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Interaction between the SH2 domains of ZAP-70 and the tyrosine-based activation motif 1 sequence of the zeta subunit of the T-cell receptor.

Abstract：

:One of the key steps involved in T-cell activation is binding of the tyrosine kinase ZAP-70 via its two SH2 domains to peptide segments termed tyrosine-based activation motifs (ITAM) which are present in three of the T-cell receptor (TCR) subunits. The crystal structure of the ZAP-70 SH2 domains complexed to phosphopeptide revealed that the amino-terminal phosphotyrosine-binding pocket is formed at the interface between the two SH2 domains. This study was designed to further characterize the binding between TCR zeta ITAM1 and the ZAP-70 SH2 domains as well as to assess the change in conformation of SH2 domain structure upon zeta ITAM1 binding. BIAcore analysis of wild type and nonfunctional single-point mutants of ZAP-70 SH2 domains demonstrated that the amino-terminal SH2 domain can bind phosphopeptide in the absence of a functional carboxyl-terminal SH2 domain. In addition, the amino-terminal SH2 domain prefers the RREEpYDVLDK sequence of zeta chain ITAM1 over the GQNQLpYNELNL sequence. To assess changes in protein conformation upon ITAM binding to ZAP-70 SH2 domains, fluorescence spectroscopy and analytical ultracentrifugation experiments were performed. A significant blue shift in the tryptophan emission spectrum of the SH2 domains was observed in the presence of saturating amounts of phosphopeptide, indicating a loss in solvent exposure for the tryptophan residues in the protein-phosphopeptide complex. This was accompanied by changes in the frictional coefficient consistent with a compacting of the protein structure. Finally, thermal denaturation experiments showed an increase in stability and cooperativity in unfolding for the protein-phosphopeptide complex relative to the protein alone.

journal_name

Arch Biochem Biophys

journal_title

Archives of biochemistry and biophysics

authors

Labadia ME,Jakes S,Grygon CA,Greenwood DJ,Schembri-King J,Lukas SM,Warren TC,Ingraham RH

doi

10.1006/abbi.1997.0118

subject

Has Abstract

pub_date

1997-06-01 00:00:00

pages

117-25

issue

eissn

0003-9861

issn

1096-0384

pii

S0003-9861(97)90118-1

journal_volume

342

pub_type

杂志文章

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Interaction between the SH2 domains of ZAP-70 and the tyrosine-based activation motif 1 sequence of the zeta subunit of the T-cell receptor.