Human Cbl-c (Cbl-3) TKB domain in complex with EGFR pY1069 peptide

Target ID:

CBLCA

Aliases:

CBL-3CBL-SLCBLCRNF57

Deposition Date:

Tuesday 31st August 2010

Families:

Signalling proteinsUbiquitin Related BiologyUbiquitylation - E3 Ligases

Structure type:

apo

Download Coordinates:

3OP0

Authors:

Chaikuad, A., Guo, K., Cooper, C., Ayinampudi, V., Krojer, T., Ugochukwu, E., Muniz, J.R.C., Vollmar, M., Canning, P., von Delft, F., Arrowsmith, C.H., Weigelt, J., Edwards, A.M., Bountra, C., Bullock, A.

Site:

Oxford

3OP0

tabs

Structure Details

Cbl-c, also known as Cbl-3 or RNF57, is the most diverse of the three mammalian Cbl proteins (for Casitas B-lineage lymphoma). The founding member Cbl was discovered first as the oncogenic protein v-Cbl, a Gag-fusion transforming protein of Cas NS-1 retrovirus, which causes pre- and pro-B lymphomas in mice. Both Cbl (or c-Cbl) and Cbl-b are widely expressed and share a common protein as well as gene structure. Cbl-c shows 50% sequence identity to the amino-terminal sequences of Cbl and Cbl-b, but lacks the leucine zipper motif and most of the proline-rich domain in the C-terminus. In this respect, Cbl-c structurally most resembles the C. elegans and Drosophila Cbl proteins. The N-terminus of the Cbl proteins is composed of a tyrosine kinase-binding (TKB) domain, also called phosphotyrosine binding (PTB) domain, a short linker region and the RING-type zinc finger. The TKB domain mediates interaction with other proteins and consists of a four-helix bundle (4H), a calcium-binding EF hand and a divergent SH2 domain (Swaminathan and Tsygankov, 2006), (Kim et al., 1999).

Cbl family proteins function as E3 ubiquitin ligases. The founder member Cbl is best known for its regulation of EGFR. Less is known about Cbl-c, but activated Src is one of its substrates, whose ubiquitination results in degradation and suppression of Src induced transformation (Kim et al., 2004). Src phosphorylation of Tyr-341 in Cbl-c decreases its binding affinity for the E2 protein UbcH5b, resulting in more rapid E2 turnover and increased Cbl-c E3 ligase activity (Swaminathan and Tsygankov, 2006), (Ryan et al., 2010). A role for Cbl-c in the control of neuron sensitivity to neurotrophic factors has also been recently described. Cbl-c acts as a switch that is triggered by the CD2-associated protein (CD2AP) to regulate protein levels of the tyrosine kinase receptor Ret, which is the receptor for the neurotrophic factor (GDNF) family of ligands critical for nervous system development and maintenance (Tsui and Pierchala, 2008).

Cbl-c is specifically expressed in epithelial cells of a variety of tissues, in particular the digestive tract, trachea, prostate, adrenal gland, and salivary gland. Despite this distinct expression pattern, knockout mice of Cbl-c do not display any apparent phenotype and show normal proliferation of epithelial cells in the epidermises and gastrointestinal tract (Griffiths et al., 2003).

Here we report the crystal structure of the N-terminal TKB domain of Cbl-c in complex with EGFR pY1069 peptide refined at 2.5 Å resolution.

Materials and Methods

Entry Clone Source: MGC

Entry Clone Accession: BC028915

SGC Construct ID: CBLCA-c012

GenBank GI number: gi|195927027

Vector: pNIC-CTHF. Details [ PDF ]; Sequence [ FASTA ] or [ GenBank ]

