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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.

SUBMITTER: Abramczyk O

PROVIDER: S-EPMC2897722 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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

Abramczyk Olga O Rainey Mark A MA Barnes Richard R Martin Lance L Dalby Kevin N KN

Biochemistry 20070721 32

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 th ...[more]

PMID: 17658891

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Project description:ERK2, a major effector of the BRAF oncogene, is a promiscuous protein kinase that has a strong preference for phosphorylating substrates on Ser-Pro or Thr-Pro motifs. As part of a program to understand the fundamental basis for ERK2 substrate recognition and catalysis, we have studied the mechanism by which ERK2 phosphorylates the transcription factor Ets-1 at Thr-38. A feature of the mechanism in the forward direction is a partially rate-limiting product release step (koff = 59 +/- 6 s(-1)), which is significant because to approach maximum efficiency substrates for ERK2 may evolve to ensure that ADP dissociation is rate-limiting. To improve our understanding of the mechanism of product release, the binding of the products to ERK2 was assessed and the reaction was examined in the reverse direction. These studies demonstrated that phospho-Ets-1 (p-Ets) binds >20-fold more tightly to ERK2 than ADP (Kd = 7.3 and 165 microM, respectively) and revealed that the products exhibit little interaction energetically while bound to ERK2 and that they can dissociate ERK2 in a random order. The overall equilibrium for the reaction in solution (Keq = 250 M(-1)) was found to be similar to that with the substrate bound to the enzyme (Kint = 525 M(-1)). To determine what limits koff, several pre-steady-state experiments were performed. A catalytic trapping approach furnished a rate constant (k-ADPa) of 61 +/- 12 s(-1) for the dissociation of ADP from the abortive ternary complex, ERK2.Ets.ADP. To examine p-Ets dissociation, the binding of a fluorescent derivative (p-Ets-F), which binds ERK2 with an affinity similar to that of p-Ets, was examined by stopped-flow kinetics. Using this approach, p-Ets-F was found to bind through a single-step mechanism, with the following parameters: k-p-Ets-F = 121 +/- 3.8 s(-1), and kp-Ets-F = (9.4 +/- 0.3) X 10(6) M(-1) s(-1). Similar results were found in the presence of a saturating ADP concentration. These data suggest that koff may be limited by the dissociation of both products and are consistent with the notion that Ets-1 has evolved to be an efficient substrate for ERK2, where ADP release is, at least, partially rate-limiting. A molecular mechanics model of the complex formed between ERK2 and residues 28-138 of Ets-1 provides insight into the role of substrate docking interactions.

Dataset Information

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

Publications

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

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