Unconstrained enhanced sampling for free energy calculations of biomolecules: a review

Miao, Yinglong; McCammon, J. Andrew

doi:10.1080/08927022.2015.1121541

Cited by 154 publications

(132 citation statements)

References 123 publications

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“…Here, we characterize the dynamics of the activation pathway of the Cas9 endonuclease through extensive enhanced MD simulations (∼15 μs in length), using a GaMD methodology (18), which allows unconstrained sampling and accurate reconstruction of the free-energy profiles and probes long timescale events over hundreds of microseconds to milliseconds (19,20).…”

Section: Discussionmentioning

confidence: 99%

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CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations

Palermo

Miao

Walker

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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154

251

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CRISPR-Cas9 has become a facile genome editing technology, yet the structural and mechanistic features underlying its function are unclear. Here, we perform extensive molecular simulations in an enhanced sampling regime, using a Gaussian-accelerated molecular dynamics (GaMD) methodology, which probes displacements over hundreds of microseconds to milliseconds, to reveal the conformational dynamics of the endonuclease Cas9 during its activation toward catalysis. We disclose the conformational transition of Cas9 from its apo form to the RNA-bound form, suggesting a mechanism for RNA recruitment in which the domain relocations cause the formation of a positively charged cavity for nucleic acid binding. GaMD also reveals the conformation of a catalytically competent Cas9, which is prone for catalysis and whose experimental characterization is still limited. We show that, upon DNA binding, the conformational dynamics of the HNH domain triggers the formation of the active state, explaining how the HNH domain exerts a conformational control domain over DNA cleavage [Sternberg SH et al. (2015) Nature, 527, 110-113]. These results provide atomic-level information on the molecular mechanism of CRISPR-Cas9 that will inspire future experimental investigations aimed at fully clarifying the biophysics of this unique genome editing machinery and at developing new tools for nucleic acid manipulation based on CRISPR-Cas9.protein-nucleic acid interactions | gene regulation | RNA dynamics | enhanced sampling | free energy L ife sciences are undergoing a transformative phase due to an emerging genome editing technology based on the RNAprogrammable CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) system (1-3). Although this facile genome editing technology is revolutionizing the fields of medicine, pharmaceutics, and even agriculture with the development of drought-resistant crops, the structural and mechanistic details underlying the CRISPR-Cas9 function remain unclear. The CRISPR-Cas9 function begins with the association of Cas9 with a guide RNA, composed of a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA), which enables the recognition and cleavage of matching sequences in double-stranded DNA (3, 4). Upon site-specific recognition of a Protospacer Adjacent Motif (PAM) within the DNA, the latter binds Cas9 matching one strand with the RNA guide (the target DNA strand, t-DNA), whereas the other strand (nontarget DNA, nt-DNA) is displaced. Subsequently, two nuclease domains-HNH and RuvC-perform site-specific cleavages of the t-DNA and nt-DNA strands, respectively.Structural studies have revealed that Cas9 is a large multidomain protein, composed of a recognition lobe, which mediates the nucleic acid binding through three regions (RECI-III), and a nuclease lobe including the RuvC and HNH catalytic cores (Fig. 1) (5). An arginine rich helix (R-rich) bridges the two lobes, whereas the protein C-terminal (Cterm) and PAM-interacting (PI) domains take part in the DNA bindi...

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Section: Discussionmentioning

confidence: 99%

“…GaMD has shown robust sampling capability and accurate reconstruction of free-energy profiles for simulations of large biomolecules. It has enabled characterization of ligand binding and conformational changes in G protein-coupled receptors (19) as well as protein folding (20).…”

Section: Significancementioning

confidence: 99%

CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations

Palermo

Miao

Walker

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

154

251

View full text Add to dashboard Cite

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“…Longer classical MD simulations of TFnucleosome complexes and comparing with similar-length simulations of free nucleosomes will provide a framework for studying the role of TF binding to nucleosome dynamics. For this, enhanced sampling techniques may be required to capture larger scale motions and longer time scales 34 .…”

Section: Discussionmentioning

confidence: 99%

Nucleosomal DNA dynamics mediate Oct4 pioneer factor binding

Huertas

MacCarthy

Schoeler

et al. 2019

Preprint

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Transcription factor (TF) proteins bind to DNA to regulate gene expression. Normally, accessibility to DNA is required for their function. However, in the nucleus the DNA is often inaccessible, wrapped around histone proteins in nucleosomes forming the chromatin.Pioneer TFs are thought to induce chromatin opening by recognizing their DNA binding sites on nucleosomes. For example, Oct4, a master regulator and inducer of stem cell pluripotency, binds to DNA in nucleosomes in a sequence specific manner. Here we reveal the structural dynamics of nucleosomes that mediate Oct4 binding. Nucleosome mobility and the amplitude of nucleosome motions such as breathing and twisting correlate with the number of Oct4 binding sites available. Moreover, the regions around the binding sites display higher local mobility. Probing different structures of Oct4-nucleosome complexes, we show that alternative configurations display stable protein-DNA interactions and are compatible with the DNA curvature and DNA-histone interactions.

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“…To make progress, two broad categories of enhanced sampling methods have been developed with the goal of increasing total 4 effective time covered by a simulation. Predefined collective variables can be exhaustively sampled (Metadynamics [20][21][22] , Umbrella Sampling [23][24][25] , Steered MD [26][27][28] ), or the potential energy function of the forcefield can be explicitly altered (scaled MD [29][30][31][32] accelerated MD (aMD) 33,34 ). The former class of methods require the degrees of freedom underpinning the motion of interest to be supplied in advance, which presupposes that the relevant motions are already known.…”

Section: Introductionmentioning

confidence: 99%

Dynamic design: manipulation of millisecond timescale motions on the energy landscape of Cyclophilin A

Juárez-Jiménez

Gupta

Karunanithy

et al. 2018

Preprint

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Proteins need to interconvert between many conformations in order to function, many of which are formed transiently, and sparsely populated. Particularly when the lifetimes of these states approach the millisecond timescale, identifying the relevant structures and the mechanism by which they inter-convert remains a tremendous challenge. Here we introduce a novel combination of accelerated MD (aMD) simulations and Markov State modelling (MSM) to explore these 'excited' conformational states . Applying this to the highly dynamic protein CypA, a protein involved in immune response and associated with HIV infection, we identify five principally populated conformational states and the atomistic mechanism by which they interconvert. A rational design strategy predicted that the mutant D66A should stabilise the minor conformations and substantially alter the dynamics whereas the similar mutant H70A should leave the landscape broadly unchanged. These predictions are confirmed using CPMG and R1ρ solution state NMR measurements. By accurately and reliably exploring functionally relevant, but sparsely populated conformations with milli-second lifetimes in silico, our aMD/MSM method has tremendous promise for the design of dynamic protein free energy landscapes for both protein engineering and drug discovery.

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Unconstrained enhanced sampling for free energy calculations of biomolecules: a review

Cited by 154 publications

References 123 publications

CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations

CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations

Nucleosomal DNA dynamics mediate Oct4 pioneer factor binding

Dynamic design: manipulation of millisecond timescale motions on the energy landscape of Cyclophilin A

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