Establishing the allosteric mechanism in <scp>CRISPR‐Cas9</scp>

Nierzwicki, Łukasz; Arantes, Pablo Ricardo; Saha, Aakash; Palermo, Giulia

doi:10.1002/wcms.1503

Cited by 41 publications

(41 citation statements)

References 71 publications

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“…This theoretical structure enabled to initiate in-depth studies of the catalysis ( Palermo, 2019 ; Casalino et al, 2020 ), the allostery ( Palermo et al, 2017 ; East et al, 2020 ; Nierzwicki et al, 2020 ) and the system’s specificity ( Mitchell et al, 2020 ; Ricci et al, 2019 ), when no structural information on the active state was available. This helped obtaining information to improve the enzyme catalytic efficiency and to reduce off-target effects, which is a key goal for biomedical applications ( Fu et al, 2013 ).…”

Section: Capturing Transitions and Short-lived Conformational Statesmentioning

confidence: 99%

Molecular Dynamics to Predict Cryo-EM: Capturing Transitions and Short-Lived Conformational States of Biomolecules

Nierzwicki

Palermo

2021

Front. Mol. Biosci.

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Single-particle cryogenic electron microscopy (cryo-EM) has revolutionized the field of the structural biology, providing an access to the atomic resolution structures of large biomolecular complexes in their near-native environment. Today’s cryo-EM maps can frequently reach the atomic-level resolution, while often containing a range of resolutions, with conformationally variable regions obtained at 6 Å or worse. Low resolution density maps obtained for protein flexible domains, as well as the ensemble of coexisting conformational states arising from cryo-EM, poses new challenges and opportunities for Molecular Dynamics (MD) simulations. With the ability to describe the biomolecular dynamics at the atomic level, MD can extend the capabilities of cryo-EM, capturing the conformational variability and predicting biologically relevant short-lived conformational states. Here, we report about the state-of-the-art MD procedures that are currently used to refine, reconstruct and interpret cryo-EM maps. We show the capability of MD to predict short-lived conformational states, finding remarkable confirmation by cryo-EM structures subsequently solved. This has been the case of the CRISPR-Cas9 genome editing machinery, whose catalytically active structure has been predicted through both long-time scale MD and enhanced sampling techniques 2 years earlier than cryo-EM. In summary, this contribution remarks the ability of MD to complement cryo-EM, describing conformational landscapes and relating structural transitions to function, ultimately discerning relevant short-lived conformational states and providing mechanistic knowledge of biological function.

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Section: Capturing Transitions and Short-lived Conformational Statesmentioning

confidence: 99%

Molecular Dynamics to Predict Cryo-EM: Capturing Transitions and Short-Lived Conformational States of Biomolecules

Nierzwicki

Palermo

2021

Front. Mol. Biosci.

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“…Multiple evidences including experiments and computations have indicated that CRISPR–Cas9 is also an intriguing “allosteric engine” 124,128–130 . Indeed, CRISPR–Cas9 requires an intricate allosteric activation to accomplish DNA cleavages.…”

Section: Applicationsmentioning

confidence: 99%

“…Multiple evidences including experiments and computations have indicated that CRISPR-Cas9 is also an intriguing "allosteric engine". 124,[128][129][130] Indeed, CRISPR-Cas9 requires an intricate allosteric activation to accomplish DNA cleavages. Biochemical experiments have indicated that the central element of the CRISPR-Cas9 allosteric signaling is the HNH domain, since its high flexibility can allow the signal transmission.…”

