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

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

Ágnes Garai et al. Sci Signal.

Abstract

Mitogen-activated protein kinases (MAPKs) have a docking groove that interacts with linear "docking" motifs in binding partners. To determine the structural basis of binding specificity between MAPKs and docking motifs, we quantitatively analyzed the ability of 15 docking motifs from diverse MAPK partners to bind to c-Jun amino-terminal kinase 1 (JNK1), p38α, and extracellular signal-regulated kinase 2 (ERK2). Classical docking motifs mediated highly specific binding only to JNK1, and only those motifs with a sequence pattern distinct from the classical MAPK binding docking motif consensus differentiated between the topographically similar docking grooves of ERK and p38α. Crystal structures of four complexes of MAPKs with docking peptides, representing JNK-specific, ERK-specific, or ERK- and p38-selective binding modes, revealed that the regions located 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 nondiscriminatory fashion, the conformation of the intervening region between the anchor points mostly determined specificity. We designed peptides with tailored MAPK binding profiles by rationally changing the length and amino acid composition of intervening regions located between anchor points. These results suggest a coherent structural model for MAPK docking specificity that reveals how short linear motifs binding to a common kinase docking groove can mediate diverse interaction patterns and contribute to correct MAPK partner selection in signaling networks.

Figures

Figure 1
Figure 1. Biochemical specificity of classical D-motifs
(A) Binding affinities of chemically synthesized D-motif peptides with JNK1, p38α and ERK2. “-” indicates no detectable binding, Kd > 100 μM; N=3 experiments. (B) These binding affinities are plotted on a three-dimensional scatter plot in which axes represent dissociation constants (Kd) for the three different MAPKs and squares correspond to individual peptides listed in the table on the left. (C) The in vivo wiring diagram for MAPKs with their partners. Black connections indicate physiologically relevant links; gray dashed lines indicate binding that does not concur to physiologically relevant connections. (D-G) Docking motifs govern the phosphorylation of critical transcription factor regions in MEF2A (D) and NFAT4 (E) by MAPKs. A representative set of phosphor imaging results of SDS-PAGE gels are shown from at least two in vitro kinase experiments using activated (*) MAPKs to phosphorylate control, “wild-type” or D-motif MEF2A (F) and NFAT4 (G) chimera reporters. AP constructs: Thr or Ser residues in target S/TP site were mutated to alanines; NoDock constructs: the basic and ΦxΦ motif residues, indicated by arrows, were mutated to alanines; DBD, DNA-binding domain.
Figure 2
Figure 2. MAPK binding specificity of MAPKAPK docking regions
(A) Domain architecture of MAPKAPKs. MAPK docking regions and phosphorylation sites on MAPKAPKs that are important for regulation of MAPKAPK activity regulation in cells are indicated with black rectangles and circles, respectively. (B) The in vivo MAPK interaction profile of the C-terminal MAPK docking regions in select MAPKAPKs. (C) Results of the GST pull down experiments (N=2) are shown on Coomassie stained SDS-PAGE gels. (D) Results of binding affinity measurements with chemically synthesized peptides are shown in table at the bottom. “-” indicates no detectable binding or Kd > 100 μM. N=3 experiments. (E) Discrimination factors of peptides were calculated by taking the logarithm of the ratio of binding affinities for ERK2 and p38α and were plotted in a bar graph.
Figure 3
Figure 3. MAPK docking grooves are compatible with different linear motif binding modes
(A) Overlay of the JNK1-pepNFAT4 and the JNK1-pepJIP1 (PDB ID: 1UKH) crystal structures (13). (B) Crystal structure of the ERK2-pepMNK1 complex. The MAPK docking groove is located on the opposite side of the kinase relative to the active site (arrow in the inset). N and C, N- and C-termini of the peptide, respectively. (C) Comparison of the ERK2-pepMNK1 complex with the ERK2-pepHePTP complex (PDB ID: 2GPH) (12). Colored arrows indicate the N→C-terminal direction of the docking peptides. The last hydrophobic contact point in the pepHePTP D-motif consensus is only inferred because this residue was modified and the slight mis-alignment at this region is likely to be the result of the forced covalent attachment of pepHePTP to ERK2 through an artificial disulfide bond. (D) Superposition of the ERK2-pepRSK1 complex with the p38α-pepMK2 (PDB ID: 2OKR) crystal structure (30). (C) and (D) show the ERK2 or the p38α surface from the ERK2-pepMNK1 and p38α-pepMK2 crystal structures, respectively. Non-cognate protein-peptide pairs do not show any clash, indicating that surface complementarity alone does not explain the selective formation of ERK2-pepRSK1 and p38α-pepMK2 complexes. New structures shown on this figure are JNK1-pepNFAT4, ERK2-pepMNK1 and ERK2-pepRSK1.
