Specificity of Linear Motifs That Bind to a Common Mitogen-Activated Protein Kinase Docking Groove

Abstract

Mitogen activated protein kinases (MAPKs) have a docking groove that interacts with linear motifs in binding partners. To determine the structural basis of binding specificity between MAPKs and docking motifs, we quantitatively analyzed the ability of fifteen linear motifs from diverse MAPK partners to bind to c-Jun N-terminal kinase 1 (JNK1), p38α and extracellular signal-regulated kinase 2 (ERK2). Classical docking motifs mediated highly specific binding only to JNK1, and only motifs with a sequence pattern distinct from the classical MAPK binding docking motif consensus could differentiate between the topographically similar docking grooves of ERK and p38. We also solved the crystal structures for four MAPK-docking peptide complexes that represented JNK-specific, ERK-specific or ERK-and p38-selective binding modes. These structures revealed that the regions located in between consensus positions in the docking motifs showed conformational diversity. Although the consensus positions in the docking motifs served as anchor points that bound to common MAPK surface features and mostly contributed to docking in a non-discriminatory fashion, specificity was determined mainly by the conformation of the intervening region between the anchor points. These insights enabled us to successfully design peptides with tailored MAPK binding profiles by rationally changing the length and amino acid composition of docking motif regions located between anchor points. We present a coherent structural model underlying MAPK docking specificity that reveals how short linear motifs

Two Hydrophobic Residues Can Determine the Specificity of Mitogen-activated Protein Kinase Docking Interactions

“…These results suggest that the precise chemical identity of a given basic residue does not dramatically influence binding efficiency and thus likewise does not determine binding specificity. This is consistent with the idea that the basic residues may be able to bind to corresponding acidic patches in the docking groove in a flexible or "fuzzy" manner, accounting for the lack of resolution of this portion of the D-site in many co-crystal structures (11,42).…”

“…Furthermore, many D-sites contain an "extended" ⌽X⌽X⌽ hydrophobic submotif. The "extra" hydrophobic residue in such D-sites can be accommodated in an additional hydrophobic pocket in the docking groove, as was first seen in the co-crystal structure of the MEF2A D-site peptide with p38␣ (57) and has since been observed in several other D-site⅐MAPK co-crystal structures, including Msg5⅐Fus3 (58), Ste7⅐Fus3 (58), JNK1⅐NFAT4 (42), and JNK3⅐ATF2 (59). A schematic depiction of the JNK1⅐NFAT4 structure is shown in Fig.…”

Mapping the binding interface of ERK and transcriptional repressor Capicua using photocrosslinking

“…Recently, the structures of ERK in complex with protein substrates PEA15 and RSK1 were solved and found to involve a DRS-D site interaction using a reverse-orientation D site (28,33). The structures of peptides corresponding to the MNK1 and RSK1 reverse D sites in complex with ERK have also been solved (32). Notably, all of these interactions feature a leucine buried in the hydrophobic pocket.…”

“…The MNK1 reverse D site peptide has a leucine residue buried in the ERK2 hydrophobic pocket (Fig. 3B) (32). Two residues upstream-in the position analogous to F1054 in Cic relative to the ERK-binding leucine-a methionine side chain is aligned with ERK T108-the amino acid in ERK that was shown to crosslink to GSTCic F1054AzF.…”

“…Crystal structures of ERK in complex with its binding partners (28)(29)(30)(31)(32)(33) provide invaluable insights, many of which formed the basis of this study. However, the ability to form crystals of a complex is not guaranteed, and most of the structures of ERK complexes use linear peptides derived from the ERK-interacting molecules instead of structured proteins.…”