Amplified construct sequence:
TTAAGAAGGAGATATACTATGGGGCGACAG
TGGGAAGAGGCCCGCGCCCTGGGCCGGGCA
GTCAGGATGCTGCAGCGCCTAGAAGAGCAA
TGCGTCGACCCCCGGCTGTCCGTGAGTCCC
CCTTCGCTGCGGGACCTGCTGCCCCGCACA
GCGCAGCTGCTTCGAGAGGTGGCCCATTCT
CGGCGGGAGGCCGGCGGAGGCGGCCCCGGG
GGTCCCGGCGGCTCTGGGGACTTTCTACTC
ATCTACCTGGCCAATCTGGAGGCCAAGAGC
AGGCAGGTGGCCGCGCTGCTGCCTCCCCGG
GGCCGAAGGAGTGCCAACGACGAGCTCTTC
CGGGCGGGCTCCAGACTCAGGCGACAGCTG
GCCAAGCTGGCCATCATCTTCAGCCACATG
CACGCAGAGCTGCACGCACTCTTCCCCGGG
GGAAAGTACTGTGGACACATGTACCAGCTC
ACCAAGGCCCCCGCCCACACCTTCTGGAGG
GAAAGTTGCGGAGCCCGGTGTGTGCTGCCC
TGGGCTGAGTTTGAGTCCCTCCTGGGCACC
TGCCACCCTGTGGAACCAGGCTGCACAGCC
CTGGCCTTGCGCACCACCATTGACCTCACC
TGCAGCGGGCACGTGTCCATCTTCGAGTTC
GACGTCTTCACCAGGCTCTTTCAGCCATGG
CCAACACTCCTCAAGAACTGGCAGCTCCTG
GCAGTCAACCACCCAGGCTACATGGCCTTC
CTCACCTATGATGAGGTCCAAGAGCGTCTG
CAGGCCTGCAGGGACAAGCCAGGCAGTTAC
ATCTTCCGGCCCAGCTGTACTCGCCTGGGG
CAGTGGGCCATCGGCTATGTGAGCTCAGAT
GGCAGCATCCTGCAGACCATCCCTGCCAAC
AAACCCCTGTCCCAGGTGCTCCTGGAGGGA
CAGAAGGACGGCTTCTACCTCTACCCAGAT
GGAAAGACCCACAACCCAGACCTGACTGAG
CTCGGCGCAGAGAACCTCTACTTCCAATC

Note a mutation was identified by DNA sequencing with the codon change from GCG to GAG (shown bold) resulting in the amino acid change A64E

Final protein sequence (tag sequence in lowercase):
MGRQWEEARALGRAVRMLQRLEEQCVDPRL
SVSPPSLRDLLPRTAQLLREVAHSRREAGG
GGPGGPGGSGDFLLIYLANLEAKSRQVAAL
LPPRGRRSANDELFRAGSRLRRQLAKLAII
FSHMHAELHALFPGGKYCGHMYQLTKAPAH
TFWRESCGARCVLPWAEFESLLGTCHPVEP
GCTALALRTTIDLTCSGHVSIFEFDVFTRL
FQPWPTLLKNWQLLAVNHPGYMAFLTYDEV
QERLQACRDKPGSYIFRPSCTRLGQWAIGY
VSSDGSILQTIPANKPLSQVLLEGQKDGFY
LYPDGKTHNPDLTELGAENLYFq*shhhhh
hdykddddk
^ TEV cleave site

Tags and additions: C-terminal TEV-cleavable (at *) his-tag with the following sequence: Q*SHHHHHHDYKDDDDK

Host: BL21 (DE3)R3-pRARE2 (Phage resistant strain)

Growth medium, induction protocol: 10ml of overnight culture from grown from a glycerol stock and finally added to 1L LB flasks with 50mg/ml of Kanamycin and 34mg/ml of chloramphenicol (total 12L). The cells were cultured at 37°C until the OD reached 0.8 and then decreased to 18°C. IPTG was added at 0.2mM (final concentration) for induction at 18°C overnight.
Binding buffer: 500 mM NaCl, 5% Glycerol, 50 mM HEPES pH 7.5, 30 mM Imidazole. Complete Protease Inhibitor Cocktail Tablets (Roche) were added (one tablet/50ml buffer).
Extraction buffer, extraction method: The cells were harvested by centrifugation at 4,000 g for 10 min. The pellet from 1 L culture was resuspended in 25 ml of extraction buffer. The sample was lysed by sonication and then centrifuged at 37505 g for 30 minutes. The supernatant was kept for further purification.

Column 1: Ni-sepharose

Column 1 Buffers:
Binding buffer: 500 mM NaCl, 5% Glycerol, 50 mM HEPES pH 7.5, 30 mM Imidazole.
Washing Buffer: 500 mM NaCl, 5% Glycerol, 50 mM HEPES pH 7.5, 30 mM Imidazole.
Elution Buffer: I: 500 mM NaCl, 5% Glycerol, 50 mM HEPES pH 7.5, 60 mM Imidazole.
Elution Buffer II: 500 mM NaCl, 5% Glycerol, 50 mM HEPES pH 7.5, 125 mM Imidazole.
Elution Buffer III: 500 mM NaCl, 5% Glycerol, 50 mM HEPES pH 7.5, 250 mM Imidazole.

