Examining docking interactions on ERK2 with modular peptide substrates.
Examining docking interactions on ERK2 with modular peptide substrates.
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检查与模块化肽底物上ERK2上的对接相互作用。
DOI：
10.1021/bi201103b
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发表时间：
2011-11-08
期刊：
影响因子：
2.9
通讯作者：
Dalby, Kevin N.
中科院分区：
文献类型：
--
作者：
Lee, Sunbae;Warthaka, Mangalika;Yan, Chunli;Kaoud, Tamer S.;Ren, Pengyu;Dalby, Kevin N.
ERK 2主要通过两个募集位点识别底物，这两个位点位于激酶的活性位点裂缝之外。这些募集位点结合称为对接位点的模块对接序列，并且是用于开发非ATP竞争性抑制剂的潜在有吸引力的位点。D-募集位点（DRS）和F-募集位点（FRS）分别结合D-位点和F-位点。例如，已经提出靶向FRS的肽抑制所有ERK 2活性（J.Biol.Chem.2001，276，965-973），然而还没有确定这种抑制是空间还是变构起源的。为了促进抑制剂设计并检查ERK 2内募集位点与其他配体识别位点的潜在偶联，使用两种新的ERK 2模块肽底物研究了ERK 2内的高能偶联。建模显示，一种肽（Sub-D）识别DRS，而另一种肽（Sub-F）结合FRS。稳态动力学分析表明，肽底物和ATP之间的热力学联系的证据很少。两种肽通过随机顺序顺序机制磷酸化，kcat/Km与Ets-1（一种真正的ERK 2底物）相当。用含有模块对接序列的肽占据FRS对ERK 2磷酸化Sub-D的内在能力没有影响。使用含有模块对接序列的肽占据DRS对ERK 2磷酸化Sub-F的内在能力有轻微影响（kcat增加1.3 ± 0.1倍）。这些数据表明，虽然在DRS和FRS的对接相互作用是大力解耦，DRS可以表现出弱的通信到活性位点。此外，他们认为结合到FRS的肽通过空间机制抑制蛋白质底物的磷酸化。建模和动力学数据表明，ERK 2通过其DRS募集到细胞位置可能有助于形成F位点选择性ERK 2信号传导复合物，而通过FRS募集可能通过空间抑制机制抑制ERK 2。这种募集可以作为ERK 2调节的额外水平。
ERK2 primarily recognizes substrates through two recruitment sites, which lie outside the active site cleft of the kinase. These recruitment sites bind modular-docking sequences called docking sites and are potentially attractive sites for the development of non-ATP competitive inhibitors. The D-recruitment site (DRS) and the F-recruitment site (FRS) bind D-sites and F-sites, respectively. For example, peptides that target the FRS have been proposed to inhibit all ERK2 activity (J. Biol. Chem. 2001, 276, 965-973), however it has not been established whether this inhibition is steric or allosteric in origin. To facilitate inhibitor design and to examine potential coupling of recruitment sites to other ligand recognition sites within ERK2 Energetic coupling within ERK2 was investigated using two new modular peptide substrates for ERK2. Modeling shows that one peptide (Sub-D) recognizes the DRS, while the other peptide (Sub-F) binds the FRS. A steady-state kinetic analysis reveals little evidence of thermodynamic linkage between peptide substrate and ATP. Both peptides are phosphorylated through a random-order sequential mechanism with a kcat/Km comparable to Ets-1, a bona fide ERK2 substrate. Occupancy of the FRS with a peptide containing a modular docking sequence has no effect on the intrinsic ability of ERK2 to phosphorylate Sub-D. Occupancy of the DRS with a peptide containing a modular docking sequence has a slight effect (1.3 ± 0.1-fold increase in kcat) on the intrinsic ability of ERK2 to phosphorylate Sub-F. These data suggest that while docking interactions at the DRS and the FRS are energetically uncoupled, the DRS can exhibit weak communication to the active site. In addition, they suggest that peptides bound to the FRS inhibit the phosphorylation of protein substrates through a steric mechanism. The modeling and kinetic data suggest that the recruitment of ERK2 to cellular locations via its DRS may facilitate the formation of F-site selective ERK2 signaling complexes, while recruitment via the FRS will likely inhibit ERK2 through a steric mechanism of inhibition. Such recruitment may serve as an additional level of ERK2 regulation.
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影响因子：
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作者：
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通讯作者：
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DOI：
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发表时间：
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影响因子：
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影响因子：
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通讯作者：
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