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Investigation of the intermolecular recognition mechanism between the E3 ubiquitin ligase Keap1 and substrate based on multiple substrates analysis

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Abstract

E3 ubiquitin ligases are attractive drug targets due to their specificity to the ubiquitin machinery. However, the development of E3 ligase inhibitors has proven challenging for the fact that they must disrupt protein–protein interactions (PPIs). The E3 ligase involved in interactome provide new hope for the discovery of the E3 ligase inhibitors. These currently known natural binding partners of the E3 ligase can benefit the discovery of other unknown substrates and also the E3 ligase inhibitors. Herein, we present a novel strategy that using multiple substrates to elucidate the molecular recognition mechanism of E3 ubiquitin ligase. Molecular dynamics simulation, molecular mechanics-generalized born surface area (MM-GBSA) binding energy calculation and energy decomposition scheme were incorporated to evaluate the quantitative contributions of sub-pocket and per-residue to binding. In this case, Kelch-like ECH-associated protein-1 (Keap1), a substrate adaptor component of the Cullin–RING ubiquitin ligases complex, is applied for the investigation of how it recognize its substrates, especially Nrf2, a master regulator of the antioxidant response. By analyzing multiple substrates binding determinants, we found that both the polar sub-pockets (P1 and P2) and the nonpolar sub-pockets (P4 and P5) of Keap1 can make remarkable contributions to intermolecular interactions. This finding stresses the requirement for substrates to interact with the polar and nonpolar sub-pockets simultaneously. The results discussed in this paper not only show the binding determinants of the Keap1 substrates but also provide valuable implications for both Keap1 substrate discovery and PPI inhibitor design.

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Abbreviations

Keap1:

Kelch-like ECH-associated protein-1

Nrf2:

Nuclear factor erythroid 2-related factor 2

MD:

Molecular dynamics

RMSD:

Root-mean square deviation

MM-GBSA:

Molecular mechanics generalized born surface area

PPI:

Protein–protein interaction

CRL:

Cullin–RING ubiquitin ligases

Cul3:

Cullin 3

Rbx1:

Ring box 1

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Acknowledgments

This work is supported by the Project 81230078 (key program), 81202463 (youth foundation), 81173087 and 91129732 of National Natural Science Foundation of China, 2014ZX09507002-005-015, 2013ZX09402102-001-005 and 2010ZX09401-401 of the National Major Science and Technology Project of China (Innovation and Development of New Drugs)

Conflict of interest

The authors declare no other conflicts of interest.

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Authors and Affiliations

  1. State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, 210009, China

    Zheng-Yu Jiang, Li-Li Xu, Meng-Chen Lu, Yang Pan, Hao-Ze Huang, Xiao-Jin Zhang, Hao-Peng Sun & Qi-Dong You

  2. Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing, 210009, China

    Zheng-Yu Jiang, Li-Li Xu, Meng-Chen Lu, Yang Pan, Hao-Ze Huang, Xiao-Jin Zhang, Hao-Peng Sun & Qi-Dong You

  3. Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, 210009, China

    Hao-Peng Sun

  4. Department of Organic Chemistry, School of Science, China Pharmaceutical University, Nanjing, 210009, China

    Xiao-Jin Zhang

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  1. Zheng-Yu Jiang
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Correspondence to Hao-Peng Sun or Qi-Dong You.

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Jiang, ZY., Xu, LL., Lu, MC. et al. Investigation of the intermolecular recognition mechanism between the E3 ubiquitin ligase Keap1 and substrate based on multiple substrates analysis. J Comput Aided Mol Des 28, 1233–1245 (2014). https://doi.org/10.1007/s10822-014-9799-y

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