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An adjacent arginine, and the phosphorylated tyrosine in the c-Met receptor target sequence, dictates the orientation of c-Cbl binding

2010; Wiley; Volume: 585; Issue: 2 Linguagem: Inglês

10.1016/j.febslet.2010.11.060

1873-3468

Qingxiang Sun, C.W.W. Ng, Graeme R. Guy, J. Sivaraman,

Glycosylation and Glycoproteins Research

c-Cbl bind to Met : shown by surface plasmon resonance (view interactions 1,2) The casitas B-lineage lymphoma (Cbl) family of proteins, epitomized by c-Cbl, has a well-documented role in a number of aspects of cell signaling. Cbl was first known as a scaffold protein and thereafter characterized as a Ubiquitin E3 ligase [1, 2]. It plays a major role in the downregulation of a number of receptor and non-receptor tyrosine kinases. Cbl proteins have multiple domains and have been demonstrated to interact with a number of other proteins via proline-rich sequences and phosphotyrosine residues [3]. To enable its role in the ubiquitin-targeting system, Cbl contains a novel embedded Src homology 2 (SH2) domain that recognizes particular phoshotyrosine containing sequences on target proteins [4]. SH2 domains are well-characterized phosphotyrosine recognizing domains that seemed to co-evolve with tyrosine phosphorylation in metazoans, with relatively strict recognition sequences specific to each SH2 domain contained within different proteins [5]. The association of 4H and EF domains with the SH2 domain on Cbl, however, appears to be unique. It was somewhat surprising that the embedded SH2 domain of Cbl appeared to have a rather wide specificity in sequence recognition. We recently addressed the question as to how the Cbl tyrosine kinase binding (TKB) domain could recognize a ‘consensus’ sequence (N/D)XpY(S/T)XXP as well as an apparently unrelated DpYR sequence [6]. We demonstrated that in recognizing the DpYR sequence on the hepatocyte growth factor receptor (Met), Cbl flips over to bind in a reverse orientation relative to targets with the canonical sequence. Binding affinity and perhaps orientation were mediated by an intrapeptidyl bond between the phosphorylated tyrosine residue and an adjacent Arg or a nearby Asp residue [7]. It is noteworthy that the epidermal growth factor receptor (EGFR), a well characterized Cbl target, had an Arg in the pY−1 position that would resemble the DpYR motif found in the Met family of receptors, but in a reversed direction. The EGFR sequence also contained a pY+1 Ser and pY+4 Pro unlike Met's sequence. We asked the question in this study: what determines the orientation of target peptides binding to the Cbl TKB domain? To accomplish this, we took the most minimal DpYR Met binding sequence and tested the binding of derivative peptides and crystallized candidates that bound, in order to understand factors governing binding motif orientation of the Cbl-TKB. We have designed two peptides based on the sequence of wild type Met (MetWT), (1) by reversing core residues of the peptide (hereafter referred as MetRD) and (2) by changing pYRA residues of MetWT peptide to pYAN (hereafter referred as MetN) (Table 1 ). These peptides were purchased from GL Biochem (Shanghai, China). χ2 is the statistical error between the experimental and theoretical models. χ2 is defined as Σ(Rf− Rx)2/(n− p), where Rf is the fitted value at a given point, Rx is the experimental value at the same point, n is the number of data points, and p is the number of fitted parameters. Surface plasmon resonance (SPR) experiments were performed with Biacore 3000 (Biacore AB, Uppsala, Sweden). c-Cbl protein (50 ng/μL in 5 mM sodium citrate pH 6.5) was immobilized onto a CM 5 Chip as per the manufacturer's recommendations. The running buffer consisted of 20 mM Na Hepes pH 7.0 and 200 mM NaCl. Different concentrations of peptides (300 nM to 27 μM in running buffer) were applied to the chip surface at a flow rate of 20 μl/min at 25 °C. Regeneration using running buffer wash for 2 min resulted in a stable baseline corresponding to the starting baseline level. Reference cells are immobilized with inactive c-Cbl TKB domain resulting from 1 min flow of 10 mM H2SO4 through the cell. The equilibrium