Structural basis for the selective inhibition of JNK1 by the scaffolding protein JIP1 and SP600125
2004; Springer Nature; Volume: 23; Issue: 11 Linguagem: Inglês
10.1038/sj.emboj.7600212
ISSN1460-2075
AutoresYong-Seok Heo, Su Kyoung Kim, Chang Il Seo, Young Kwan Kim, B.-J. Sung, Hye Shin Lee, Jae Il Lee, Sam‐Yong Park, Jin Hwan Kim, Kwang Yeon Hwang, Young-Lan Hyun, Young Ho Jeon, Seonggu Ro, Joong Myung Cho, Tae Gyu Lee, Chul‐Hak Yang,
Tópico(s)Cancer Mechanisms and Therapy
ResumoArticle13 May 2004free access Structural basis for the selective inhibition of JNK1 by the scaffolding protein JIP1 and SP600125 Yong-Seok Heo Yong-Seok Heo The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Molecular Enzymology Laboratory, School of Chemistry and Molecular Engineering, Seoul National University, Seoul, Korea Search for more papers by this author Su-Kyoung Kim Su-Kyoung Kim The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Search for more papers by this author Chang Il Seo Chang Il Seo The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Search for more papers by this author Young Kwan Kim Young Kwan Kim The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Search for more papers by this author Byung-Je Sung Byung-Je Sung The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Search for more papers by this author Hye Shin Lee Hye Shin Lee The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Search for more papers by this author Jae Il Lee Jae Il Lee The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Search for more papers by this author Sam-Yong Park Sam-Yong Park Protein Design Laboratory, Yokohama City University, Suechiro-cho, Tsurumi, Yokohama, Japan Search for more papers by this author Jin Hwan Kim Jin Hwan Kim The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Search for more papers by this author Kwang Yeon Hwang Kwang Yeon Hwang The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Search for more papers by this author Young-Lan Hyun Young-Lan Hyun The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Search for more papers by this author Young Ho Jeon Young Ho Jeon The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Search for more papers by this author Seonggu Ro Seonggu Ro The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Search for more papers by this author Joong Myung Cho Joong Myung Cho The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Search for more papers by this author Tae Gyu Lee Corresponding Author Tae Gyu Lee The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Search for more papers by this author Chul-Hak Yang Corresponding Author Chul-Hak Yang Molecular Enzymology Laboratory, School of Chemistry and Molecular Engineering, Seoul National University, Seoul, Korea Search for more papers by this author Yong-Seok Heo Yong-Seok Heo The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Molecular Enzymology Laboratory, School of Chemistry and Molecular Engineering, Seoul National University, Seoul, Korea Search for more papers by this author Su-Kyoung Kim Su-Kyoung Kim The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Search for more papers by this author Chang Il Seo Chang Il Seo The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Search for more papers by this author Young Kwan Kim Young Kwan Kim The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Search for more papers by this author Byung-Je Sung Byung-Je Sung The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Search for more papers by this author Hye Shin Lee Hye Shin Lee The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Search for more papers by this author Jae Il Lee Jae Il Lee The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Search for more papers by this author Sam-Yong Park Sam-Yong Park Protein Design Laboratory, Yokohama City University, Suechiro-cho, Tsurumi, Yokohama, Japan Search for more papers by this author Jin Hwan Kim Jin Hwan Kim The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Search for more papers by this author Kwang