CRISPR-Cas12a is a genome-editing system, recently also harnessed for nucleic acid detection, which is promising for the diagnosis of the SARS-CoV-2 coronavirus through the DETECTR technology. Here, ...a collective ensemble of multimicrosecond molecular dynamics characterizes the key dynamic determinants allowing nucleic acid processing in CRISPR-Cas12a. We show that DNA binding induces a switch in the conformational dynamics of Cas12a, which results in the activation of the peripheral REC2 and Nuc domains to enable cleavage of nucleic acids. The simulations reveal that large-amplitude motions of the Nuc domain could favor the conformational activation of the system toward DNA cleavages. In this process, the REC lobe plays a critical role. Accordingly, the joint dynamics of REC and Nuc shows the tendency to prime the conformational transition of the DNA target strand toward the catalytic site. Most notably, the highly coupled dynamics of the REC2 region and Nuc domain suggests that REC2 could act as a regulator of the Nuc function, similar to what was observed previously for the HNH domain in the CRISPR-associated nuclease Cas9. These mutual domain dynamics could be critical for the nonspecific binding of DNA and thereby for the underlying mechanistic functioning of the DETECTR technology. Considering that REC is a key determinant in the system’s specificity, our findings provide a rational basis for future biophysical studies aimed at characterizing its function in CRISPR-Cas12a. Overall, our outcomes advance our mechanistic understanding of CRISPR-Cas12a and provide grounds for novel engineering efforts to improve genome editing and viral detection.
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
Gaussian accelerated molecular dynamics (GaMD) and its applications to a wide range of biological systems.
CRISPR-Cas9 is the forefront technology for editing the genome. In this system, the Cas9 protein is programmed with guide RNAs to process DNA sequences that match the guide RNA forming an RNA:DNA ...hybrid structure. However, the binding of DNA sequences that do not fully match the guide RNA can limit the applicability of CRISPR-Cas9 for genome editing, resulting in the so-called off-target effects. Here, molecular dynamics is used to probe the effect of DNA base pair mismatches within the RNA:DNA hybrid in CRISPR-Cas9. Molecular simulations revealed that the presence of mismatched pairs in the DNA at distal sites with respect to the Protospacer Adjacent Motif (PAM) recognition sequence induces an extended opening of the RNA:DNA hybrid, leading to novel interactions established by the unwound nucleic acids and the protein counterpart. On the contrary, mismatched pairs upstream of the RNA:DNA hybrid are rapidly incorporated within the heteroduplex, with minor effect on the protein-nucleic acid interactions. As a result, mismatched pairs at PAM distal ends interfere with the activation of the catalytic HNH domain, while mismatches fully embedded in the RNA:DNA do not affect the HNH dynamics and enable its activation to cleave the DNA. These findings provide a mechanistic understanding to the intriguing experimental evidence that PAM distal mismatches hamper a proper function of HNH, explaining also why mismatches within the heteroduplex are much more tolerated. This constitutes a step forward in understanding off-target effects in CRISPR-Cas9, which encourages novel structure-based engineering efforts aimed at preventing the onset of off-target effects.
Allostery is a ubiquitous biological mechanism in which a distant binding site is coupled to and drastically alters the function of a catalytic site in a protein. Allostery provides a high level of ...spatial and temporal control of the integrity and activity of biomolecular assembles composed of proteins, nucleic acids, or small molecules. Understanding the physical forces that drive allosteric coupling is critical to harnessing this process for use in bioengineering, de novo protein design, and drug discovery. Current microscopic models of allostery highlight the importance of energetics, structural rearrangements, and conformational fluctuations, and in this review, we discuss the synergistic use of solution NMR spectroscopy and computational methods to probe these phenomena in allosteric systems, particularly protein-nucleic acid complexes. This combination of experimental and theoretical techniques facilitates an unparalleled detection of subtle changes to structural and dynamic equilibria in biomolecules with atomic resolution, and we provide a detailed discussion of specialized NMR experiments as well as the complementary methods that provide valuable insight into allosteric pathways in silico. Lastly, we highlight two case studies to demonstrate the adaptability of this approach to enzymes of varying size and mechanistic complexity.
