1. Volume 14, Issue 1, Pages 43-55 (April 2004)
Docking Motif Interactions in MAP Kinases Revealed by Hydrogen Exchange Mass Spectrometry 
Thomas Lee, Andrew N Hoofnagle, Yukihito Kabuyama, James Stroud, Xiaoshan Min, Elizabeth J Goldsmith, Lin Chen, Katheryn A Resing, Natalie G Ahn 
Molecular Cell 
Volume 14, Issue 1, Pages (April 2004)
DOI: /S (04)

2. Figure 1
HX-MS Reveals Binding Interactions between p38α MAPK and MKK3b-DEJL
(A) Sequence coverage map of p38α MAPK, numbered from the N-terminal His6 tag, with secondary structure as described by Chang et al. (2002). Indicated are 38 peptides observed by HX-MS, colored red, green, or white to indicate, respectively, exchange rates that increased, decreased, or did not change upon binding MKK3b-DEJL peptide. Peptides colored cyan were observed disulfide crosslinked to a single Cys residue on the MKK3b-DEJL peptide.
Molecular Cell  , 43-55DOI: ( /S (04) )

3. Figure 2 HX-MS Reveals Conserved DEJL Interactions in ppERK2
(A) Sequence coverage map of ppERK2, with residue numbering and secondary structure as described (Canagarajah et al., 1997). Indicated are 29 peptides observed by HX-MS, colored red, green, or white to indicate, respectively, exchange rates that increased, decreased, or did not change upon binding Elk1-DEJL peptide.
(B) Structural representation of ppERK2 (2ERK, Canagarajah et al., 1997) indicating peptides that showed significant changes in exchange rate between the presence (○) and absence (•) of 100 μM Elk1-DEJL peptide, with coloring scheme as in (A). Significant protection was observed in regions around β7-β8 and αD-αE, confirming that solution interaction of the DEJL motif occurs in ppERK2 within the same region observed in p38α MAPK. Results of nonlinear least squares fitting are summarized in Supplemental Table S2.
(C) Surface views of ppERK2 and p38α comparing proposed DEJL docking sites. These overlap the DEJL docking site in p38α identified by X-ray crystallography (Chang et al., 2002).
Molecular Cell  , 43-55DOI: ( /S (04) )

4. Figure 3 HX-MS Reveals DEF Motif Interactions with ppERK2
(A) Sequence coverage map of ppERK2 showing 29 peptides observed by HX-MS, colored as in Figure 2 to indicate time courses of exchange for peptides that varied upon binding Elk1-DEF peptide.
Molecular Cell  , 43-55DOI: ( /S (04) )

5. Figure 4
Modeling DEF Peptide Interactions with a Hydrophobic Molecular Surface in ppERK2
(A) Molecular surface representation of ppERK2 (2ERK), showing surface-accessible areas of the regions with greatest solvent protection by Elk1-DEF peptide. Peptides 23/L, 24/25, and 30 are colored red, green, and cyan, respectively, pT183 and pY185 residues are orange, and the catalytic nucleophile, D147, is blue.
(B) Representation of ppERK2, in which surface hydrophobic residues are colored yellow, reveals a hydrophobic pocket overlapping the solvent protected region.
(C) Docking interactions between ppERK2 and a peptide sequence derived from Elk1 (PRSPAKLSFQFPS, red) modeled with O. The conformation of the Elk1 peptide is constrained to orient the hydroxyl side chain of the first Ser residue within hydrogen bonding distance from D147. In this model, the P−2 peptide Pro residue interacts with hydrophobic residues W190 and Y191, and the P+1 peptide Pro residue interacts with hydrophobic/nonpolar residues V186 and A187.
(D) Rotation of the image in (C), highlighting interactions between the Elk1 peptide DEF motif and the putative hydrophobic binding pocket. Close interactions are apparent between the two peptide Phe residues and hydrophobic residues pY185, M197, L198, Y231, and L232 within ppERK2. L235 is buried deep within the pocket, and Y261 is located in the MAP kinase insert in proximity to the peptide C-terminal Pro.
Molecular Cell  , 43-55DOI: ( /S (04) )