Yeon Hwang Kwang Yeon Hwang The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Search for more papers by this author Young-Lan Hyun Young-Lan Hyun The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Search for more papers by this author Young Ho Jeon Young Ho Jeon The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Search for more papers by this author Seonggu Ro Seonggu Ro The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Search for more papers by this author Joong Myung Cho Joong Myung Cho The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Search for more papers by this author Tae Gyu Lee Corresponding Author Tae Gyu Lee The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea Search for more papers by this author Chul-Hak Yang Corresponding Author Chul-Hak Yang Molecular Enzymology Laboratory, School of Chemistry and Molecular Engineering, Seoul National University, Seoul, Korea Search for more papers by this author Author Information Yong-Seok Heo1,2, Su-Kyoung Kim1, Chang Il Seo1, Young Kwan Kim1, Byung-Je Sung1, Hye Shin Lee1, Jae Il Lee1, Sam-Yong Park3, Jin Hwan Kim1, Kwang Yeon Hwang1, Young-Lan Hyun1, Young Ho Jeon1, Seonggu Ro1, Joong Myung Cho1, Tae Gyu Lee 1 and Chul-Hak Yang 2 1The Division of Drug Discovery, CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejon, Korea 2Molecular Enzymology Laboratory, School of Chemistry and Molecular Engineering, Seoul National University, Seoul, Korea 3Protein Design Laboratory, Yokohama City University, Suechiro-cho, Tsurumi, Yokohama, Japan *Corresponding authors. CrystalGenomics, Inc., Daeduk Biocommunity, Jeonmin-dong, Yuseong-gu, Daejeon 305-390, Korea. Tel.: +82 42 866 9320; Fax: +82 42 866 9301; E-mail: [email protected] Enzymology Laboratory, School of Chemistry and Molecular Engineering, Seoul National University, NS60, Seoul 151-742, Korea. Tel.: +82 2 878 8545; Fax: +82 2 889 1568; E-mail: [email protected] The EMBO Journal (2004)23:2185-2195https://doi.org/10.1038/sj.emboj.7600212 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The c-jun N-terminal kinase (JNK) signaling pathway is regulated by JNK-interacting protein-1 (JIP1), which is a scaffolding protein assembling the components of the JNK cascade. Overexpression of JIP1 deactivates the JNK pathway selectively by cytoplasmic retention of JNK and thereby inhibits gene expression mediated by JNK, which occurs in the nucleus. Here, we report the crystal structure of human JNK1 complexed with pepJIP1, the peptide fragment of JIP1, revealing its selectivity for JNK1 over other MAPKs and the allosteric inhibition mechanism. The van der Waals contacts by the three residues (Pro157, Leu160, and Leu162) of pepJIP1 and the hydrogen bonding between Glu329 of JNK1 and Arg156 of pepJIP1 are critical for the selective binding. Binding of the peptide also induces a hinge motion between the N- and C-terminal domains of JNK1 and distorts the ATP-binding cleft, reducing the affinity of the kinase for ATP. In addition, we also determined the ternary complex structure of pepJIP1-bound JNK1 complexed with SP600125, an ATP-competitive inhibitor of JNK, providing the basis for the JNK specificity of the compound. Introduction The mitogen-activated protein kinase (MAPK) groups play critical roles in many physiological processes including cell growth, oncogenic transformation, cell differentiation, apoptosis, and the immune response by mediating extracellular stresses to cellular signals. The MAPKs are activated by cascade modules that consist of the upstream kinases of MAPKs, MAPK kinases (MKKs), and the upstream kinase of MKKs, MAPK kinase kinases (MKKKs). Of the MAPK families, Erk, p38 MAPKs, and JNKs have been studied extensively as major stress-activated MAPKs. The diverse combination of MAPKs, MKKs, and MKKKs seems to cause extreme complexity in the cellular responses to a wide range of stimuli. Then, what gives the ability of specific regulation of the MAPK pathways? The specificity is achieved, in part, by the use of scaffolding proteins to coordinate the interaction