constant (Kd) was determined by the 1:1 Langmuir binding fitting model provided by the Biacore 3000 instrument software. Cloning, expression and purification of c-Cbl was performed as described previously [6]. Phosphopeptides were reconstituted in 20 mM Hepes, 200 mM NaCl and pH 7.0. MetRD was incubated with purified c-Cbl TKB in fivefold molar excess, and concentrated to 5 mg/ml using Amicon ultrafiltration devices (Millipore, Billerica, MA). TKB: MetRD complex crystallized in 100 mM Bis–tris propane pH 6.5, 50 mM ammonium sulfate by mixing 1 μl of reservoir solution with 2 μl of protein using hanging drop vapor diffusion method at room temperature. The mother liquor supplemented with 10% glycerol as cryo-protectant. The X-ray diffraction data was collected using in-house Bruker X-8 PROTEUM system and processed with HKL2000 [7]. The structure was solved by using molecular replacement method with the program MolRep [8] using c-Cbl–TKB domain as a search model (pdb code: 3BUX). The resulting model with the electron density map was examined in the program COOT [9], and necessary manual model building was performed. Several cycles of map fitting and alternated with refinement using the program Refmac5 [10] led to the convergence of R-values (Table 2 ). Coordinates and structure factors of the MetRD: Cbl-TKB complex was deposited at RCSB Protein Data Bank with the code 3PLF. A peptide from the Cbl–TKB atypical binding motif (DpYR) in the Met receptor (MetWT) chosen in our previous study was crystallised in complex with the Cbl–TKB domain [6]. This peptide had the sequence SNESVDpYRATFPE, and bound in a reverse direction on the TKB domain relative to other peptides of the typical binding motif [NXpY(S/T)XXP]. In an initial experiment to determine factors responsible for the orientation reversal of the Met atypical sequence, two more peptides: (1) SNESVRpYDATFPE (MetRD), where conserved residues flanking the phosphorylated tyrosine were reversed, (2) SNESVDpYANTFPE (MetN) where the conserved arginine in the wildtype sequence was replaced by alanine, were constructed. The affinities of these peptides binding to the TKB domain were assessed by SPR and the data obtained is shown in Table 1 and Fig. 1 . Two distinct observations were apparent: (1) The MetRD binding affinity was similar to the wildtype peptide affinity, and (2) no binding was observed when the conserved arginine in the wild type Met sequence was substituted by alanine. It was previously determined that the minimum TKB binding sequence for MetWT is DpYR, suggesting little contribution by any other residues flanking the DpYR motif. The lack of binding of the MetN peptide to the TKB domain indicates that the conserved arginine is essential for binding and possibly orientation of the peptide. While the MetRD and MetWT peptides bind with similar affinity, we did not know the structural changes implicated by reversal of the DpYR motif in the MetRD peptide. To ascertain the binding orientation of MetRD and to investigate its mode of binding to the Cbl-TKB, we proceeded to crystallize the MetRD: Cbl-TKB complex and compared it with the previously determined MetWT: Cbl-TKB structure [6]. The c-Cbl-TKB: MetRD complex was crystallized in P21 spacegroup with two complex molecules in an asymmetric unit. The structure was refined to a final R factor 0.159 (R free = 0.180) (Table 2, Fig. 2 ). Four residues (RpYDA) of MetRD peptide were clearly observed in the electron density map. The remaining residues of this peptide were disordered in the crystal. The mass spectrometric experiment on these crystals confirmed the presence of intact 13mer peptide in the complex crystal (Supplementary Fig. 1). When MetRD: Cbl-TKB was superimposed with MetWT: Cbl-TKB (Fig. 3 A), the phosphotyrosines of both peptides occupy the same pocket on the TKB domain in the same orientation, with the side chains of phosphotyrosine and arginine from the two complexes well superimposed. However, MetRD binds in the reverse direction compared with MetWT such that the N- and C-terminus of the MetWT and the MetRD peptide are 180° rotated with respect to each other. Closer examination of MetRD and MetWT reveal that the peptide backbones