Deciphering and engineering chromodomain-methyllysine peptide recognition

Hard, Ryan L.; Li, Nan; Wang, He; Ross, Brian; Mo, Gary; Qin, Peng; Stein, Richard S.; Komives, Elizabeth A.; Wang, Yingxiao; Zhang, Jin; Wang, Wei

doi:10.1126/sciadv.aau1447

Cited by 18 publications

(30 citation statements)

References 44 publications

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“…As powerful end-point binding free energy calculation tools, MM/PBSA and MM/GBSA have also been widely used in many other fields besides small-molecule drug design. For example, a very useful application of MM/PB(GB)SA is to predict the interactions between macromolecules, such as protein–protein, ,,,− protein–peptide, ,,− and protein–nucleic acid interactions. ,,− At present, calculations of the absolute binding free energies for these problems remain very challenging for alchemical methods.…”

Section: Applications In Macromolecular Interactionsmentioning

confidence: 99%

End-Point Binding Free Energy Calculation with MM/PBSA and MM/GBSA: Strategies and Applications in Drug Design

et al. 2019

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Molecular mechanics Poisson−Boltzmann surface area (MM/PBSA) and molecular mechanics generalized Born surface area (MM/GBSA) are arguably very popular methods for binding free energy prediction since they are more accurate than most scoring functions of molecular docking and less computationally demanding than alchemical free energy methods. MM/PBSA and MM/GBSA have been widely used in biomolecular studies such as protein folding, protein−ligand binding, protein−protein interaction, etc. In this review, methods to adjust the polar solvation energy and to improve the performance of MM/PBSA and MM/GBSA calculations are reviewed and discussed. The latest applications of MM/GBSA and MM/PBSA in drug design are also presented. This review intends to provide readers with guidance for practically applying MM/PBSA and MM/GBSA in drug design and related research fields. CONTENTS 1. Introduction 9478 2. Methodology 9480 3. Assessing the Performance of MM/PBSA and MM/GBSA 9484 4. The Polar Solvation Energy and Entropy Terms in MM/PB(GB)SA Calculations 9485 4.1. The Polar Solvation Energy Term in MM/ PBSA 9485 4.2. The Polar Energy Solvation Term in MM/ GBSA 9486 4.3. Theory, Implementation, and Limitations of the Variable Dielectric Model in MM/GBSA 9487 4.4. Comparison between PB and GB 9488 4.5. Efficient Entropy Calculation Methods To Estimate the Entropy Change upon Ligand Binding 9488 5.

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Section: Applications In Macromolecular Interactionsmentioning

confidence: 99%

End-Point Binding Free Energy Calculation with MM/PBSA and MM/GBSA: Strategies and Applications in Drug Design

et al. 2019

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“…If a protein-target interaction has a low affinity which results in a low phage ELISA signal, the affinity can be increased based on the published variants with a higher affinity. For instance, it was documented that the Cbx1 double mutant (K43A/D59F; Albanese et al, 2020 ) had a four-fold higher affinity, and the triple mutant (V22E/K25E/D59S; Hard et al, 2018 ) had ten-fold high affinity compared to the wild type Cbx1. These two mutants may have higher ELISA signals than the wild type.…”

Section: Resultsmentioning

confidence: 99%

“…The affinity clamping technology has been developed by which a low-affinity peptide-binding domain is combined with a second domain to enhance affinity ( Hard et al, 2018 ) dramatically. For example, a fibronectin type III domain (FN3) was attached to Grb2 SH2 so that a target peptide can be “clamped” with the improved affinity ( Yasui et al, 2014 ).…”

Section: Resultsmentioning

confidence: 99%

“…The surface-displayed proteins include antibody derivatives [e.g., scFv ( Tadayoni et al, 2021 ) and Fab ( Smith and Kaiser, 2016 )] and some proteins that bind to specific targets. For example, ADP-ribose binding macro domain protein Af1521 was engineered with a 1,000-fold increased affinity towards ADP-ribose ( Nowak et al, 2020 ), the Fyn SH2 domain was engineered with over 100-fold increased affinity towards phosphotyrosine (pY; Kaneko et al, 2012 ; Li et al, 2021 ), and H3K9me3-binding protein Cbx1 was engineered to specifically recognize H3K9me3 with enhanced affinity ( Hard et al, 2018 ; Albanese et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

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Development in Detection Methods for the Expression of Surface-Displayed Proteins

Jiang

Zhao

et al. 2022

Front. Microbiol.