of the three components of the MAPK modules via direct protein–protein interactions. Scaffolding of multicomponent regulatory systems is now recognized as a major mechanism for controlling signal transduction pathways (Mochly-Rosen, 1995; Pawson, 1995; Faux and Scott, 1996; Pawson and Scott, 1997). Assembly of MAPK modules by scaffolding proteins makes it possible to segregate MAPK signaling components into units that are responsive to independent stimuli, and therefore obtain appropriate subcellular targeting by being insulated from similar modules (Elion, 1998; Garrington and Johnson, 1999). JNK-interacting protein-1 (JIP1) was identified as a scaffolding protein of the JNK module in yeast two-hybrid analysis, enhancing the correspondent signaling through the pathway (Whitmarsh et al, 1998). However, the overexpression of either the domain within JIP1 that binds to JNK, or the full-length protein, potently inhibits JNK signaling in the cell (Dickens et al, 1997). This is because JIP1 blocks nuclear translocation of JNK with its ability to retain JNK in the cytoplasm and sequesters JNK module components into different JIP1 complexes when it is overexpressed. It has been proposed that JIP1 facilitates mixed-lineage kinase (MLK)-dependent JNK signal transduction by aggregating the three components of the module, MLK, MKK7, and JNK (Tournier et al, 1999; Davis, 2000). JIP1 consists of 707 amino acids and contains an N-terminal JNK-binding domain (residues 1–282) and C-terminal MLK- and MKK7-binding domain (residues 283–660). The C-terminal MLK- and MKK7-binding domain contains a putative phosphotyrosine interaction domain and an Src homology 3 (SH3) domain (Dickens et al, 1997; Whitmarsh et al, 1998). JIP1 is highly concentrated in the adult brain, and particularly enriched in the cerebral cortex and hippocampus (Kim et al, 1999). The minimal region of JIP1 has been identified as retaining the JNK-inhibitory property (Dickens et al, 1997; Bonny et al, 2001; Barr et al, 2002). This peptide, pepJIP1 (a peptide version of JIP1), inhibited JNK activity in vitro toward recombinant c-jun, Elk, and ATF2 up to 90% with significant selectivity of no inhibition of the related Erk and p38 MAPKs. MAPK docking sites have been identified for substrate transcription factors, MKKs, and scaffolding proteins. It is interesting that the docking sites of substrate transcription factors, MKKs, and scaffolding proteins of MAPKs have a consensus in sequences, (R/K)2–3-X1–6-ϕA-X-ϕB, where ϕA and ϕB are hydrophobic residues such as Leu, Ile, or Val (Sharrocks et al, 2000). The variability in the number and position of hydrophobic and basic residues within the docking site is known to determine specificity. The number and spacing of basic residues, in particular, make an important contribution but do not appear to be the only specificity determinants (Smith et al, 2000; Tanoue et al, 2001). As a counterpart of the docking sites, MAPKs have surfaces (docking grooves) for docking with the docking sites of MAPK-interacting molecules, which regulate the docking specificity. The docking groove is located on the opposite side of the substrate recognition site. Besides the location of the docking grooves, the docking interaction is different from the transient enzyme–substrate recognition for substrate phosphorylation in the sense that the Ser/Thr-Pro sites, which are phosphorylated in substrates by MAPKs, do not seem to possess a clear consensus motif as found in docking sites. In addition, peptide versions of the Ser/Thr-Pro sites in substrates do not mimic the phosphorylation kinetics of the whole protein, whereas peptides of the minimal regions in docking sites retain properties similar to those of the whole protein (Kemp and Pearson, 1991; Dickens et al, 1997; Bardwell et al, 2001; Barr et al, 2002). Three distinct genes encoding JNKs, Jnk1, Jnk2, and Jnk3, have been identified and at least 10 different splicing isoforms exist in mammalian cells (Gupta et al, 1996). The JNK isoforms differ in their binding activity with