Results Three-dimensional structure of PEA-15 The structure of PEA-15 was determined from a total of 2937 NMR-derived restraints, including 2522 nuclear Overhauser effects (NOEs), 308 dihedral angle restraints and 107 3J NHα coupling constants (Figure 1). The 20 conformers exhibit good geometry, with no violations of distance restraints >0.5 Å and no dihedral angle violations >5° (Table I). The structure of the region corresponding to the DED is well defined by the NMR data. The atomic r.m.s.ds about the mean coordinate positions of the backbone atoms (N, C α and C′) and all heavy atoms for residues 1–89 are 0.16 ± 0.02 and 0.62 ± 0.02 Å, respectively. The C-terminus of the protein does not adopt a regular structure in solution. This is evidenced by the absence of NOEs between non-sequential amino acids and by C α and C β chemical shifts close to their random coil values. Residues 98–112 or 114–126 can be superimposed independently over the backbone atoms to <1.0 Å r.m.s.d.; however, no long-range NOEs were observed between the C-terminus and the DED to define their relative orientation. | Fig. 1. Three-dimensional structure of PEA-15. (A) Stereoview of the backbone atoms (N, Cα and C′) of 20 NMR conformers of PEA-15 superimposed over residues 1–97. (B) Ribbon representation of the averaged (more ...) |
| Table I. NMR structural statistics of PEA-15 |
The 3D structure of PEA-15 consists of an N-terminal DED comprised of six antiparallel amphipathic α-helices closely packed around a central hydrophobic core, followed by a long C-terminal tail (Figures 1A and B). The C-terminal tail is irregular in structure, with the exception of residues 120–123, which appear to form a single turn of a 3 10-helix. The α-helices in the DED are connected by short loops, two of which contain β-turns (α2–α3 and α4–α5). The α-helices are arranged in a Greek key topology, with helices α1 and α2 being centrally located, α3 and α4 on one side and α5 and α6 on the other. This fold represents the core structure of the death motif superfamily that also includes the DD and CARD ( Fesik, 2000). The overall fold of the PEA-15 DED closely resembles the structure of the FADD DED ( Eberstadt et al., 1998), the only other DED structure which has been determined to date. The two structures are very similar, with an r.m.s.d. of 1.8 Å for C α atoms of residues 1–83, and differ primarily in the length of helix α6 (Figure 1C). Helix α6 of PEA-15 is seven residues longer than that of the FADD DED, and numerous NOEs were observed between residues in α1 and α6 of PEA-15 to define their relative orientation. The N-terminus of the FADD DED, on the other hand, is oriented away from α6 and the core of the protein as compared with PEA-15. PEA-15 also lacks the two hydrophobic patches observed on the surface of FADD DED. One of these patches in FADD consists of several residues in helix α2 that have been implicated in FADD’s apoptotic activity and interaction with the DEDs of procaspase-8 ( Eberstadt et al., 1998). In contrast, many charged residues are present on the surface of the PEA-15 DED, suggesting that electrostatic interactions may mediate molecular recognition of ERK1/2. PEA-15 DED and tail are required for ERK interaction To explore the ERK-binding surface of PEA-15, complexes of 15N-labeled PEA-15 and unphosphorylated ERK2 were prepared, and the effect of ERK2 interaction on backbone N and H N chemical shifts (the NMR ‘footprint’) was examined. At the protein concentrations used in this study, unphosphorylated ERK2 is predicted to be dimeric ( Khokhlatchev et al., 1998), resulting in a PEA-15–ERK2 complex of ~115 kDa. As expected, the NMR spectrum of PEA-15 broadened away upon the addition of a stoichiometric amount of ERK2, consistent with the formation of a large, slowly tumbling complex in solution. At substoichiometric levels of ERK2, however, differential broadening of PEA-15 NMR resonances was observed, providing clues to where ERK2 may form direct contacts with PEA-15 (Figure 2A). Residues implicated in ERK binding were located in helices α1, α2, α5 and α6 of the DED, as well as in the C-terminal tail (Figure 2B and C). | Fig. 2. Identification of the ERK2-binding surface of PEA-15 by NMR ‘footprinting’. (A) Superposition of the 1H–15N HSQC spectra of PEA-15 in the free (black) and ERK2-bound form (red) at an ERK2:PEA-15 ratio of 0.7:1. (more ...) |
