn”, right is “zoom in and out” and the middle is “drag” around. Once you have mastered the basic commands of PyMol and the image on the screen has given you a good impression of the interaction between SH2 and phosphotyrosine, you save your work.

To do this, try to get an as big representation as possible (cover the screen). Then click on “Ray”. Once the picture looks smooth you go to file and “Save Session” as “Grb2-SH2-pTyr image”. You next “Save Image” as “Grb2-SH2-pTyr image”.

You now will have to answer a number of questions.
For instance, how many amino acids constitutes the SH2 domain you see on the screen and what is its sequence? What is the molecular weight of an SH2 domain? How many amino acids constitute the pTyr-peptide that is bound to the SH2 domain?

A quick answer can be found by clicking on “Sequence Details” in the top navigation bar of PDB website (see below). You will also find information about the precise composition of the different protein folds (for the code of the different amino acids see document “basic protein folds.jpg” in the casier).

For a quick view of protein folds, please click here. For a reminder of what amino acids are, click here.

Is the whole protein, Grb2, represented or only a segment of the protein (compare amino acid numbers of the blue line with that of the red line in the section “mapping to UniProt entry P62993). When you click on P62993 you will get all the information of human Grb2 you will need for the next question (from UniProtKB/Swiss-Prot).

In “SwissProt” you will also find a link with databases that contain information about protein domains, their composition, how a consensus sequence was derived and how you could use these consensus sequences to predict protein domains when you only have a gene sequence. Move down to Pfam and click on Pfam graphical view of domain structure. Pfam stands for protein families database of alignment and HMMs and it provides information about the different domains that constitute the protein. This kind of information gives a first impression of the biological function of proteins.

The screen you will get (see below) informs you about the whole protein, until now we have only seen one of its domains (SH2) and you will realise that Grb2 consists of one SH2 domain and two SH3 domains. By domain analogy the image informs you about the localisation and length of each domain. For instance the first SH3 domain extents from amino acid 1 to 56, followed by a very short linker region, an SH2 domain etc.

You can continue your search by clicking on Pfam SH2 to find out more about this particular domain: its elementary structure, literature references and it offers you the possibility to study other proteins that also possess an SH2 domain (by sequence alignment studies).

When you go down to “Alignment” you will see a “Full” list of 1058 proteins (23 Nov 2004) that contain an SH2 domain. It suffices to see a seed of 58 to get an impression of the different proteins that contain such domain. Click on “Get alignment” and you will find the level of analogy of protein domains that are classified as “SH2 domain”. Realise that these sequences are not all identical but together they form the same structural folds and have highly conserved residues at the right places.

this is the Grb2 sequence for SH2

Another way to study proteins with SH2 domains is to click on “Domain Organisation” in the Pfam page (see above) and this will provide you images of domain maps in the same proteins that you have observed using the “alignment” option but this time you see other domains as well. .

If you go down the list you will come across LCK MOUSE, a proto-oncogene (meaning: can cause cell transformation but has not been discovered yet as a mutant protein in human cancers). The domain organisation shows you an SH3, SH2 and Pkinase domain. What is the role of the SH2 domain in this particular protein (check with your lecture on protein phosphorylation).

This is the end of the GRB2 SH2 exploration. Assure to have saved some crucial images and information that you may find useful for the oral presentation at the end of this course.

We repeat the session by exploring the GRB2-SH3 domain using the PDB entry 1GBQ. This sequence is bound to a proline rich peptide obtained from the GTP exchange protein Sos.

-- The GTPase Ras --

In this section you are going to study three different conformations:

1. Ras in its GDP bound state (PDB entry 1LF5), then
2. Ras in its GTP bound state (PDB entry 1LF0) and finally
3. Ras complexed with Sos (PDB entry 1BKD), the exchange factor that facilitates the release of GDP and hence the fixation of GTP.

