CRISPR–Cas9 is a bacterial immune system with exciting applications for genome editing. In spite of extensive experimental characterization, the active site chemistry of the RuvC domainwhich ...performs DNA cleavageshas remained elusive. Its knowledge is key for structure-based engineering aimed at improving DNA cleavages. Here, we deliver an in-depth characterization by using quantum-classical (QM/MM) molecular dynamics (MD) simulations and a Gaussian accelerated MD method, coupled with bioinformatics analysis. We disclose a two-metal aided architecture in the RuvC active site, which is poised to operate DNA cleavages, in analogy with other DNA/RNA processing enzymes. The conformational dynamics of the RuvC domain further reveals that an “arginine finger” stably contacts the scissile phosphate, with the function of stabilizing the active complex. Remarkably, the formation of a catalytically competent state of the RuvC domain is only observed upon the conformational activation of the other nuclease domain of CRISPR–Cas9i.e., the HNH domainsuch allowing concerted cleavages of double stranded DNA. This structure is in agreement with the available experimental data and remarkably differs from previous models based on classical mechanics, demonstrating also that only quantum mechanical simulations can accurately describe the metal-aided active site in CRISPR–Cas9. This fully catalytic structurein which both the HNH and RuvC domains are prone to perform DNA cleavagesconstitutes a stepping-stone for understanding DNA cleavage and specificity. It calls for novel experimental verifications and offers the structural foundations for engineering efforts aimed at improving the genome editing capability of CRISPR–Cas9.
The spliceosome (SPL) is a majestic macromolecular machinery composed of five small nuclear RNAs and hundreds of proteins. SPL removes noncoding introns from precursor messenger RNAs (pre-mRNAs) and ...ligates coding exons, giving rise to functional mRNAs. Building on the first SPL structure solved at near–atomic-level resolution, here we elucidate the functional dynamics of the intron lariat spliceosome (ILS) complex through multi-microsecond-long molecular-dynamics simulations of ∼1,000,000 atoms models. The ILS essential dynamics unveils (i) the leading role of the Spp42 protein, which heads the gene maturation by tuning the motions of distinct SPL components, and (ii) the critical participation of the Cwf19 protein in displacing the intron lariat/U2 branch helix. These findings provide unprecedented details on the SPL functional dynamics, thus contributing to move a step forward toward a thorough understanding of eukaryotic pre-mRNA splicing.
We performed density functional calculations aimed at identifying the atomistic and electronic structure origin of the valence and conduction band, and band gap tunability of halide perovskites ABX3 ...upon variations of the monovalent and bivalent cations A and B and the halide anion X. We found that the two key ingredients are the overlap between atomic orbitals of the bivalent cation and the halide anion, and the electronic charge on the metal center. In particular, lower gaps are associated with higher negative antibonding overlap of the states at the valence band maximum (VBM), and higher charge on the bivalent cation in the states at the conduction band minimum (CBM). Both VBM orbital overlap and CBM charge on the metal ion can be tuned over a wide range by changes in the chemical nature of A, B and X, as well as by variations of the crystal structure. On the basis of our results, we provide some practical rules to tune the valence band maximum, respectively the conduction band minimum, and thus the band gap in this class of materials.
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.
CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat and associated Cas9 protein) 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, multi-microsecond molecular dynamics is combined with solution NMR and graph theory-derived models to probe the allosteric role of key specificity-enhancing mutations. We show that 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 allosteric connectivity of the HNH domain, exerting the highest perturbation on the signaling transfer, while K810A and K848A result in more moderate effects on the allosteric communication. This differential perturbation of the allosteric signal correlates to the order of specificity enhancement (K855A > K848A ~ K810A) observed in biochemical studies, with the mutation achieving the highest specificity most strongly perturbing the signaling transfer. These findings suggest that alterations of the allosteric communication from DNA recognition to cleavage are critical to increasing the specificity of Cas9 and that allosteric hotspots can be targeted through mutational studies for improving the system's function.
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.
CRISPR–Cas9 is a widely employed genome-editing tool with functionality reliant on the ability of the Cas9 endonuclease to introduce site-specific breaks in double-stranded DNA. In this system, an ...intriguing allosteric communication has been suggested to control its DNA cleavage activity through flexibility of the catalytic HNH domain. Here, solution NMR experiments and a novel Gaussian-accelerated molecular dynamics (GaMD) simulation method are used to capture the structural and dynamic determinants of allosteric signaling within the HNH domain. We reveal the existence of a millisecond time scale dynamic pathway that spans HNH from the region interfacing the adjacent RuvC nuclease and propagates up to the DNA recognition lobe in full-length CRISPR–Cas9. These findings reveal a potential route of signal transduction within the CRISPR–Cas9 HNH nuclease, advancing our understanding of the allosteric pathway of activation. Further, considering the role of allosteric signaling in the specificity of CRISPR–Cas9, this work poses the mechanistic basis for novel engineering efforts aimed at improving its genome-editing capability.
CRISPR-Cas12a is a powerful RNA-guided genome-editing system that generates double-strand DNA breaks using its single RuvC nuclease domain by a sequential mechanism in which initial cleavage of the ...non-target strand is followed by target strand cleavage. How the spatially distant DNA target strand traverses toward the RuvC catalytic core is presently not understood. Here, continuous tens of microsecond-long molecular dynamics and free-energy simulations reveal that an α-helical lid, located within the RuvC domain, plays a pivotal role in the traversal of the DNA target strand by anchoring the crRNA:target strand duplex and guiding the target strand toward the RuvC core, as also corroborated by DNA cleavage experiments. In this mechanism, the REC2 domain pushes the crRNA:target strand duplex toward the core of the enzyme, while the Nuc domain aids the bending and accommodation of the target strand within the RuvC core by bending inward. Understanding of this critical process underlying Cas12a activity will enrich fundamental knowledge and facilitate further engineering strategies for genome editing.
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