Section: Allosteric Effects Across the Crispr-cas9 Complexmentioning

confidence: 99%

Gaussian accelerated molecular dynamics: Principles and applications

Wang

Arantes

Bhattarai

et al. 2021

WIREs Comput Mol Sci

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Gaussian accelerated molecular dynamics (GaMD) is a robust computational method for simultaneous unconstrained enhanced sampling and free energy calculations of biomolecules. It works by adding a harmonic boost potential to smooth biomolecular potential energy surface and reduce energy barriers. GaMD greatly accelerates biomolecular simulations by orders of magnitude. Without the need to set predefined reaction coordinates or collective variables, GaMD provides unconstrained enhanced sampling and is advantageous for simulating complex biological processes. The GaMD boost potential exhibits a Gaussian distribution, thereby allowing for energetic reweighting via cumulant expansion to the second order (i.e., “Gaussian approximation”). This leads to accurate reconstruction of free energy landscapes of biomolecules. Hybrid schemes with other enhanced sampling methods, such as the replica‐exchange GaMD (rex‐GaMD) and replica‐exchange umbrella sampling GaMD (GaREUS), have also been introduced, further improving sampling and free energy calculations. Recently, new “selective GaMD” algorithms including the Ligand GaMD (LiGaMD) and Peptide GaMD (Pep‐GaMD) enabled microsecond simulations to capture repetitive dissociation and binding of small‐molecule ligands and highly flexible peptides. The simulations then allowed highly efficient quantitative characterization of the ligand/peptide binding thermodynamics and kinetics. Taken together, GaMD and its innovative variants are applicable to simulate a wide variety of biomolecular dynamics, including protein folding, conformational changes and allostery, ligand binding, peptide binding, protein–protein/nucleic acid/carbohydrate interactions, and carbohydrate/nucleic acid interactions. In this review, we present principles of the GaMD algorithms and recent applications in biomolecular simulations and drug design. This article is categorized under: Structure and Mechanism > Computational Biochemistry and Biophysics Molecular and Statistical Mechanics > Molecular Dynamics and Monte‐Carlo Methods Molecular and Statistical Mechanics > Free Energy Methods

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“…Allostery is a fundamental property of the CRISPR-Cas9 gene editing tool. 32,33 In this system, the allosteric relay is critical for transferring the information of DNA binding from the recognition (REC) lobe to the nuclease domains for cleavage and specificity. 4–8 Biochemical, 4 structural 9 and biophysical 5–8 approaches have shown that the HNH domain is the pivot of this allosteric regulation, holding a striking flexibility that facilitates the signal transduction 9,10 and controls DNA cleavage.…”

Section: Discussionmentioning

confidence: 99%

Enhanced Specificity Mutations Perturb Allosteric Signaling in CRISPR-Cas9

Palermo

Nierzwicki

East

et al. 2021

Preprint

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CRISPR-Cas9 is a molecular tool with transformative genome editing capabilities. At the molecular level, an intricate allosteric signaling is critical for DNA cleavage, but its role in the specificity enhancement of the Cas9 endonuclease is poorly understood. Here, solution NMR is combined with multi-microsecond molecular dynamics and graph theory-derived models to probe the allosteric role of key enhancement specificity mutations. We show that the mutations responsible for increasing the specificity of Cas9 alter the allosteric structure of the catalytic HNH domain, impacting the signal transmission from the DNA recognition region to the catalytic sites for cleavage. Specifically, the K855A mutation strongly disrupts the HNH domain allosteric structure, exerting the highest perturbation on the signaling transfer, while K810A and K848A result in more moderate effects on the allosteric intercommunication. This differential perturbation of the allosteric signaling reflects the different capabilities of the single mutants to increase Cas9 specificity, with the mutation achieving the highest specificity also strongly perturbing the signaling transfer. These outcomes reveal that the allosteric regulation is critical for the specificity enhancement of the Cas9 enzyme, and are valuable to harness the signaling network to improve the system specificity.

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Establishing the allosteric mechanism in CRISPR‐Cas9

Cited by 41 publications

References 71 publications

Molecular Dynamics to Predict Cryo-EM: Capturing Transitions and Short-Lived Conformational States of Biomolecules

Molecular Dynamics to Predict Cryo-EM: Capturing Transitions and Short-Lived Conformational States of Biomolecules

Gaussian accelerated molecular dynamics: Principles and applications

Enhanced Specificity Mutations Perturb Allosteric Signaling in CRISPR-Cas9

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