The segments of PEA-15 in which selective broadening was observed were functionally probed by site-directed mutagenesis and analyzed for binding to GST–ERK2 in an in vitro pull-down assay (Figure 3; Table II). Mutants were constructed in which surface-exposed residues were substantially altered in side chain length and/or charge, while minimizing the likelihood of altering the protein structure. Five out of 23 mutations introduced into the DED displayed either a reduced or complete loss of binding to ERK2 (Figure 3A; Table II). Three of these mutations were located in the α1–α2 loop (Asn14, Thr16 and Glu18) and the remaining two mutations in the α5–α6 loop (Arg71 and Asp74). Together, these five residues form a contiguous, predominantly negatively charged surface on the PEA-15 DED which defines part of the binding site for ERK2 (Figure 3D and E). In contrast, extensive mutagenesis of other regions of the DED had no effect on the interaction with ERK2 (Figure 3D; Table II). | Fig. 3. ERK2 recognition by PEA-15. (A) Effects of point mutations of PEA-15 on interaction with ERK2, determined by the binding activity of [35S]methionine-labeled wild-type (WT) or mutant PEA-15 to GST–ERK2 in a pull-down (more ...) |
To assess the structural integrity of the five DED mutant proteins that demonstrated reduced binding to ERK, the 2D 1H- 15N HSQC spectrum of each mutant was compared with that of wild-type PEA-15 (Figure 4). The sensitivity of the chemical shift to local environment permits the assessment of local and global changes in the structure of PEA-15 with amino acid resolution. Thus, the peak pattern and linewidth are diagnostic of the similarity in protein fold between the wild-type and mutant proteins. It is evident that the N14R, T16R, E18R and R71A mutant proteins are folded correctly, with only minor chemical shift perturbations observed for a few residues spatially proximal to the mutated site. The fifth mutant protein, D74A, displayed several differences in the N and H N chemical shifts compared with wild-type PEA-15 (Figure 4E). However, complete backbone chemical shift assignment of the D74A mutant revealed that neither significant local nor global structural changes had occurred. First, secondary chemical shift deviations for C α and C β ( Wishart et al., 1992) established that the wild-type and D74A proteins possessed identical helical segments, indicating that the secondary structure of D74A was unchanged by the mutation. Secondly, the absolute values of the C α and C β chemical shifts are nearly identical between D74A and wild type, establishing that the global fold of the mutant protein is intact. These data strongly suggest that the D74A mutant was not significantly altered in structure. More probably, the removal of an exposed negative charge from a highly charged region of the protein (Figure 3E) altered the local electronic environment, resulting in the perturbation of several backbone N and H N resonances. Since all of the DED mutants were folded correctly, the loss of ERK binding observed for these mutants was most probably the consequence of a disruption in the interaction surface of PEA-15 for the enzyme. | Fig. 4. Analysis of the structural integrity of the PEA-15 DED mutant proteins. (A–E) Superpositions of the 1H–15N HSQC spectra of wild-type (black) and mutant (red) PEA-15 proteins N14R, T16R, E18R, R71A and D74A. |
Regions outside the DED of PEA-15 have previously been implicated in ERK2 binding, as overexpression of the PEA-15 DED alone did not activate ERK ( Ramos et al., 2000; Formstecher et al., 2001). NMR ‘footprinting’ also suggested the involvement of the C-terminal tail in binding to ERK2 (Figure 2). Furthermore, neither the DED nor C-terminal tail of PEA-15 bound ERK well in isolation (Figure 3B), and deletion of only 11 amino acids from the tail was sufficient to disrupt interaction with ERK (Figure 3C). Site-directed mutagenesis within the tail revealed that Ile121, Lys122, Leu123 (all in the 3 10-helix) in addition to Lys128 and Lys129 were important for ERK interaction (Figure 3A; Table II). The irregular structure of the tail and its lack of interaction with the DED suggests that none of the tail mutants would have affected the integrity of the protein. Although two hydrophobic residues were changed to arginine, which could have disrupted the putative 3 10-helix (I121R and L123R), mutation of these residues to alanine displayed an identical phenotype to that of the arginine mutants (Table II). Thus, the functional defect observed