ATF2, Elk-1, and c-jun transcription factors. So, individual members of the JNK group may therefore selectively target specific transcription factors in vivo. JNK1 and JNK2 are widely expressed in a variety of tissues, whereas JNK3 is selectively expressed in the brain and to a lesser extent in the hearts and testis (Mohit et al, 1995; Martin et al, 1996). Activation of the JNK pathway has been documented in a number of diseases, providing the rationale for targeting this pathway for drug discovery (Manning and Davis, 2003). In rheumatoid arthritis, cartilage and bone erosion is promoted by inducible expression of matrix metalloproteinases (MMPs), which is regulated by activation of AP-1 via the JNK pathway (Gum et al, 1997; Han et al, 2002). The JNK pathway is activated in vulnerable neurons in patients with Alzheimer's disease (AD), suggesting that the JNK pathway is involved in the pathophysiology and pathogenesis of AD (Troy et al, 2001; Bozyczko-Coyne et al, 2002; Zhu et al, 2002). And it has been reported that JNK is a crucial mediator of obesity and insulin resistance and a potential target for type II diabetes (Waeber et al, 2000; Hirosumi et al, 2002). In the hope that therapeutic inhibition of JNK may provide clinical benefit in diverse diseases, JNK inhibitors have been discovered and characterized. As a reversible ATP-competitive JNK inhibitor, SP600125 was reported to inhibit JNK with high potency and selectivity (Bennett et al, 2001). To investigate the mechanism of selective regulation of JNK by JIP1, we have determined the crystal structure of JNK1 in complex with pepJIP1, the docking site peptide of JIP1 (residues 153–163), at a resolution of 2.35 Å. It is the first structure demonstrating the way by which MAPKs are selectively regulated by their scaffolding proteins. In this structure, it is found that the docking site of the scaffolding protein also binds at the same docking grooves of JNK1 as the upstream kinase and the substrate transcription factor do in p38 MAPK (Chang et al, 2002), but with quite different interaction modes. Interestingly, the dramatic interdomain rearrangement between the N- and C-terminal domains of JNK1 upon pepJIP1 binding distorts the ATP-binding pocket, and thus inhibits the catalytic activity of JNK due to the failure of productive ATP binding. We also determined the structure of the ternary complex JNK1–pepJIP1–SP600125 at a resolution of 2.7 Å. Despite the small size of SP600125 (molecular weight=220), it effectively occupies the hydrophobic pocket of the ATP-binding site in JNK1, and the variations of the crucial hydrophobic residues in other MAP kinases give SP600125 the JNK specificity. These structural studies will facilitate the discovery of more potent and selective JNK inhibitors in the future. Results Overall structure of JNK1 complexed with pepJIP1 As a result of the high homology of sequences with 92% identity, the overall architecture of JNK1 is substantially equal to that of JNK3 (PDB entry 1JNK; Xie et al, 1998). The N-terminal domain (residues 9–112 and 347–363) contains seven β strands (β1, β2, β3, β4, β5, β6, and β7) and two α helices (α1 and α14). The C-terminal domain (residues 113–337) consists of a bundle of seven α helices (α2, α3, α5, α6, α8, α11, and α12) with five short 310-helices (α4, α7, α9, α10, and α13) and three β strands (β8, β9, and β10). The N-terminal domain and the C-terminal domain are linked by two loops, one (residues 108–112) of which is located between β7 and β8 and the other (residues 331–351) is a long connector of α14 to α13 of the C-terminal domain, and some part of the loop is disordered in our model as in the case of the JNK3 structure, implying the high flexibility of the loop (Figure 1A). Figure 1.Overall structure of JNK1 complexed with pepJIP1 at a resolution of 2.35 Å. (A) JNK1 is shown in a ribbon model and the disordered regions of JNK1 (residues 173–189, 282–286, and 337–348) are expressed as dotted lines. The bound pepJIP1 is shown in a stick model of atomic color. The regions for the N- and