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Structure of the amino-terminal domain of Cbl complexed to its binding site on ZAP-70 kinase

Nature volume 398, pages 84–90 (1999)Cite this article

Abstract

Cbl is an adaptor protein that functions as a negative regulator of many signalling pathways that start from receptors at the cell surface1,2,3,4. The evolutionarily conserved amino-terminal region of Cbl (Cbl-N) binds to phosphorylated tyrosine residues and has cell-transforming activity. Point mutations in Cbl that disrupt its recognition of phosphotyrosine also interfere with its negative regulatory function and, in the case of v-cbl, with its oncogenic potential5. In T cells, Cbl-N binds to the tyrosine-phosphorylated inhibitory site of the protein tyrosine kinase ZAP-706. Here we describe the crystal structure of Cbl-N, both alone and in complex with a phosphopeptide that represents its binding site in ZAP-70. The structures show that Cbl-N is composed of three interacting domains: a four-helix bundle (4H), an EF-hand7 calcium-binding domain, and a divergent SH2 domain8 that was not recognizable from the amino-acid sequence of the protein. The calcium-bound EF hand wedges between the 4H and SH2 domains and roughly determines their relative orientation. In the ligand-occupied structure, the 4H domain packs against the SH2 domain and completes its phosphotyrosine-recognition pocket. Disruption of this binding to ZAP-70 as a result of structure-based mutations in the 4H, EF-hand and SH2 domains confirms that the three domains together form an integrated phosphoprotein-recognition module.

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Figure 1: Cbl domain structure and sequence comparisons.
Figure 2: Stereo diagram showing the experimental electron density map in the region of the calcium-binding site.
Figure 3: Structure of the Cbl-N / ZAP-70 pY292 complex.
Figure 4: Mutations in the phosphotyrosine-binding pocket, the calcium-binding site and the 4H domain disrupt recognition of ZAP-70 by Cbl-N.

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Acknowledgements

We thank C. Dahl for synthesis and purification of the ZAP-70 phosphopeptide, the staff at MacCHESS for assistance with data collection, and S. Harrison and T. Roberts for comments on the manuscript. M.J.E. is a recipient of a Burroughs–Wellcome Career Award in the Biomedical Sciences. Diffraction data were recorded at the Cornell High Energy Synchrotron Source (CHESS), which is supported by grants from the NSF and NIH.

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Authors and Affiliations

  1. Departments of Biological Chemistry and Molecular Pharmacology and Cancer Biology,

    Wuyi Meng & Michael J. Eck

  2. Departments of Pediatrics, and Departments and Oncology, Harvard Medical School, Dana-Farber Cancer Institute, 44 Binney Street, Boston, 02115, Massachusetts, USA

    Sansana Sawasdikosol & Steven J. Burakoff

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  1. Wuyi Meng
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Correspondence to Michael J. Eck.

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Meng, W., Sawasdikosol, S., Burakoff, S. et al. Structure of the amino-terminal domain of Cbl complexed to its binding site on ZAP-70 kinase. Nature 398, 84–90 (1999). https://doi.org/10.1038/18050

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