Protospacer Adjacent Motif-Induced Allostery Activates CRISPR-Cas9

Palermo, Giulia; Ricci, Clarisse Gravina; Fernando, Amendra; Basak, Rajshekhar; Jinek, Martin; Rivalta, Ivan; Batista, Víctor S.; McCammon, J. Andrew

doi:10.1021/jacs.7b05313

Cited by 122 publications

(207 citation statements)

References 20 publications

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“…The SPL: A Protein-Directed Ribozyme. To characterize the SPL functional dynamics and the leading role of Spp42, the largest and the most important protein of the SPL, we have computed the cross-correlation scores (CSs) for each pair of SPL components (i.e., RNAs and proteins, explicitly considering the Spp42 domains and N-terminal motifs), which enable to qualitatively measure their interdependent dynamical coupling (27)(28)(29). The CSs histogram for ILS-1 model (Fig.…”

Section: Cwf15mentioning

confidence: 99%

All-atom simulations disentangle the functional dynamics underlying gene maturation in the intron lariat spliceosome

Casalino

Palermo

Spinello

et al. 2018

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

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106

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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 () the leading role of the Spp42 protein, which heads the gene maturation by tuning the motions of distinct SPL components, and () 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.

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Section: Cwf15mentioning

confidence: 99%

All-atom simulations disentangle the functional dynamics underlying gene maturation in the intron lariat spliceosome

Casalino

Palermo

Spinello

et al. 2018

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

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106

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“…for understanding the molecular basis of the CRISPR-Cas9 function. (13)(14)(15)(16)(17) Among other studies, our group has successfully applied MD simulations to disclose a mechanism for the conformational activation of Cas9, clarifying the activation process by which the HNH domain is repositioned for cleavage in good agreement with structural and smFRET experiments. (18) Remarkably, MD simulations as well as experimental studies have indicated that the conformational changes underlying the CRISPR-Cas9 function occur over and beyond micro-to-millisecond time scales.…”

Section: Introductionmentioning

confidence: 81%

“…(18,34) As well, L1/L2 exert an allosteric control on the activity of HNH and RuvC, enabling the information transfer for concerted cleavages of the two DNA strands. (15,35) In order to understand the role of these two loops in the interaction with the hybrid, we specifically measured the interactions established by L1/L2 and the DNA:RNA hybrid. As a result, the interactions of L1 do not show relevant differences among the investigated systems ( Fig.…”

Section: Effect Of Off-target Binding On the Catalytic Domainsmentioning

confidence: 99%

Molecular mechanism of off-target effects in CRISPR-Cas9

Ricci

Chen

Miao

et al. 2018

Preprint

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CRISPR-Cas9 is the state-of-the-art technology for editing and manipulating nucleic acids. However, the occurrence of off-target mutations can limit its applicability. Here, allatom enhanced molecular dynamics (MD) simulations -using Gaussian accelerated MD (GaMD) -are used to decipher the mechanism of off-target binding at the molecular level.GaMD reveals that base pair mismatches in the target DNA at specific distal sites with respect to the Protospacer Adjacent Motif (PAM) induce an extended opening of the RNA:DNA heteroduplex, which leads to newly discovered interactions between the unwound nucleic acids and the protein counterpart. The conserved interactions between the target DNA strand and the L2 loop of the catalytic HNH domain constitute a "lock" effectively decreasing the conformational freedom of the HNH domain and its activation for cleavage. Remarkably, depending on their position at PAM distal sites, DNA mismatches leading to off-target cleavages are unable to "lock" the HNH domain, thereby identifying the ability to "lock" HNH as a key determinant. Consistently, off-target sequences hampering the catalysis have been shown to "trap" somehow the HNH domain in an inactive "conformational checkpoint" state (Dagdas et al. Sci Adv, 2017). As such, this mechanism identifies the molecular basis underlying off-target cleavages and contributes in clarifying a long-lasting open issue of the CRISPR-Cas9 function. It also poses the foundation for designing novel and more specific Cas9 variants, which could be obtained by magnifying the "locking" interactions between HNH and the target DNA in the presence of any incorrect off-target sequence, thus preventing undesired cleavages.In this respect, a promising strategy to fight off-target cleavages is the molecular engineering of highly specific Cas9 proteins,(6-8) which would enable safer and easy-to-use applications of the CRISPR technology on a genome-wide scale.(9) However, rational design of CRISPR-Cas9 requires detailed knowledge of the molecular bases underlying off-target effects, which are not yet understood. (10, 11) At the molecular level, off-target effects are the unselective cleavages of DNA sequences that do not fully match the guide RNA, bearing base pair mismatches at PAM distal sites of the DNA:RNA hybrid (Fig. 1). Kinetic and single molecule (sm) Förster Resonance Energy Transfer (FRET) experiments have shown that the occurrence of off-target cleavages is directly related to the conformational state adopted by the catalytic HNH domain.(7, 12) Upon DNA binding, HNH undergoes a conformational change from an inactive state, in which the catalytic H840 is far away from the cleavage site on the TS, to an activated state that is prone for catalysis (Fig. 1A). The inactive state of the HNH domain has been identified as a "conformational checkpoint" between DNA binding and cleavage, in which the RNA:DNA complementarity is recognized before the HNH domain assumes an activated conformation.(12) Sm FRET experiments have shown that the presence of DNA mismatc...

