Allosteric regulation plays an important role in many biological processes, such as signal transduction, transcriptional regulation, and metabolism. Allostery is rooted in the fundamental physical properties of macromolecular systems, but its underlying mechanisms are still poorly understood. A collection of contributions to a recent interdisciplinary CECAM (Center Européen de Calcul Atomique et Moléculaire) workshop is used hereto provide an overview of the progress and remaining limitations in the understanding of the mechanistic foundations of allostery gained from computational and experimental analyses of real protein systems and model systems. The main conceptual frameworks instrumental in driving the field are discussed. We illustrate the role of these frameworks in illuminating molecular mechanisms and explaining cellular processes, and describe some of their promising practical applications in engineering molecular sensors and informing drug design efforts.

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...

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

The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system recently emerged as a transformative genome-editing technology that is innovating basic bioscience and applied medicine and biotechnology. The endonuclease Cas9 associates with a guide RNA to match and cleave complementary sequences in double stranded DNA, forming an RNA:DNA hybrid and a displaced non-target DNA strand. Although extensive structural studies are ongoing, the conformational dynamics of Cas9 and its interplay with the nucleic acids during association and DNA cleavage are largely unclear. Here, by employing multi-microsecond time scale molecular dynamics, we reveal the conformational plasticity of Cas9 and identify key determinants that allow its large-scale conformational changes during nucleic acid binding and processing. We show how the “closure” of the protein, which accompanies nucleic acid binding, fundamentally relies on highly coupled and specific motions of the protein domains, collectively initiating the prominent conformational changes needed for nucleic acid association. We further reveal a key role of the non-target DNA during the process of activation of the nuclease HNH domain, showing how the nontarget DNA positioning triggers local conformational changes that favor the formation of a catalytically competent Cas9. Finally, a remarkable conformational plasticity is identified as an intrinsic property of the HNH domain, constituting a necessary element that allows for the HNH repositioning. These novel findings constitute a reference for future experimental studies aimed at a full characterization of the dynamic features of the CRISPR-Cas9 system, and—more importantly—call for novel structure engineering efforts that are of fundamental importance for the rational design of new genome-engineering applications.

CONSPECTUS: Two-metal-ion-dependent nucleases cleave the phosphodiester bonds of nucleic acids via the two-metal-ion (2M) mechanism. Several high-resolution X-ray structures portraying the two-metal-aided catalytic site, together with mutagenesis and kinetics studies, have demonstrated a functional role of the ions for catalysis in numerous metallonucleases. Overall, the experimental data confirm the general mechanistic hypothesis for 2M-aided phosphoryl transfer originally reported by Steitz and Steitz ( Proc. Natl. Acad. Sci. U.S.A. 1993 , 90 ( 14 ), 6498 - 6502 ). This seminal paper proposed that one metal ion favors the formation of the nucleophile, while the nearby second metal ion facilitates leaving group departure during RNA hydrolysis. Both metals were suggested to stabilize the enzymatic transition state. Nevertheless, static X-ray structures alone cannot exhaustively unravel how the two ions execute their functional role along the enzymatic reaction during processing of DNA or RNA strands when moving from reactants to products, passing through metastable intermediates and high-energy transition states. In this Account, we discuss the role of multiscale molecular simulations in further disclosing mechanistic insights of 2M-aided catalysis for two prototypical enzymatic targets for drug discovery, namely, ribonuclease H (RNase H) and type II topoisomerase (topoII). In both examples, first-principles molecular simulations, integrated with structural data, emphasize a cooperative motion of the bimetal motif during catalysis. The coordinated motion of both ions is crucial for maintaining a flexible metal-centered structural architecture exquisitely tailored to accommodate the DNA or RNA sugar-phosphate backbone during phosphodiester bond cleavage. Furthermore, our analysis of RNase H and the N-terminal domain (PAN) of influenza polymerase shows that classical molecular dynamics simulations coupled with enhanced sampling techniques have contributed to describe the modulatory effect of metal ion concentration and metal uptake on the 2M mechanism and efficiency. These aspects all point to the emerging and intriguing role of additional adjacent ions potentially involved in the modulation of phosphoryl transfer reactions and enzymatic turnover in 2M-catalysis, as recently observed experimentally in polymerase η and homing endonuclease I-DmoI. These computational results, integrated with experimental findings, describe and reinforce the nascent concept of a functional and cooperative dynamics of the catalytic metal ions during the 2M-dependent enzymatic processing of DNA and RNA. Encouraged by the insights provided by computational approaches, we foresee further experiments that will feature the functional and joint dynamics of the catalytic metal ions for nucleic acid processing. This could impact the de novo design of artificial metallonucleases and the rational design of potent metal-chelating inhibitors of pharmaceutically relevant enzymes.

