Expanding the repertoire of an ERK2 recruitment site: cysteine footprinting identifies the D-recruitment site as a mediator of Ets-1 binding.
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.
Division of Medicinal Chemistry, University of Texas at Austin, Texas 78712, USA.
Olga
O
Abramczyk
Mark A
MA
Rainey
Richard
R
Barnes
Lance
L
Martin
Kevin N
KN
Dalby
D000478:Alkylation; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive / Q000235:genetics; D002384:Catalysis; D003545:Cysteine / Q000378:metabolism*; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D023302:Protein Footprinting; D025941:Protein Interaction Mapping; D021381:Protein Transport / Q000235:genetics; D050784:Proto-Oncogene Protein c-ets-1 / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity / Q000235:genetics
D050784:Proto-Oncogene Protein c-ets-1; D019950:Mitogen-Activated Protein Kinase 1; D003545:Cysteine