for these mutants was not likely to be a consequence of the introduction of a conformational change in the tail. These results demonstrate that electrostatic interactions involving residues in the DED as well as hydrophobic and charged residues in the tail are critical for PEA-15 binding to ERK2. Functional characterization of PEA-15 mutants To correlate the in vitro ERK binding deficiency of the PEA-15 mutants with their functional activity in vivo, the ability of wild-type or mutant PEA-15 to inhibit ERK nuclear signaling was assessed (Figure 5A). Wild-type PEA-15 markedly attenuated the ERK-stimulated transcriptional activation of Elk-1 ( Formstecher et al., 2001), whereas mutants of PEA-15 that lacked the capacity to bind ERK2 in vitro (Figure 3A) also displayed re duced inhibition of Elk-1-dependent transcription in vivo (Figure 5A). Mutations in the DED, such as E18R, were clearly less effective at reversing the inhibition of transcription compared with mutations in the C-terminal tail (e.g. I121R, L123R and K129E). These results correlate well with the residual ERK-binding activity of the DED and tail mutants observed in the pull-down assay (Figure 3A). Thus, the capacity of PEA-15 to bind ERK correlates with its ability to block the phosphorylation and transcriptional activity of ERK nuclear substrates. | Fig. 5. In vivo analysis of PEA-15 mutants. (A) Elk-1-dependent transcriptional activity was measured in serum-stimulated CHO-K1 cell lysates transfected with expression plasmids encoding either wild-type PEA-15, mutant PEA-15 or control vector. (more ...) |
The effects of PEA-15 on transcription were associated with an alteration in the nuclear translocation of ERK (Figure 5B). Mutations that completely abrogated ERK binding (e.g. I121R) failed to block ERK nuclear translocation. A similar outcome was shown previously for the D74A mutant in the DED ( Formstecher et al., 2001). In contrast, mutants that retained some ability to bind ERK2 in vitro, such as E18R and K122E, also substantially retained ERK in the cytoplasm. Thus, the ERK-binding activity of PEA-15 is required for its biological effects on ERK localization. A common binding surface in the death motif mediates diverse protein–protein interactions A comparison of the ERK-binding surface of PEA-15 with that observed for Tube DD in the crystal structure of the Tube–Pelle DD complex ( Xiao et al., 1999) revealed an unexpected similarity in the binding motifs of Tube and PEA-15 (Figure 6). The interaction between Tube and the serine/threonine kinase Pelle through their N-terminal DDs is required for the activation of dorsal–ventral patterning genes during Drosophila embryogenesis ( Belvin and Anderson, 1996). The molecular determinants by which Tube recognizes Pelle include a compact surface within the Tube DD as well as a distinct set of interactions formed by its irregularly structured C-terminal tail ( Xiao et al., 1999). These elements of Tube contact adjacent sites on the Pelle DD, forming an almost continuous interaction surface. The similarity to the PEA-15–ERK2 interaction resides in the interaction surface of Tube DD, formed by the α1–α2 and α5–α6 loops and α6 (Figure 6). Mutations introduced into the these regions of PEA-15 were functionally deficient for ERK2 binding, thereby restoring ERK2 nuclear translocation and consequent stimulation of transcription. Mutants within the corresponding loops and helix of Tube DD rendered the protein inactive ( Xiao et al., 1999), indicative of an important role for these regions in the interaction between the Tube and Pelle DDs. Since the targets of Tube and PEA-15 are structurally and functionally unrelated, the similarity between the DD and DED surfaces implicated in protein–protein interaction is suggestive of a conserved binding epitope inherent to the common fold of the DD and DED of these proteins. While the irregularly structured C-terminal tails of Tube and PEA-15 both appear to play critical roles in the recognition of their binding partners, a correspondence between their respective roles cannot be drawn in the absence of the 3D structure of the PEA-15–ERK2 complex. | Fig. 6. Comparison of the PEA-15 DED, Tube DD and Pelle DD binding epitopes. (A) Backbone representations of the death motifs of PEA-15, Tube and Pelle (Xiao et al., 1999; PDB accession code 1D2Z) with residues important for binding their target (more ...) |
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