Expanding the repertoire of an ERK2 recruitment site: cysteine footprinting identifies the D-recruitment site as a mediator of Ets-1 binding

Abstract

Many substrates of ERK2 contain a D-site, a sequence recognized by ERK2 that is used to promote catalysis. Despite lacking a canonical D-site, the substrate Ets-1 is displaced from ERK2 by peptides containing one. This suggests that Ets-1 may contain a novel or cryptic D-site. To investigate this possibility a protein footprinting strategy was developed to elucidate ERK2-ligand interactions. Using this approach, single cysteine reporters were placed in the D-recruitment site (DRS) of ERK2 and the resulting ERK2 proteins subjected to alkylation by iodoacetamide. The ability of residues 1-138 of Ets-1 to protect the cysteines from alkylation was determined. The pattern of protection observed is consistent with Ets-1 occupying a hydrophobic binding site within the DRS of ERK2. Significantly, a peptide derived from the D-site of Elk-1, which is known to bind the DRS, exhibits a similar pattern of cysteine protection. This analysis expands the repertoire of the DRS on ERK2 and suggests that other targeting sequences remain to be identified. Furthermore, cysteine-footprinting is presented as a useful way to interrogate protein-ligand interactions at the resolution of a single amino acid.

Figure 1

Structure of EtsΔ138 and ERK2. A. Ribbon diagram of activated ERK2 (PDB 4ERK) showing the D-recruitment site (DRS) and the F-recruitment site (FRS). The activation segment (¹⁶⁵DFG–APE¹⁹⁵) is colored red. B. The DRS of ERK2 is composed of three segments: segment 1, helix αD–loop-8–helix αE (green); segment 2, strand β7–loop-11–strand β8 (cyan); and segment 3, loop-16 which contains the CD-site (Asp-316 and Asp-319) (plum). Several amino acids that have been shown to be important for recognition of various ERK2 ligands are indicated. C. A ribbon diagram depicting the NMR structure of Ets-1 (29–138) (PDB 1BQV) showing Thr-38 and Pro-39 in the flexible N-terminal tail, as well as Phe-120 in the PNT domain.

Figure 2

Differences in the binding modes of D-site peptides to the DRS of ERK2. A. Binding of the D-site peptide RRLKQGNLPVR (consensus; (R/K)_2–3-X_4–6-Φ_A-X-Φ_B) derived from MAP kinase phosphatase-3 to unactivated ERK2. The Φ_A residue occupies the hydrophobic site Ø₂. B. The binding of the D-site peptide RLQERRGSNVALMLDV, derived from hematopoietic protein tyrosine phosphatase, to unactivated ERK2. The Φ_A and Φ_B residues of hematopoietic protein tyrosine phosphatase occupy the hydrophobic Ø₁ and Ø₂ sites, respectively. Surface representations: red, oxygen; orange, carboxylate oxygen; blue, neutral nitrogen; cyan, ε lysine nitrogen, or arginine guanidino nitrogen.

Scheme 1

Model Describing an Equilibrium between the Folded State and Unfolded Exposed States That Determines the Rate of Cysteine Alkylation

Scheme 2 a

a ERK2 proteins containing single cysteine residues are incubated with iodoacetamide (IAA) to produce alkylated ERK2, I. Unalkylated ERK2 is cyanylated with NTCB to form the cyanylated intermediate II, which in the presence of ammonium hydroxide undergoes β-elimination to form III or polypeptide cleavage to form IV.

Figure 3

Cysteine footprinting of the ERK2 and EtsΔ138·ERK2 complexes. C-terminally radiolabeled single cysteine ERK2 mutants (A, C159; B, H123C; C, L113C; and D, Q117C) were alkylated by iodoacetamide at pH 8.3 and 23 °C in the absence and presence of 1.45 mM EtsΔ138. At the times indicated above each lane, 30 μL aliquots were quenched with excess DTT, and the ERK2 was separated from excess EtsΔ138 (where necessary), denatured, and then cyanylated with excess (25 mM) 2-nitro-5-thiocyanobenzoic acid (NTCB). The cyanylated proteins were precipitated, washed, then resuspended and cleaved in 0.6 M NH₄OH. Samples were then fractionated by 15% SDS-PAGE, the gels dried, and the bands quantified on a Phosphorimager (see Experimental Procedures). The lanes correspond to the time when an aliquot was quenched. The upper band corresponds to uncleaved ERK2 (see II and III in Scheme 2), and the lower band corresponds to the fragment C-terminal to the single cysteine (IV in Scheme 2).