Column 1 Procedure: The column was packed with 6ml of Ni-sepharose slurry and equilibrated with 15ml of binding buffer. The supernatant was loaded onto the column and the flow through was collected. The column was washed sequentially with 10ml of washing buffer followed by a second 20 ml wash fraction. The protein was then eluted with a step gradient of 10ml fractions of elution buffer I, II and III, respectively.

Enzymatic treatment: 400µl of TEV protease (6mg/ml) were added into the sample from Ni-purification (fractions wash II, elute I, II and III). The sample was incubated at 4°C overnight.

Column 2: Superdex 200 Hiload 16/60

Column 2 Buffers: 500 mM NaCl, 5% Glycerol, 50 mM HEPES pH 7.5, 0.5 mM TCEP.

Column 2 Procedure: After TEV cleavage, the sample was concentrated to 3ml before loading onto an ÄKTA Purifier. Gel filtration was run at 4°C. Elution fractions were analyzed by SDS-PAGE and the most purified fractions were collected.

Column 3: Ni-sepharose

Column 3 Buffers: 500 mM NaCl, 5% Glycerol, 50 mM HEPES pH 7.5, 0.5 mM TCEP.

Column 3 Procedure: The cleaved sample was loaded onto the column (packed from 0.5ml of Ni-sepharose slurry). The flow through was collected and the column was then washed with 5ml of the buffer (also collected).

Protein concentration: Protein was concentrated to 10 mg/ml using an Amicon 10 kDa cut-off concentrator.

Mass spectrometry characterization: Masses of purified proteins were confirmed by LC-MS, using an Agilent LC/MSD TOF system with reversed-phase HPLC coupled to electrospray ionisation and an orthogonal time-of-flight mass analyser. Proteins were desalted prior to mass spectrometry by rapid elution off a C3 column with a gradient of 5-95% isopropanol in water with 0.1% formic acid. A mass of 35952.2 was observed suggesting that the N-terminal methionine was lost.

Crystallisation: Crystals were obtained at 4°C in 150 nl sitting drops set up at a ratio of 1:1 with mother liquor and 12.3mg/ml protein with 1mM of EGFR peptide (pY1069). The mother liquor consisted of 0.1 M HEPES pH 7.5 and 20% (v/v) jeffamine M-600. On mounting crystals were cryo-protected with an additional 10% PEG400.

Structure Determination:
Resolution: 2.5 Å resolution.
X-ray source: Diamond Light Source, station I02, using monochromatic radiation at wavelength 0.9795 Å.

References

Griffiths, E.K., Sanchez, O., Mill, P., Krawczyk, C., Hojilla, C.V., Rubin, E., Nau, M.M., Khokha, R., Lipkowitz, S., Hui, C.C., et al. (2003). Cbl-3-deficient mice exhibit normal epithelial development. Mol Cell Biol 23, 7708-7718.
Kim, M., Tezuka, T., Suziki, Y., Sugano, S., Hirai, M., and Yamamoto, T. (1999). Molecular cloning and characterization of a novel cbl-family gene, cbl-c. Gene 239, 145-154.
Kim, M., Tezuka, T., Tanaka, K., and Yamamoto, T. (2004). Cbl-c suppresses v-Src-induced transformation through ubiquitin-dependent protein degradation. Oncogene 23, 1645-1655.
Ryan, P.E., Sivadasan-Nair, N., Nau, M.M., Nicholas, S., and Lipkowitz, S. (2010). The N terminus of Cbl-c regulates ubiquitin ligase activity by modulating affinity for the ubiquitin-conjugating enzyme. J Biol Chem 285, 23687-23698.
Swaminathan, G., and Tsygankov, A.Y. (2006). The Cbl family proteins: ring leaders in regulation of cell signaling. J Cell Physiol 209, 21-43.
Tsui, C.C., and Pierchala, B.A. (2008). CD2AP and Cbl-3/Cbl-c constitute a critical checkpoint in the regulation of ret signal transduction. J Neurosci 28, 8789-8800.