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“…Over the past decade, MD simulations have become increasingly important in solving the atomiclevel mechanisms of protein allostery, often revealing missing features that purely experimental techniques could not provide (16)(17)(18)(19). However, a lack of sufficient sampling of events that take place on very large timescales constitutes a significant limitation when employing standard MD simulations to explore allosteric mechanisms.…”

Section: Introductionmentioning

confidence: 99%

Mechanism of allosteric activation of human mRNA cap methyltransferase (RNMT) by RAM: Insights from accelerated molecular dynamics simulations

Bueren-Calabuig

Bage

Cowling

et al. 2019

Preprint

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The RNA guanine-7 methyltransferase (RNMT) in complex with RNMT-Activating Miniprotein (RAM) catalyses the formation of a N7-methylated guanosine cap structure on the 5' end of nascent RNA polymerase II transcripts. The mRNA cap protects the transcript from exonucleases and recruits capbinding complexes that mediate RNA processing, export and translation. By using microsecond standard and accelerated molecular dynamics simulations, we provide for the first time a detailed molecular mechanism of allosteric regulation of RNMT by RAM. We show that RAM selects the RNMT active site conformations that are optimal for binding of substrates (AdoMet and the cap), thus enhancing their affinity. Furthermore, our results strongly suggest the likely scenario in which the cap binding promotes the subsequent AdoMet binding, consistent with the previously suggested cooperative binding model. By employing the dynamic network and community analyses, we revealed the underlying long-range allosteric networks and paths that are crucial for allosteric regulation by RAM.Our findings complement and explain previous experimental data on RNMT activity. Moreover, this study provides the most complete description of the cap and AdoMet binding poses and interactions within the enzyme's active site. This information is critical for the drug discovery efforts that consider RNMT as a promising anti-cancer target.Recently, we reported the crystal structure of the catalytic domain (residues 165-476) of human RNMT in complex with the RNMT activation domain of RAM (residues 2-45) and AdoHcy (PDB ID: 5E8J) (8). Human RNMT consists of a catalytic domain (residues 121-476), homologous to other eukaryotic cap methyltransferases, and an N-terminal regulatory domain (residues 1-120) that facilitates the recruitment to RNA polymerase II transcription initiation sites (9, 10) and regulates RNMT activity (11). The presence of RAM permitted the stabilization and crystallization of a modular lobe of RNMT (residues 416-456), which was unresolved in previously reported structures of isolated RNMT (PDB IDs: 3BGV, 5E9W). This strongly suggested that the lobe is disordered in the absence of RAM. . The role of the RAM N-terminal region in the RNMT activation was investigated by simulating RNMT in complex with a truncated form of RAM (residues 20 to 45), in which the N-terminal region was removed. apo-RNMT +RAMAll models were prepared with the LEaP utility from the AMBER14 suite of programs (26) using the ff14SB force field (27). The N-terminus of RNMT was capped with an acetyl group (ACE), and the Nand C-termini of RAM were capped with ACE and a methylated amino group (NME), respectively.The geometries of the ligands were refined with Gaussian03 (28) at the HF/6-31G* level. The optimized geometries were used to calculate the electrostatic potential-derived (ESP) charges using the RESP methodology (29), as implemented in the antechamber module in Amber14 (26). The force field parameters for AdoMet were generated with antechamber module, using the general AMBER fo...

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Protospacer Adjacent Motif-Induced Allostery Activates CRISPR-Cas9

Cited by 122 publications

References 20 publications

All-atom simulations disentangle the functional dynamics underlying gene maturation in the intron lariat spliceosome

All-atom simulations disentangle the functional dynamics underlying gene maturation in the intron lariat spliceosome

Molecular mechanism of off-target effects in CRISPR-Cas9

Mechanism of allosteric activation of human mRNA cap methyltransferase (RNMT) by RAM: Insights from accelerated molecular dynamics simulations

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