Figure 4

Alkylation of ERK2-CTs versus time in the presence and absence of Ets¢138. Representative plots (A–D) showing the time course for the disappearance of unalkylated ERK2-CTs (measured by the presence of NTCB-mediated cleavage product) in the presence (●) and absence (■) of 1.45 mM EtsΔ138. The relative density of each gel quadrant (reproducible to within 20% after normalizing for variability in background radioactivity) containing a band associated with the ERK2-CT cleavage product was determined at various time points by phosphorimage analysis of gels similar to those shown in Figure 3 (see Experimental Procedures). The line through the data corresponds to the best fit to a first-order rate law (reproducible to within 30%). Endpoints used to determine rate constants for each alkylation reaction were determined after more than 10 half-lives. Some variability in the relative densities of the endpoints is seen between experiments, which mainly reflects variability in the background radioactivity of the gel quadrants used to determine the relative densities. Replicate experiments were performed on a minimum of two different preparations of each ERK2-CT. The sensitivity and reproducibility of the footprinting analysis is such that a 2-fold difference in a rate constant for alkylation may be readily determined. Rate constants and errors are reported in Table 1.

Figure 5

Fooprinting patterns within the DRS exhibited by Ets-1 and a D-site peptide. A. Footprinting EtsΔ138. B. Footprining D-site peptide. Residues protected are shown in yellow circles, while those not protected are shown in blue circles. C. Alignment of DRS backbone residues for unactivated ERK2 (PDB 1ERK), activated ERK2 (PDB 2ERK), and unactivated ERK2 complexed to RRLQKGNLPVR (PDB 2FYS).

Figure 6

Cysteine footprinting of a peptide·ERK2 complex. C-terminally radiolabeled single cysteine ERK2 mutants (A, C159; B, H123C; C, L113C; and D, L119C) were alkylated by iodoacetamide at pH 8.3 and 23 °C in the absence and presence of 0.625 mM peptide; QKGRKPRDLELPLSPSL. At the times indicated above each lane, 30 μL aliquots were quenched with excess DTT and the ERK2 was separated from excess EtsΔ138 (where necessary), denatured, and then cyanylated with excess (25 mM) 2-nitro-5-thiocyanobenzoic acid (NTCB). The cyanylated proteins were precipitated, washed, then resuspended and cleaved in 0.6 M NH₄OH. Samples were then fractionated by 15% SDS–PAGE, the gels dried, and the bands quantified on a Phosphorimager (see Experimental Procedures). The lanes correspond to the time when an aliquot was taken. The upper band corresponds to uncleaved ERK2 (see II and III in Scheme 2), and the lower band corresponds to the fragment C-terminal to the single cysteine (IV in Scheme 2).

Figure 7

Alkylation of ERK2-CTs versus time in the presence and absence of a D-site peptide. Representative plots (A–D) showing the time course for the disappearance of unalkylated ERK2-CTs (measured by the presence of NTCB-mediated cleavage product) in the presence (●) and absence (■) of 0.625 mM of peptide QKGRKPRDLELPLSPSL. The relative density of each gel quadrant (reproducible to within 20% after normalizing for variability in background radioactivity) containing a band associated with the ERK2-CT cleavage product was determined at various time points by phosphorimage analysis of gels similar to those shown in Figure 6 (see Experimental Procedures). The line through the data corresponds to the best fit to a first-order rate law (reproducible to within 30%). Endpoints used to determine rate constants for each alkylation reaction were determined after more than 10 half-lives. Some variability in the relative densities of the endpoints is seen between experiments, which mainly reflect variability in the background radioactivity of the gel quadrants used to determine the relative densities. Replicate experiments were performed on a minimum of two different preparations of each ERK2-CT. The sensitivity and reproducibility of the footprinting analysis are such that a 2-fold difference in a rate constant for alkylation may be readily determined. Rate constants and errors are reported in Table 1.

Figure 8

Fold protection for the alkylation of ERK2-CTs by IAA in the presence of 0.625 mM QKGRKPRDLELPLSPSL or 1.45 mM EtsΔ138.