C3d is a hallmark protein of the complement system, whose presence is critical to measure the progression of several immune diseases. Here, we propose to directly target C3d through small peptides ...mimicking the binding of its natural ligand, the complement regulator Factor H (FH). Through iterative computational analysis and binding affinity experiments, we establish a rationale for the structure-based design of FH-inspired peptides, leading to low-micromolar affinity for C3d and stable binding over microsecond-length simulations. Our FH-inspired peptides call now for further optimization toward high-affinity binding and suggest that small peptides are promising as novel C3d biomarkers and therapeutic tools.
The complement system is assembled from a network of proteins that function to bring about the first line of defense of the body against invading pathogens. However, complement deficiencies or ...invasive pathogens can hijack complement to subsequently increase susceptibility of the body to infections. Moreover, invasive pathogens are increasingly becoming resistant to the currently available therapies. Hence, it is important to gain insights into the highly dynamic interaction between complement and invading microbes in the frontlines of immunity. Here, we developed a mathematical model of the complement system composed of 670 ordinary differential equations with 328 kinetic parameters, which describes all three complement pathways (alternative, classical, and lectin) and includes description of mannose-binding lectin, collectins, ficolins, factor H-related proteins, immunoglobulin M, and pentraxins. Additionally, we incorporate two pathogens: (type 1) complement susceptible pathogen and (type 2)
located in either nasopharynx or bloodstream. In both cases, we generate time profiles of the pathogen surface occupied by complement components and the membrane attack complex (MAC). Our model shows both pathogen types in bloodstream are saturated by complement proteins, whereas MACs occupy <<1.0% of the pathogen surface. Conversely, the MAC production in nasopharynx occupies about 1.5-10% of the total
surface, thus making nasal MAC levels at least about eight orders of magnitude higher. Altogether, we predict complement-imbalance, favoring overactivation, is associated with nasopharynx homeostasis. Conversely, orientating toward complement-balance may cause disruption to the nasopharynx homeostasis. Thus, for sporadic meningococcal disease, our model predicts rising nasal levels of complement regulators as early infection biomarkers.
Poxviruses are dangerous pathogens, which can cause fatal infection in unvaccinated individuals. The causative agent of smallpox in humans,
, is closely related to the bovine
, yet the molecular ...basis of their selectivity is currently incompletely understood. Here, we examine the role of the electrostatics in the selectivity of the smallpox protein SPICE and vaccinia protein VCP toward the human and bovine complement protein C3b, a key component of the complement immune response. Electrostatic calculations, in-silico alanine-scan and electrostatic hotspot analysis, as introduced by Kieslich and Morikis (
. 2012), are used to assess the electrostatic complementarity and to identify sites resistant to local perturbation where the electrostatic potential is likely to be evolutionary conserved. The calculations suggest that the bovine C3b is electrostatically prone to selectively bind its VCP ligand. On the other hand, the human isoform of C3b exhibits a lower electrostatic complementarity toward its SPICE ligand. Yet, the human C3b displays a highly preserved electrostatic core, which suggests that this isoform could be less selective in binding different ligands like SPICE and the human Factor H. This is supported by experimental cofactor activity assays revealing that the human C3b is prone to bind both SPICE and Factor H, which exhibit diverse electrostatic properties. Additional investigations considering mutants of SPICE and VCP that revert their selectivity reveal an "electrostatic switch" into the central modules of the ligands, supporting the critical role of the electrostatics in the selectivity. Taken together, these evidences provide insights into the selectivity mechanism of the complement regulator proteins encoded by the
and
to circumvent the complement immunity and exert their pathogenic action. These fundamental aspects are valuable for the development of novel vaccines and therapeutic strategies.
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