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Directed evolution is a widely-used engineering strategy for improving the stabilities or biochemical functions of proteins by repeated rounds of mutation and selection. A protein of interest is selected as the template and expressed on a molecular display platform such as a bacteriophage for engineering. Initially, the surface-displayed protein template needs to be checked against the desired target via ELISA to examine whether the functions of the displayed template remain intact. The ELISA signal is subject to the protein-target binding affinity. A low-affinity results in a weak ELISA signal which makes it difficult to determine whether the weak signal is because of low affinity or because of poor expression of the protein. Using a methyllysine-binding chromodomain protein Cbx1 that weakly binds to the histone H3K9me3 peptide, we developed and compared three different approaches to increase the signal-to-background ratio of ELISA measurements. We observed that the specific peptide-binding signal was enhanced by increasing the Cbx1 phage concentration on the ELISA plate. The introduction of previously known gain-of-function mutations to the Cbx1 protein significantly increased the ELISA signals. Moreover, we demonstrated that the H3K9me3-specific binding signal was enhanced by fusing Cbx1 with a high-affinity phosphotyrosine-binding protein and by coating the ELISA plate with a mixture of H3K9me3 and phosphotyrosine peptides. This approach also worked with binding to a lower affinity momomethyllysine peptide H3K9me1. These approaches may help improve ELISA experiments when dealing with low-affinity ligand-protein interactions.

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“…3b, 3c and 3d). To the best of our knowledge, the CBX1 double mutant is the tightest H3K9me3 reader protein reported to date 35 . Analogous to HP1α, the mutations did not significantly perturb global folding or thermal stability, and the T m measured for the engineered CBX readers was more than 15 °C greater than the engineered HP1α.…”

Section: H3k9 Methylation Is Recognized More Efficiently By the Engineered Hp1α Chromodomainmentioning

confidence: 99%

Engineered Reader Proteins for Enhanced Detection of Methylated Lysine on Histones

et al. 2019

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Histone post-translational modifications (PTMs) are crucial for many cellular processes including mitosis, transcription, and DNA repair. The cellular readout of histone PTMs is dependent on both the chemical modification and histone site, and the array of histone PTMs on chromatin is dynamic throughout the eukaryotic life cycle. Accordingly, methods that report on the presence of PTMs are essential tools for resolving open questions about epigenetic processes and for developing therapeutic diagnostics. Reader domains that recognize histone PTMs have shown potential as advantageous substitutes for anti-PTM antibodies and engineering efforts aimed at enhancing reader domain affinities would advance their efficacy as antibody alternatives. Here we describe engineered chromodomains from D. melanogaster and humans that bind more tightly to H3K9 methylation (H3K9me) marks and result in the tightest reported reader:H3K9me interaction to date. Point mutations near the binding interface of the HP1 chromodomain were screened in a combinatorial fashion, and a triple mutant was found that binds 20-fold tighter than the native scaffold without any loss in PTM-site selectivity. The beneficial mutations were then translated to a human homolog, CBX1, resulting in an even tighter interaction with H3K9me3. Furthermore, we show that these engineered readers (eReaders) increase detection of H3K9me marks in several biochemical assays and outperform a commercial anti-H3K9me antibody in detecting H3K9me-*

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Deciphering and engineering chromodomain-methyllysine peptide recognition

Abstract: Chromodomain-peptide recognition specificity is decided by physiochemical properties defined by posttranslational modifications.

Cited by 18 publications

References 44 publications

End-Point Binding Free Energy Calculation with MM/PBSA and MM/GBSA: Strategies and Applications in Drug Design

End-Point Binding Free Energy Calculation with MM/PBSA and MM/GBSA: Strategies and Applications in Drug Design

Development in Detection Methods for the Expression of Surface-Displayed Proteins

Engineered Reader Proteins for Enhanced Detection of Methylated Lysine on Histones

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