CRISPR-Cas9 is a genome editing technology with major impact in life sciences. In this system, the endonuclease Cas9 generates double strand breaks in DNA upon RNA-guided recognition of a complementary DNA sequence, which strictly requires the presence of a protospacer adjacent motif (PAM) next to the target site. Although PAM recognition is essential for cleavage, it is unknown whether and how PAM binding activates Cas9 for DNA cleavage at spatially distant sites. Here, we find evidence of a PAM-induced allosteric mechanism revealed by microsecond molecular dynamics simulations. PAM acts as an allosteric effector and triggers the interdependent conformational dynamics of the Cas9 catalytic domains (HNH and RuvC), responsible for concerted cleavage of the two DNA strands. Targeting such an allosteric mechanism should enable control of CRISPR-Cas9 functionality.

Understanding the conformational dynamics of CRISPR (clustered regularly interspaced short palindromic repeat)–Cas9 is of the utmost importance for improving its genome editing capability. Here, molecular dynamics simulations performed using Anton-2 – a specialized supercomputer capturing micro-to-millisecond biophysical events in real time and at atomic-level resolution – reveal the activation process of the endonuclease Cas9 toward DNA cleavage. Over the unbiased simulation, we observe that the spontaneous approach of the catalytic domain HNH to the DNA cleavage site is accompanied by a remarkable structural remodeling of the recognition (REC) lobe, which exerts a key role for DNA cleavage. Specifically, the significant conformational changes and the collective conformational dynamics of the REC lobe indicate a mechanism by which the REC1–3 regions ‘sense’ nucleic acids, ‘regulate’ the HNH conformational transition, and ultimately ‘lock’ the HNH domain at the cleavage site, contributing to its catalytic competence. By integrating additional independent simulations and existing experimental data, we provide a solid validation of the activated HNH conformation, which had been so far poorly characterized, and we deliver a comprehensive understanding of the role of REC1–3 in the activation process. Considering the importance of the REC lobe in the specificity of Cas9, this study poses the basis for fully understanding how the REC components control the cleavage of off-target sequences, laying the foundation for future engineering efforts toward improved genome editing.

Group II introns are Mg(2+)-dependent ribozymes that are considered to be the evolutionary ancestors of the eukaryotic spliceosome, thus representing an ideal model system to understand the mechanism of conversion of premature messenger RNA (mRNA) into mature mRNA. Neither in splicing nor for self-cleaving ribozymes has the role of the two Mg(2+) ions been established, and even the way the nucleophile is activated is still controversial. Here we employed hybrid quantum-classical QM(Car-Parrinello)/MM molecular dynamics simulations in combination with thermodynamic integration to characterize the molecular mechanism of the first and rate-determining step of the splicing process (i.e., the cleavage of the 5'-exon) catalyzed by group II intron ribozymes. Remarkably, our results show a new RNA-specific dissociative mechanism in which the bulk water accepts the nucleophile's proton during its attack on the scissile phosphate. The process occurs in a single step with no Mg(2+) ion activating the nucleophile, at odds with nucleases enzymes. We suggest that the novel reaction path elucidated here might be an evolutionary ancestor of the more efficient two-metal-ion mechanism found in enzymes.