C-terminal domains are indicated. (B) The sigma-A-weighted 2Fo–Fc electron density map calculated with a final refined model without pepJIP1 at a level of 1.2σ. The peptide used in the experiment is RPKRPTTLNLF, but the starting residue, Arg153, is not shown in the density. (C) Surface representation of JNK1 in complex with pepJIP1, colored by electrostatic potential. (D) The docking site sequences of the scaffolding protein (JIP1), the upstream kinase (MKK7), and the substrate transcription factor (c-jun), which are components of the JNK signaling regulated by JIP1 scaffolding protein. The basic residues and ϕ residues are shown in blue and violet letters, respectively. Download figure Download PowerPoint PepJIP1 used in this complex study is an 11-mer docking site peptide (residues 153–163 of JIP1 with the sequence of RPKRPTTLNLF) containing the ϕA-X-ϕB motif, where ϕA and ϕB are Leu160 and Leu162 of JIP1, respectively. The peptide docks to the docking groove of JNK1, a surface of the C-terminal domain covering α2, α3, α13, and β8, which is away from the ATP pocket or substrate recognition site, implying an allosteric inhibition mechanism of pepJIP1. The peptide version of the JIP1 scaffolding protein used in the study has a consensus motif with the upstream kinase (MKK7) and the substrate transcription factor (c-jun) (Figure 1D). Unexpectedly, the side chain of Lys155 (ϕA−5) of pepJIP1, which is also conserved among the basic residues of the docking sites in both MKK7 and c-jun, is not shown in the electron density map due to the lack of interaction with the docking groove of JNK1 (Figure 1B), implying that the basic residues crucial for binding cannot be predicted only by the sequence comparison at the docking sites. Interactions of the complex The complex forms a continuous interface that buries a total surface area of 1034 Å2 (Figure 1C). All of the buried area is contributed by interactions from only the C-terminal domain of JNK1. The interface is mostly nonpolar, relying on hydrophobic interaction. Leu160 and Leu162 of the ϕA-X-ϕB motif in pepJIP1 are extensively surrounded by many hydrophobic residues of JNK1 including Met121, Val118, Leu115, Ala113, Leu123, Val59, Leu131, and Cys163 (Figure 2). Asn161 of pepJIP1, the X residue of ϕA-X-ϕB motif, does not have any interaction with JNK1 and this is consistent with the fact that the X residues have high diversity among the proteins containing the docking site (Yang et al, 1998; Holland and Cooper, 1999). The side chains of Thr158 (ϕA−2) and Thr159 (ϕA−1) are also oriented to the opposite side of the interface, resulting in no interaction. The backbone amide of Leu160 (ϕA) makes a hydrogen bond with the backbone carbonyl group of Ser161 of JNK1. The oxygen atom of the backbone carbonyl of Thr159 (ϕA−1) hydrogen bonds to the N ε atom of Arg127. Pro157 (ϕA−3) has van der Waals contact with the side chains of Tyr130, Glu126, and Trp324, and Pro154 (ϕA−6) makes a weak interaction with Val323. Finally, Arg156 (ϕA−4) interacts with Glu329 with a bidentate salt bridge of length 2.70 Å. Arg153 (ϕA−7) and Phe163 (ϕB+1) are outside of the complex interface and make little or no contribution to the interaction, so it is not surprising that Arg153 (ϕA−7) is not shown in the electron density map. In the previous study of glycine or alanine replacement and truncation (Dickens et al, 1997; Barr et al, 2002), Arg156 (ϕA−4), Pro157 (ϕA−3), Leu160 (ϕA), and Leu162 (ϕB) were identified as critical mediators of the JNK inhibition, and a single residue removal from the N-terminus or the C-terminus of pepJIP1 caused no difference in JNK inhibition. The structural features of the JNK1–pepJIP1 complex are completely consistent with the results of alanine scanning replacement and truncation analysis within pepJIP1 to identify the crucial residues for JNK inhibition. To confirm the importance of Arg127 and Glu329 of JNK1 for pepJIP1 binding, we have used isothermal titration calorimetry (ITC) to measure Kd values of pepJIP1 to wild-type JNK1 and mutants of R127A and