6. Figure 5
Mutation of Hydrophobic Residues in ERK2 Define the DEF Motif Binding Pocket
(A) Disruption of Elk1-DEF binding by mutagenesis of ERK2. Hydrophobic residues in ERK2 (Figure 4D) were mutated singly and in combination, followed by diphosphorylation and purification of kinase. Top: Immunoreactivity of WT versus mutant forms of ppERK2 bound to GST-Elk1(307–428) in pull-down experiments. Bottom: Relative binding of ppERK2 to GST-Elk1(307–428), indicating averages and standard deviations of three independent experiments.
(B) Disruption of pS383 phosphorylation by mutagenesis of ERK2. Top: Specific activity of WT and mutant ppERK2 toward GST-Elk1(307–428), indicating average and standard deviation of three independent experiments. Bottom: Total phosphorylation of GST-Elk1(307–428) (lanes 1 and 3–12) or GST-Elk1-ΔF (lane 2) by WT or mutant ppERK2 measured by 32P incorporation after 5 min. Amounts of ppERK2 were adjusted according to specific activity as described in the Experimental Procedures. Relative levels of pS383 were then visualized by Western blotting probed with anti-pS383 antibody.
(C) Alignment characterized DEF motif sequences in substrates, underlining proximal phosphorylation sites, with sequences from nucleoporins 153 and 214, showing potential DEF motifs within FG repeat domains that have been previously shown to bind ERK2.
(D) Disruption of GST-nucleoporin 153c interactions by mutagenesis of ERK2. ppERK2 mutants described in (A) were assayed for binding to GST-Nup153c in pull-down experiments and visualized for ppERK2 immunoreactivity. The results verify that mutation of hydrophobic residues within the putative binding pocket in ERK2 significantly interfere with DEF motif interactions in Elk1 and nucleoporin 153.
Molecular Cell  , 43-55DOI: ( /S (04) )

7. Figure 6
DEF Docking Motif Interactions Are Enhanced by Phosphorylation and Activation of ERK2
(A) Molecular representation of the DEF motif binding site, comparing ppERK2 with ERK2 (2ERK, Canagarajah et al., 1997; 1ERK, Zhang et al., 1994). Left: Surface hydrophobic residues in ppERK2 are colored in yellow, showing the hydrophobic pocket for binding the Elk1-DEF peptide (red) and positions of activation lip residues F181 and L182 (green). Right: Surface residues in ERK2, showing movement of F181 and L182 upon rearrangement of the activation lip. F181 moves by 22.7 Å between these structures. The pocket in ppERK2 found to be involved in DEF motif binding is occupied by intramolecular interactions with the activation lip residues, predicting that the inactive form of ERK2 should have lower binding affinity for the DEF motif.
(B) GST pull-down assays demonstrating higher affinity of Elk1 for ppERK2 than ERK2. WT-ERK2 and ppERK2 were incubated with either GST-Elk1(307–428) or GST-Elk1-ΔF (FQF to AAA), and levels of binding were analyzed by Western blotting. Graphs represent averages and maximal/minimal values for two independent experiments.
(C) GST pull-down assays demonstrating higher affinity of nucleoporin 153 for ppERK2 than ERK2. WT-ERK2 and ppERK2 were incubated with GST-Nup153c, and levels of binding were analyzed by Western blotting. Top: Representative Western blots showing greater binding of ppERK2 than ERK2 to GST-Elk1 and GST-Nup153c. “Bkg” represents nonspecific binding of ERK2 to GSH-Sepharose in the absence of GST-Elk1 or GST-Nup153c. Bottom: Quantitation of ppERK2 and ERK2 binding to GST-Nup153c, representing averages and standard deviations for three independent experiments.
Molecular Cell  , 43-55DOI: ( /S (04) )