Download the files according to the instructions presented on the first page (click on “Download/Display file” and when the image arrives on the screen you click on the right hand mouse button and save the file as “1LF5 Ras-GDP” or “1LFO Ras-GTP” or “1BKD Ras-Sos”. You next open “PyMol”and follow instructions as provided for SH2 above

In this session it is important that you get familiar with the structural changes that occur in Ras when GDP is replaced by GTP, in particular with respect to the effector loop (comprising residues 32-40). You will also get an impression of the changes brought about by Sos when comparing Ras bound to Sos with Ras bound to GDP (open1LF5 and 1BKD simultaneously).

Ras GDP (1LF5)

How many amino acids constitute Ras? The structural studies are performed with a recombinant protein, which amino acids make up this protein? (see above where to find information about proteins)
Which method has been used to determine the structure of Ras.GDP?

The conserved domains or Ras are stretches of amino acids that interact with the nucleotide GDP or GTP. These stretches are named G1-5 and consist of aa 10-17, 31-35, 57-60, 116-119 and 142-146. In order to find the conserved regions you execute the next lines of command: select 180 {enter}, you have now selected GDP, click on “Display” and select “sticks”. This will give you a bright red nucleotide. To highlight the conserved regions you type select 10-17 {enter} and you click on “Display” and “cartoon”, followed by clicking on “Colours” and select “CPK”. Repeat this for the other sequences.

Two amino acids stand out for their importance in GDP and/or Mg2+.GTP binding. Serine 17 binds both, whereas threonine 35 reinforces GTP binding only. Execute the following commands: select 17 {enter} and click on “Display” followed by “Ball and stick”. Repeat this for threonine 35.

which of the two amino acids (ser 17 or thr35) is close to GDP?

Ras GTP (1LFO)

Open Ras GTP in the same window. You can put them next to each other by pushing the right mouse button and dragging the newly imported molecule to the left (or the right). Repeat the same command lines as given for Ras GDP

which of the two amino acids (ser 17 or thr35) is close to the gamma-phosphate of GTP?
which of the two amino acids shows a strong change in position?

The effector loop, the site where for instance Raf interacts with Ras, comprises aa 32-40. The strong change in position of thr 35 demonstrates nicely the “switch-on” action of GTP-loading and the “switch-off” action of GTP-hydrolysis.

Position the two molecules such that you see the difference in orientation of the effector loop in the different states and save the image for your report. You may prefer another background, for instance grey, this can be achieved by executing the following command: type “background grey” {enter}.

Ras Sos (1BKD)

Close RasGTP and open Ras-Sos (1BKD) in the same window. Put the two molecules next to each other (right mouse button), click on “Display” and “Cartoon”, click on “Colours” and “Chain”.

How many chains are presented in this structural analysis, and how are they abbreviated?
Which stretch of amino acids of Sos is represented in this image? Which functional domain does this protein fragment contain and how many amino acids does it constitute? What is the molecular weight of the entire protein Sos and what other domains does it contain?

It may be helpful to present Sos in a transparent way so that you get an impression of where the two proteins interact but without losing sight of the molecular details of Ras. In PyMol this can be achieved by selecting Sos and then choose a sphere representation. You next click on Setting, then on Transparency and choose 80%. Whith respect to colour, choose Greys at 90%.

1. You next select :r {enter} and you select the following amino acides Asp 30 (in conserved region G2), display these amino acids in “Ball & Stick” and colour them with CPK. Repeat this exercise for Thr 35 (G2 switch1) and Ala 146 (in conserved region G5).
2. You now repeat the same exercise for Sos. First select :s {enter} and then select Glu 942 and present as “ball & stick” in CPK colour. (This amino acid binds to Ser 17 and prevents its interaction with GDP or GTP.

You next click twice on RasGDP (or you click on “file” and select 1LF5), then click on “edit” and “select all”, you now have the RasGDP protein back to its original state. This is followed by a display as cartoon and colour in CPK. Repeat the above indicated procedure for amino acids Glu 31 and Ala 146.

what is the structural difference between Ras bound to GDP or bound to Sos? How does Sos activate Ras?

Present the two Ras molecules such that you clearly see the difference between the two and safe the file on your disk (to be used for your practical project)

You now have obtained some molecular insight into the role of protein domains in the formation of a receptor signalling complex around the EGF receptor which constitutes the activation pathway of the monomeric GTPase Ras.