E329A. The Kd values for pepJIP1 to bind to wild type, R127A, and E329A were found to be 0.42±0.13, 6.4±2.2, and 9.1±3.4 μM, respectively (Figure 2C–E). These Kd values derived from ITC agree well with the above structural description, demonstrating that Arg127 and Glu329 of JNK1 are key residues for pepJIP1 binding. Figure 2.Interactions between JNK1 and pepJIP1. (A) Stereoview of the interactions between JNK1 (violet) and pepJIP1 (green). The residues of JNK1 involved in the interactions are shown in white labels and those of pepJIP1 in green labels. The hydrogen bonds in the interactions are shown as thin white lines. (B) Schematic expression of the interactions. (C) The binding affinity of pepJIP1 to wild-type JNK1 was measured by ITC. The upper panel shows the raw data, a trace of power with time. The lower panel shows the integrated heats from each injection, and the line through the measured points shows the best-fit model for a single binding site. (D, E) The respective Kd values of pepJIP1 to JNK1 mutants, R127A and E329A, were derived from the ITC data. Download figure Download PowerPoint pepJIP1 as a JNK-specific inhibitor In the comparison of the complex structure of JNK1–pepJIP1 with that of p38-pepMEF2A or p38-pepMKK3b (PDB entries 1LEW and 1LEZ, respectively; Chang et al, 2002), the overall conformations and binding positions of the bound peptides are quite different from each other when the structures of JNK1 and p38 are superimposed, although the difference between those of pepMEF2A and pepMKK3b is relatively insignificant (Figure 3A). The most notable difference is that the ϕA-X-ϕB motif of pepJIP1, especially Leu162 (ϕB), is shifted by the structural difference between JNK1 and p38 in the region containing the hydrophobic residues critical for the ϕA-X-ϕB motif binding. In this region, JNK1 forms a one-turn helix (α2), but a two-turn helix in p38. The structural difference of this region can be predicted from the sequential mismatch due to one insertion in the sequence of p38 (Figure 3D). Another notable difference in the conformation between pepJIP1 and pepMEF2A is that Arg156 (ϕA−4) interacts with Glu329 of JNK1 through a bidentate salt bridge, which is critical for pepJIP1 binding to JNK1, but the corresponding ϕA−4 residue in pepMEF2A, which is proline, cannot make such an ionic interaction. Although the residues of p38 and Erk corresponding to Glu329 of JNK1 are also glutamates, they could not interact with Arg156 (ϕA−4) of pepJIP1 due to structural differences (Figure 3A). And the fact that the structure of Glu329 of JNK1 complexed with pepJIP1 is actually identical to that of Glu367 of free JNK3 indicates that the position of the side chain of Glu329 is not changed to interact with Arg156 (ϕA−4), suggesting the high specificity of JNK1 to recognize JIP1 (Figure 3A). Although it has been suggested that the CD domain (common docking domain), which is composed of acidic residues in the docking groove of MAPK, is indispensable for binding of the docking site (Tanoue and Nishida, 2002), no possible interaction was observed between the acidic residues of p38 and the basic residues of the MEF2A or MKK3b docking site. However, the distinct interaction between Glu329 of JNK1 and Arg156 of JIP1 shows the importance of the CD domain for the selective binding of a specific docking site. In Figures 1D and 3E, Lys155 (ϕA−5) is conserved in the docking sites while Arg156 (ϕA−4) is not. But the key interaction with the CD domain of JNK1 is made only by the unconserved basic residue, Arg156 (ϕA−4), implying that the number and position of basic residues in the docking sites are important determinants of specificity. Figure 3.Structural comparison of the docking site peptides bound in the docking grooves of MAPKs. (A) Stereoview of the overlaid structures of pepJIP1 (saturated yellow) bound in JNK1 (whitened yellow) and pepMEF2A (saturated red) or pepMKK3b (saturated blue) bound in p38 MAPK (whitened blue) when the docking grooves of JNK1 and p38 are superimposed. The residues of