Structure of the Ets-1 pointed domain and mitogen-activated protein kinase phosphorylation site

Abstract

The Pointed (PNT) domain and an adjacent mitogen-activated protein (MAP) kinase phosphorylation site are defined by sequence conservation among a subset of ets transcription factors and are implicated in two regulatory strategies, protein interactions and posttranslational modifications, respectively. By using NMR, we have determined the structure of a 110-residue fragment of murine Ets-1 that includes the PNT domain and MAP kinase site. The Ets-1 PNT domain forms a monomeric five-helix bundle. The architecture is distinct from that of any known DNA- or protein-binding module, including the helix-loop-helix fold proposed for the PNT domain of the ets protein TEL. The MAP kinase site is in a highly flexible region of both the unphosphorylated and phosphorylated forms of the Ets-1 fragment. Phosphorylation alters neither the structure nor monomeric state of the PNT domain. These results suggest that the Ets-1 PNT domain functions in heterotypic protein interactions and support the possibility that target recognition is coupled to structuring of the MAP kinase site.

Figure 1

The PNT domain is defined by a region of sequence conservation found in a subset of ets proteins. (A) Schematic diagram of the murine Ets-1 protein showing the locations of the PNT domain, MAP kinase phosphorylation site (Thr-38 ∗), and DNA-binding ETS domain with flanking autoinhibitory sequences (1). The central region of the protein contains putative transactivation domain(s). (B) Alignment of the sequences of PNT domains and preceding N-terminal regions from the ets family members murine Ets-1, Ets-2, GABPα, and Fli-1, human Erg, Tel, and Ese, and Drosophila PNT-P2, Elg, and Yan. The positions of highly conserved amino acids are highlighted in black (seven or more members having blosum62 substitution scores ≥1), and those of moderately conserved residues in gray (six or more members having blosum62 substitution scores ≥0). The MAP kinase phosphorylation sites identified in Ets-1, Ets-2, and PNT-P2 are boxed. Based on a consensus MAP kinase substrate sequence P-X-T/S-P, Tel also contains a potential phosphorylation site (underlined). Positions of tryptic cleavage in Ets-1^(29–138) under conditions of partial proteolysis are denoted by ▾. The five α-helices (cylinders) in the Ets-1 PNT domain were identified by NMR methods. The fractional solvent accessibilities of the side chains in a low-energy structure of Ets-1^(29–138) are illustrated (one • = 0–25%).

Figure 2

(A) The PNT domain (Ser-54 to Glu-135) is an independently folded structural module as evidenced by well-dispersed peaks in the ¹H-¹⁵N HSQC spectrum of Ets-1^(29–138). Residues N-terminal to this domain, including the MAP kinase substrate site, adopt a disordered conformation with ¹H^N chemical shifts that cluster near 8.2 ppm. Aliased peaks are identified by ∗. (B) Hydrogen-deuterium exchange studies identify amide protons that are protected from the solvent caused by hydrogen bonding and/or burial within Ets-1^(29–138). • indicate residues with resolved ¹H-¹⁵N HSQC cross peaks that have exchange rates >10³ slower than expected for a random coil polypeptide. (C) NMR relaxation measurements provide information about the global tumbling and fast internal motions of Ets-1^(29–138). Analysis of the amide ¹⁵N T₁ and T₂ lifetimes and heteronuclear ¹⁵N NOE values according to the model-free formalism (17) yields an overall rotational correlation time of 6.9 nsec for the protein and squared order parameters (S²) for each individual nonproline with a resolved cross peak. This correlation time is consistent with that expected for a monomeric protein of ≈12.5 kDa. Relatively uniform relaxation parameters, including NOEs > 0.5 and S² values > 0.7, indicate that the residues comprising the PNT domain are generally well ordered. In contrast, residues 31–34, 36–37, 43–45, and 47–49 at the N terminus and 135–138 at the C terminus of Ets-1^(29–138) are motionally disordered on a nano- to picosecond time scale as evident by NOEs < 0.5. Mutation of Leu-36 to Pro and phosphorylation of Thr-38 does not significantly change the relaxation properties of Ets-1^(29–138) (not shown). (D) Chemical shift perturbations indicate that the effects of the Leu-36 to Pro mutation and the subsequent phosphorylation of Thr-38 are localized to the MAP kinase substrate site in the disordered N-terminal region of Ets-1^(29–138). Shown are the absolute values of the changes in the amide ¹⁵N and ¹H^N chemical shifts (ppm) caused by the mutation and phosphorylation plotted versus residue number. The small changes observed for residues in the PNT domain reflect subtle differences in experimental conditions.

Figure 3

The tertiary structure of Ets-1^(29–138) was determined by NMR methods. (A) Superimposition of the main chain atoms from 28 NMR-derived structures of Ets-1^(29–138) aligned by using residues 63–133. The five α-helices in the PNT domain are colored (H1: residues 54–62; H2: 75–87; H3: 102–107; H4: 110–116; H5: 123–132), whereas the remainder of the main chain is shown in gray. The N and C termini of the molecule (residues 29–49 and 135–138) are disordered as evidenced by both high structural rms deviations and ¹⁵N NMR relaxation data. (B) Ribbon diagram (37) of a representative low-energy structure calculated for Ets-1^(29–138). Only a single conformation is shown for the flexible N and C termini. (C) A low-energy structure of residues 26–132 of Ets-1^(29–138) showing the positions of the side chains that are highly conserved (Fig. 1B) among the PNT domains of 10 ets proteins (green = hydrophobic, red = acidic, blue = basic, dark gray = polar).

Figure 4

Views of the van der Waals surface of the Ets-1 PNT domain (residues 54–135), illustrating potential protein–protein association interfaces centered on (A) helices H4 and H5, and (B) the loop connecting helices H2 and H3. The side chains are colored as green = hydrophobic, red = acidic, blue = basic, and gray = polar (and main chain).