Figure 4
Figure 4. Topography of paralogous MAPK docking grooves
(A-C) Structural basis for the ability of JNK to discriminate between certain D-motifs and RevD-motifsPeptide-bound docking surfaces of ERK2 (A), p38α (B) and JNK1 (C) are presented in the same orientation. Arrows on (C) indicate the critical region in JNK that determines the width of the CD groove, which in turn also influences the size of the upper pocket. The insets show the sequence logo of the corresponding regions in MAPK homologs from sponges to human. Stars denote the tentative position of the pepMKK6 basic region in the p38α CD groove. (D) Structural determinants of p38-ERK discrimination. Arrows indicate conserved amino acids in the pepMNK1 sequence logo that are not part of the revD-motif consensus, which are colored in magenta on the human MAPK:revD-motif complex structure below. Superimposed p38α is colored in light gray and shown as a Cα-trace. (E) The evolutionarily conserved nature of the base, top and hinge is shown for ERK1/2 and p38 homologs from sponges to human from the KinBase database (73) as sequence logos. (F) The impact of amino acid swaps in the ERK2 docking groove on pepRSK1 or pepMK2 binding is shown as a bar diagram. Amino acid swaps were done in the base (m2), in the base and the top (m4) and in all three regions (m6) of ERK2. New structures shown on this figure are p38α-pepMKK6, JNK1-pepNFAT4 and ERK2-pepMNK1.
Figure 5
Figure 5. Sequence-specific intra-peptide hydrogen bonds in revD-motifs contribute to their MAPK specificity
(A-D) Sequence-specific inter-molecular and intra-peptide H-bonds in MAPK:revD-motif crystal structures. Peptide sequence specific inter-molecular H-bonds are gray, whereas sequence specific, intra-peptide H-bonds are red. Selected side-chains of the top region and the base are orange for ERK2 and salmon for p38α. The schematic insets on the upper left corners emphasize the intervening region between two anchor points (ΦL and ΦU; hydrophobic anchor points are shown as pentagons). These also depict the top and the base regions because they mediate important differential inter-molecular H-bonding interactions. Light blue circles indicate peptide amino acids adopting a 310 helical conformation and red dotted lines depict H-bond staples. Small insets in the upper right corners of the last two panels show the sequence logo of RSK1/2 or MK2/3 revD-motifs. The logos were based on ten sequences from five different vertebrate organisms (Danio rerio, Xenopus laevis, Gallus gallus, Mus musculus and Homo sapiens). Evolutionarily conserved amino acids that are involved in H-bond stapling are highlighted in red. New structures are ERK2-pepSynth-revD (A), ERK2-pepMNK1 (B) and ERK2-pepRSK1 (C). (E) Role of H-bond staples in mediating MAPK:revD motif binding specificity. The table shows binding affinities of revD-motifs with different H-bond staples to ERK2 and p38α (left). Amino acids involved in H-bond stapling or replaced by alanines are bolded and revD-motif defining amino acids are underlined. (F) Discrimination factors of peptides were calculated by taking the logarithm of the ratio of binding affinities for ERK2 and p38α and were plotted in a bar graph on the right. Heights for bars indicated with a “>” sign are low estimates because the experimental assay cannot measure binding affinities weaker than 100 μM.
Figure 6
Figure 6. Modification and design of MAPK-docking motif interaction profiles
(A-B) Manipulation of MAPK interaction profiles for natural D-motif peptides. The JNK-specific docking peptide of NFAT4 was made more promiscuous (A) and the promiscuous motif found in MKK4 was made more selective (B) with appropriate mutations. Binding affinities of peptides are plotted on a three-dimensional scatter plot where axes represent dissociation constants (Kd) for JNK1, p38α and ERK2. Arrows (m) indicate the impact of amino acid replacements in the MAPK ligand space. Amino acid replacements are indicated below the original peptide sequence. Amino acids in consensus motif defining positions are underlined. (C-D) Design of artificial motifs with tailored MAPK specificity profiles. Peptides containing a minimal D-motif (pepSynth-D) or a revD-motif (pepSynth-revD) are promiscuous or ERK/p38 selective, respectively. Amino acid replacements in the ERK/p38 selective pepSynth-revD are shown under the original peptide sequence (C), and their effect on the discrimination factor for ERK2 compared to p38α binding is shown in the bar graph (D). ↑AS indicates a two amino acid insertion of alanine and serine residues.

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