pepJIP1, pepMEF2A, and pepMKK7 are labeled black, red, and blue, respectively. And the structure of E327 of JNK1 is compared with those of the corresponding glutamates of JNK3 (green), p38 (blue), and Erk2 (pink). (B) The structural deviation between pepJIP1 (yellow) and pepMEF2A (pink) when their ϕA-X-ϕB motifs are superimposed. (C) The structural comparison between pepJIP1 (yellow) and pepMKK3b (blue). In (B, C), the conformational conservations of the ϕA-X-ϕB motifs are highlighted by gray rectangles. (D) The differences of the sequences in the α2 helices of the docking grooves between JNK1 and p38 MAPK. The residues participating in hydrophobic interactions with the docking site peptides are shown in green letters. (E) The sequences of docking site peptides represented in (A–C). The ϕ residues are shown in violet letters. Download figure Download PowerPoint In conclusion, the two major differences, the structural difference in α2 helix and the involvement of the CD domain in docking interaction, provide the rationale for the predominant selectivity of pepJIP1 in inhibition of JNK, but not the related Erk or p38 MAPKs. Conformational conservation of the ϕA-X-ϕB motif Despite the considerable differences in the overall conformation of the docking site peptides, it is shown that the conformations in the ϕA-X-ϕB motifs are conserved (Figure 3B and C). When the ϕA-X-ϕB motif of pepJIP1 is superimposed on that of pepMEF2A or pepMKK3b, the major torsional differences are found at the bond outside of the ϕA-X-ϕB motifs, and thus the relative orientation of the side chains in the ϕA-X-ϕB motifs can be conserved. This result implicates that any conformational change inside of the ϕA-X-ϕB motif may decrease the hydrophobic interactions of the two key residues, ϕA and ϕB, with the docking grooves of MAPKs, therefore inducing failure in the effective binding of docking sites to MAPKs. In conclusion, the docking site can be characterized by not only the sequential similarity of the ϕA-X-ϕB motifs but also the conformational conservation of the motifs in their structures. Interdomain rearrangement mediated by pepJIP1 binding The most interesting result of the pepJIP1 binding to JNK1 is the rotation of the N-terminal domain relative to the C-terminal domain by approximately 15°, thereby distorting the ATP-binding site and inducing disorder of the phosphorylation loop (Figure 4). Figure 4.Distortion of the ATP-binding site caused by interdomain rearrangement upon pepJIP1 binding. (A) Structural comparison between JNK3 (green) and pepJIP1-bound JNK1 (violet) when the C-terminal domains of the kinases are superimposed. The conformational differences of the N-terminal domains can be easily distinguished when the conventional view of kinases is rotated by 45° along the horizontal axis. The yellow circle indicates the interaction between the α1 helix and the phosphorylation loop in JNK3, but not existing in JNK1 complexed with pepJIP1. (B) Comparison of ATP-binding sites between the JNK1–pepJIP1 (violet) and JNK3–AMPPNP (green) complexes. The AMPPNP bound in JNK3 is shown in a ball-and-stick model. The residues of JNK3 involved in the hydrogen bonding with AMPPNP are labeled. The side chains of the residues in the glycine-rich loop including E75 and A74 of JNK3 are omitted for clarity because the backbone amide groups only are involved in the hydrogen bonds with the phosphate groups of AMPPNP. (C) The structural comparison of the residues crucial for the catalytic activity between the JNK1–pepJIP1 (violet) and JNK3–AMPPNP (green) complexes. The residues in JNK1 and JNK3 are labeled red and black, respectively. In (B, C), hydrogen bonds are indicated by dashed lines. Download figure Download PowerPoint The root-mean-square (r.m.s.) deviations for Cα atoms of the N- and C-terminal domain between the structures of JNK1–pepJIP1 and JNK3 are 1.27 and 0.79 Å, respectively. These values show that there is no significant structural difference between the corresponding domains. Ho
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