Laboratory of Molecular Biophysics
Next: Hydrophobic potential., Up: Protein kinases, ..., Return to: Contents.
Molecular interactions at the -chain of the T-cell receptor are responsible for initiating a number of downstream signalling events following stimulation of the receptor in response to a foreign antigen. The intracellular portion of the -chain polypeptide contains three ITAMs of sequence YXXLX7-8YXXL/I. Following T-cell activation, the tyrosines of this motif become phosphorylated allowing recruitment of the protein tyrosine kinase ZAP-70. Subsequent activation of this kinase initiates various downstream signalling pathways. Recent work has also identified the -chain in complexes formed with the HIV accessory protein Nef. Signalling from these complexes is believed to involve a different non-receptor protein kinase.
Our aim is to characterise the structural and thermodynamic basis of these two -chain interactions. Through our work on full length ZAP-70, we hope to obtain novel insights into the connection between localisation and activation for this key signalling molecule. Characterisation of the Nef interaction will hopefully offer a picture of how a promiscuous pathogenic protein can interfere with multiple host signalling pathways.
The 70kDa Zeta Associated Protein (ZAP-70) is a protein tyrosine kinase of fundamental importance to signal transduction within T-cells. The protein, expressed only in T-cells and natural killer cells , is vital for T-cell maturation and activation. This is evidenced by the fact that ZAP-70 abnormalities result in severe combined immunodeficiency (SCID) . Recognition of foreign antigens at the TCR leads to tyrosine phosphorylation of the ITAM motifs of the -chain by Src family kinases. This provides the basis for recruitment of ZAP-70 via a pair of SH2 domains which form a co-operative unit at the N-terminus of the molecule . ZAP-70 kinase activity is subsequently up-regulated through phosphorylation of its activation loop tyrosines by membrane-localised Src-family kinases including lck.
A baculovirus construct for the expression of a hexa-histidine tagged ZAP-70 fusion protein has been supplied by Roche pharmaceuticals, and used as a source of protein for preparative scale expression. Small scale (50ml) expression experiments using sf9 cells in stirrer flasks have allowed determination of the optimum MOI and time course for in-house expression.
The Roche construct is histidine-tagged at the N-terminus to allow for metal affinity purification. Subsequent gel-filtration on an analytical scale Sephadex 200 column demonstrates a very clean profile for all our in-house preparations with a single large, symmetrical peak corresponding to the ZAP-70 monomer being observed in each case (Figure 1). A number of small, broad peaks are usually observed at retention values suggestive of larger contaminants or aggregates, but these are easily resolved from the principal ZAP-70 fractions.
Protein produced in this manner exhibits considerably more activity than truncated kinase domain constructs produced in-house by Roche. Furthermore, the behaviour in gel-filtration indicates that the purified protein is compact, globular, non-aggregated and predominantly monomeric.
Attempts have also been made to use the ZAP-70 SH2 domains in affinity purification. Expression of a GST-ITAM fusion in a bacterial strain co-expressing the broad specificity elk tyrosine kinase has produced the ITAM as multiple phosphorylated variants. These expression products, when immobilised on glutathione sepharose, have been shown to be effective in an affinity purification of ZAP-70 (Figure 2), although work is still required to optimise expression of the doubly phosphorylated ITAM.
Sufficient quantities of protein have been available for preliminary screening of ZAP-70 crystallisation conditions. Concentration of the purified protein to 12mg/ml followed by a limited screen of 24 conditions at 20oC produced only precipitate. A second screen, conducted at a lower protein concentration (6mg/ml) and temperature (4oC) over the full range of the Hampton Research Crystal Screen I and II conditions, produced a broad spectrum of protein phases and indicated that the ZAP-70 was not exhibiting non-specific, concentration- or time-dependent aggregation. A number of very encouraging, apparently microcrystalline phases were observed.
The nef gene of human and simian immunodeficiency viruses is crucial for progression of the viral infection to the onset of the Acquired Immunodeficiency Syndrome (AIDS). The gene product is an accessory protein that is expressed early in the viral life cycle. The means by which nef increases HIV virulence and viral titre is still a subject of much research. The protein lacks enzymatic activity although a number of protein binding partners have been identified. It is thus thought to be through formation of complexes that Nef exerts its influence. Recent evidence indicates that a complex based at the -chain of the TCR plays a significant role in Nef action [4-7]. This complex, believed to comprise Nef, PAK, VAV and rac, is formed in association with -chain ITAM sequences. Downstream signalling from the complex has been proposed to result in cytoskeletal rearrangements and activation induced cell death/apoptosis .
HIV-1 SF2 Nef has been cloned into the pGEX-6P-2 expression vector both as a full length construct and in a truncated form lacking the first 60 N-terminal residues and the C-terminal cysteine. This truncated form is designed on the basis of previous crystal structures to provide a core domain amenable to crystallisation. The truncated construct has been expressed as a glutathione-S-transferase (GST) fusion in E.coli and purified on glutathione sepharose. The GST tag can be cleaved from the purified fusion using a 3C viral protease (Figure 3A) and the free Nef purified by gel-filtration on a superdex-75 column (Figure 3B).
This protein has been concentrated to 5mg/ml for use in co-crystallisation experiments with a doubly phosphorylated peptide corresponding to ITAM 1 of the human TCR -chain. A number of microcrystalline phases have been produced together with several protein pseudo-crystals (Figure 4). Although these do not diffract, initial results confirm that they are solid and derived from protein rather than salt. Screening around these conditions will be an early priority of the project.
1. Chan, A.C., Iwashima, M., Turck, C.W., and Weiss, A., ZAP-70: a 70 kd protein-tyrosine kinase that associates with the TCR zeta chain. Cell, 1992. 71(4), 649-62.
2. Chan, A.C., Kadlecek, T.A., Elder, M.E., Filipovich, A.H., Kuo, W.L., Iwashima, M., Parslow, T.G., and Weiss, A., ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency. Science, 1994. 264(5165), 1599-601.
3. Hatada, M.H., Lu, X., Laird, E.R., Green, J., Morgenstern, J.P., Lou, M., Marr, C.S., Phillips, T.B., Ram, M.K., Theriault, K., et al., Molecular basis for interaction of the protein tyrosine kinase ZAP-70 with the T-cell receptor. Nature, 1995. 377(6544), 32-8.
4. Bell, I., Ashman, C., Maughan, J., Hooker, E., Cook, F., and Reinhart, T.A., Association of simian immunodeficiency virus Nef with the T-cell receptor (TCR) zeta chain leads to TCR down-modulation. J Gen Virol, 1998. 79(Pt 11), 2717-27.
5. Howe, A.Y., Jung, J.U., and Desrosiers, R.C., Zeta chain of the T-cell receptor interacts with nef of simian immunodeficiency virus and human immunodeficiency virus type 2. J Virol, 1998. 72(12), 9827-34.
6. Xu, X.N., Laffert, B., Screaton, G.R., Kraft, M., Wolf, D., Kolanus, W., Mongkolsapay, J., McMichael, A.J., and Baur, A.S., Induction of Fas ligand expression by HIV involves the interaction of Nef with the T cell receptor zeta chain. J Exp Med, 1999. 189(9), 1489-96.
7. Schaefer, T.M., Bell, I., Fallert, B.A., and Reinhart, T.A.,
The T-cell receptor zeta chain contains two homologous domains with which
simian immunodeficiency virus nef interacts and mediates down- modulation.
J Virol, 2000. 74(7), 3273-83.
Next: Hydrophobic potential., Up: Protein kinases, ..., Return to: Contents.