PDBsum entry 1b47

Signal transduction

PDB id: 1b47

Contents

Protein chains

304 a.a. *

Metals

_CA ×3

Waters ×666

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Structure of the amino-Terminal domain of cbl complexed to its binding site on zap-70 kinase.

Authors

W.Meng, S.Sawasdikosol, S.J.Burakoff, M.J.Eck.

Ref.

Nature, 1999, 398, 84-90. [DOI no: 10.1038/18050]

PubMed id

10078535

Abstract

Cbl is an adaptor protein that functions as a negative regulator of many signalling pathways that start from receptors at the cell surface. 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 potential. In T cells, Cbl-N binds to the tyrosine-phosphorylated inhibitory site of the protein tyrosine kinase ZAP-70. 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-hand calcium-binding domain, and a divergent SH2 domain 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.

Figure 1.
Figure 1: Cbl domain structure and sequence comparisons. a, Ribbon diagram of unliganded Cbl-N. The N-terminal 4H domain is coloured yellow, the EF-hand domain green, and the SH2 domain blue. Secondary-structure elements are labelled A– D in the 4H domain and by established conventions for the EF-hand and SH2 domains. The bound Ca^2+ ion is indicated by a red sphere. Arginine 294 is universally conserved in SH2 domains and participates in phosphotyrosine coordination. b, Diagram of c-Cbl domain structure. The Cbl-N region and adjacent RING finger domain are conserved in all Cbl homologues. The C-terminal region, which contains proline-rich segments and tyrosine phosphorylation sites, is more variable and is completely absent in D-Cbl. A putative leucine zipper has been found near the C terminus of Cbl. c, Aligned sequences of the Cbl-N portion of human c-Cbl, human Cbl-b, Drosophila D-Cbl, and Sli-1. Residues that are identical in at least three of the sequences are shaded yellow. Secondary-structure elements are shown above the sequence and are coloured as in a and b. Black squares indicate residues that coordinate calcium. Red circles mark residues that interact with the bound ZAP-70 peptide. d, Structure-based sequence alignment of Cbl and Lck^23 SH2 domains. Seventy structurally equivalent residues are shaded yellow; -carbons of these seventy residues superimpose with an r.m.s.d. of 1.47 Å. The secondary-structure elements that are present in Lck and other SH2 domains, but not in the Cbl SH2 domain, are indicated by open boxes. e, Superposition of the Cbl SH2 domain (blue) with the Lck SH2 domain (yellow). The structural elements that are absent in the Cbl domain are red.

Figure 3.
Figure 3: Structure of the Cbl-N / ZAP-70 pY292 complex. a, Stereo diagram showing an -carbon trace of the complex. The bound ZAP-70 phosphopeptide is shown in magenta. b, Stereo diagram showing the interactions with the ZAP-70 phosphopeptide. The bound peptide is shown in white. Red spheres represent ordered water molecules that bridge Cbl-N and the bound peptide. Thin blue lines represent hydrogen bonds. In the phosphotyrosine pocket, Tyr 274 in Cbl makes an 'edge-face' interaction with the phosphotyrosine ring, and its hydroxyl group hydrogen-bonds to the carbonyl oxygen of Gly 291 in the ZAP-70 peptide. An arginine residue found in this position in most SH2 domains makes an 'amino–aromatic' interaction with the phosphotyrosine ring and also hydrogen-bonds with the carbonyl of the pY-1 residue of the bound peptide^8. C-terminal to the phosphotyrosine, the proline at position pY+4 in the ZAP-70 peptide binds in a hydrophobic cleft formed by Tyr 307, Phe 336 and Tyr 337, and the glutamic acid residue at pY+3 hydrogen-bonds with the backbone amide of His 320. c, Superposition of the liganded (yellow) and unliganded (blue) Cbl-N structures reveals a shift in the position of the SH2 domain upon phosphopeptide binding. The conformation of the 4H and EF-hand domains is essentially identical in the two structures. In the absence of phosphopeptide, the SH2 domain makes little contact with the 4H domain and its position is likely to vary, as we observe slightly different conformations among the three molecules in the asymmetric unit. Phosphopeptide binding induces a domain 'closure', in which the SH2 domain rotates to pack against the helical domain, completing the phosphotyrosine-binding pocket, as in d. d, Molecular surface representation of the Cbl-N domain, coloured by domain. The 4H domain (yellow) forms a portion of the phosphotyrosine-binding pocket. Residues 289–297 of the bound ZAP-70 phosphopeptide are shown as a stick model. The three N-terminal residues in the peptide are disordered and are not included. In the liganded structure, about 1, 200 Å^2 of the SH2 domain is buried as a result of interaction with the other two domains; 500 Å^2 is buried in the interface with the 4H domain, and 700 Å^2 is buried in the interface with the EF hand. The 4H and EF-hand domains share a solvent-excluding interface of 800 Å^2.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (1999, 398, 84-90) copyright 1999.