do not match well. All residues of the MetRD peptide are almost linear while the MetWT peptide is bent approximately 90° about the phosphotyrosine. Although orientation of the peptides are different, the same residues from c-Cbl-TKB, namely Ser80, Pro81, Tyr274, Ser296, Arg294, Cys297, Thr298 and Gln316 are involved in binding (Fig. 3A, Table S1). If the EGFR (DSFLQRpYSSDPTG), MetWT and MetRD peptides are compared, it appears that the position of the conserved arginine residue dictates directionality of binding to the TKB. Electrostatic surface potential shows that the charge distribution of the phosphotyrosine binding pocket is an important factor in orientating the conserved residues (Fig. 3B and C). pTyr1003 binds to a highly positively charged region while Arg1002 binds to a negatively charged region as indicated by the arrows in Fig. 3B. Arg294 of c-Cbl forms a salt bridge with phosphate group of pTyr1003 while the Ser80 side chain hydroxyl group and Pro81 backbone carbonyl group interact with Arg1002. In all of the TKB complex structures determined so far, the tyrosine ring of the phosphotyrosine is part of a large hydrophobic cluster, consisting of Tyr274 and Ile318 from the c-Cbl–TKB. Based on these observations, we suggest that charge distribution and spatial architecture of the phosphotyrosine binding pocket collectively direct the DpYR conserved residues to be able to bind only in one orientation, with the phosphotyrosine specifically interacting with Arg294. From this and the previous study [6], it is apparent that sequences derived from Cbl–TKB binding partners dictate both the affinity and orientation of the interacting moieties. These facets of binding are determined by: (1) key residues that insert into the phosphotyrosine binding pocket and (2) accessory residues (other than pY or pY±1 residues, e.g. well-conserved Pro at pY+4 position) that contact the surrounding TKB surface. Because these accessory residues are not apparent in the electron density map of the TKB: MetRD complex structure, we conclude they are not critically important in determining binding and binding orientation. Although the entire peptide orientation is reversed in MetRD, the positions of Arg1002 and pTyr003 remain unchanged in the phosphotyrosine binding pocket of c-Cbl-TKB relative to MetWT. It appears that in the case of MetWT binding to TKB, both the binding strength and the orientation are primarily determined by the interaction of the phosphotyrosine and the adjacent arginine residue via their intrapeptide bond. It is noteworthy that there was contribution to binding affinity by other conserved amino acids in peptides conforming to the ‘canonical’ c-Cbl TKB sequence; N/DXpYS/TXXP tested previously. The ‘theoretical’ MetRD and MetN peptides used in this study enabled us to determine factors that determine the orientation of c-Cbl on the Met receptor. These sequences are not likely to exist in cells but are useful tools to study biophysical properties of Cbl–TKB: substrate interaction. While we have also referred to the substrate peptide changing its orientation, it would be more plausible that the cytoplasmic c-Cbl changes orientation instead in comparison to other receptors such as the EGFR, while binding to the transmembrane c-Met receptor in cells. Collated data from this and our recent studies demonstrates that the only necessary binding and orientation elements required in a Cbl–TKB target are N/DXpY or RpY. Other residues deemed to be conserved in some families of TKB recognition sequences, such as the +4 Proline in the previously designated ‘typical’ binding sequence only add to the strength of binding. If the sequence is pYR the binding will reverse to essentially become in effect RpY. This work is supported by the (BMRC), A*STAR Singapore (R154000362305). Qingxiang Sun is a Ph.D student of National University of Singapore (NUS), receiving Singapore Millennium Foundation (SMF) Scholarship. Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2010.11.060. Supplementary Figure 1. Supplementary Figure 1 Supplementary data. Hydrogen bond contacts between different peptides and TKB domain residues. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.