Guided example 2: does sheep ZAP70 have an active tyrosine kinase?

Scenario

Melissa is working on a drug discovery programme to create new inhibitors of ZAP70 for use in autoimmune disease. Greg, a medicinal chemist in her group,  has synthesised a series of protein tyrosine kinase inhibitors. She needs to devise a cellular assay to see which ones inhibit the ZAP70 tyrosine kinase. She has ready access to primary T cells from the following animals:

  • Mouse
  • Rat
  • Sheep

To decide which of her models has a ZAP70 protein most closely related to the human one, she has decided to do a quick multiple sequence alignment. Using EBI search, she located the canonical sequences (isoform 1) for the human, mouse, rat and sheep ZAP70 proteins. For each one, she downloaded the protein sequence in FASTA format. She then performed a multiple sequence alignment of these proteins using the Clustal Omega tool. You can repeat her experiment if you like.

On the basis of this alignment, Melissa has decided to use sheep T cells for her assay but she wants to double check that sheep ZAP70 has an active tyrosine kinase domain before wasting time on developing an assay. For this we’re going to use a tool called InterProScan. This tool compares your chosen sequence with all the sequences in InterPro, a data resource that provides functional analysis of protein sequences by classifying them into families and predicting the presence of domains and important sites. If you’d like to learn a bit more about InterPro before continuing, you can read the InterPro Quick Tour or follow the InterPro tutorial. Go straight to the section on sequence searching if you’re short of time.

Step 1 - Searching for the ZAP70 sequence on InterProScan

Go to the InterProScan webpage and paste your sequence into the search box, or copy and paste it from here (Figure 17):

>tr|W5PW03|W5PW03_SHEEP Non-specific protein-tyrosine kinase (Fragment) OS=Ovis aries GN=ZAP70 PE=4 SV=1
PRPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHEVRFHHFPI
ERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRKPCNRPSGLEPQPGVFDNLRDA
MVRDYVRQTWKLEGEALEQAIISQAPQVEKLIATTAHERMPWYHSSLTREEAERKLYSGS
QTDGKFLLRPRKEPGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEYLKL
KADGLIYCLKEACPNSSASSGAAAPTLPAHPSTFTQPQRRIDTLNSDGYTPEPVRLVSSE
KPRTMPMDTSVYESPYSDPEELKNKKLFLKRENLLMADIELGCGNFGSVRQGVYRMRKKQ
IDVAIKVLKQSTEKGDKDEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLH
KFLVGKKEEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLS
KALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEAFSYGQKPYKKM
KGPEVMAFIEQGKRMECPPEC

Click on the search button. Your search will take some time to run; be patient.

Pasting in the sequence into the InterProScan search box

Figure 17 Pasting in a sequence into the InterProScan search box.

Step 2 - Interpreting the search results

When your search is complete, your search results will look like this (Figure 18).

Search results for ZAP70 on InterProScan

Figure 18 Search results for ZAP70 on InterProScan.

At the top of the results, you will see the reassuring news that sheep ZAP70 is indeed a member of the tyrosine protein kinase family (A), and that it has two SH2 domains and a protein tyrosine kinase domain. However, some members of this family have mutations in the active site (B), or in the ATP-binding site, that render them enzymatically inactive. In this case, however, we can see that sheep ZAP70 contains the protein profile for both the ATP-binding site and the active site. Melissa should be safe to use sheep cells for her assay!