Abstract
The binding interaction between the Nck2 SH2 domain and the phosphorylated ephrinB initiates a critical pathway for the reverse signaling network mediated by Eph receptor-ephrinB. Previously, the NMR structure and Tyr phosphorylations of the human ephrinB cytoplasmic domain have been studied. To obtain a complete story, it would be of significant interest to determine the structure of the Nck2 SH2 domain that shows a low sequence identity to other SH2 domains with known structures. Here, we report the determination of the solution structure of the human Nck2 SH2 domain and investigate its interactions with three phosphorylated ephrinB fragments by NMR spectroscopy. The results indicate that: 1) although the human Nck2 SH2 domain adopts a core tertiary fold common to all SH2 domains, it owns some unique properties such as a shorter C-terminal helix and unusual electrostatic potential surface. However, the most striking finding is that the C-terminal tail of the human Nck2 SH2 domain adopts a short antiparallel beta-sheet that, to the best of our knowledge, has never been identified in other SH2 domains. The truncation study suggests that one function of the C-terminal tail is to control the folding/solubility of the SH2 domain. 2) In addition to [Tyr(P)304]ephrinB2(301-322) and [Tyr(P)316]ephrinB2(301-322), here we identified [Tyr(P)330]ephrinB2(324-333) also capable of binding to the SH2 domain. The detailed NMR study indicated that the binding mechanisms for the three ephrinB fragments might be different. The binding with [Tyr(P)304]-ephrinB2(301-322) and [Tyr(P)316]ephrinB2(301-322) might be mostly involved in the residues over the N-half of the SH2 domain and provoked a significant increase in the backbone and side chain dynamics of the SH2 domain on the microsecond-millisecond time scale. In contrast, binding with [Tyr(P)330]ephrinB2(324-333) might have most residues over both halves engaged but induced less profound conformational dynamics on the mus-ms time scale.
Highlights
The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
It has been demonstrated that phosphorylation of any of the three tyrosine residues in the 22-residue motif dramatically abolished its well packed -hairpin structure and peptides with either Tyr304 or Tyr316 phosphorylated were able to bind to the human Nck2 SH2 domain [20]
Protein Cloning and Expression of the Full-length and Truncated SH2 Domains—Previously, we failed to obtain a sample with a concentration high enough for three-dimensional NMR experiments because of the limited solubility of the human Nck2 SH2 domain prepared by thrombin cleavage of the glutathione S-transferase fusion protein [20]
Summary
Cloning and Expression of the Full-length and Truncated Human Nck SH2 Domains—The DNA fragment encoding the full-length SH2 domain corresponding to residues 283 to 380 of the human Nck protein was subcloned into a modified pET32a (Novagen) vector with S-tag and thioredoxin genes removed with BamHI and XhoI restriction sites by using two primers (forward) 5Ј-GGGGGATCCAGAGAGTGGTACTACGGG-3Ј and (reverse) 5Ј-GGGCTCGAGTCACTGCAGGGCCCTGACG-3Ј. The full-length SH2 domain protein was purified with nickel-nitrilotriacetic acid resin (Qiagen) under native conditions, whereas the truncated one was purified under the denaturing condition. A set of manually assigned unambiguous NOE restraints together with dihedral angle restraints predicted by the TALOS program [26] based on five chemical shift values (15N, C␣, C, CO, and H␣) was used to calculate initial structures of the human Nck SH2 domain by the CYANA program [27]. With the availability of the initial structure, more NOE cross-peaks in the two NOESY spectra were automatically assigned by the CYANA program followed by manual confirmation. The NMR structures of the human Nck SH2 domain were deposited in the Protein Data Bank, with the PDB ID of 1Z3K. The perturbed residues were assigned by superimposing the two HSQC spectra in the absence and presence of the peptides
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