Unveiling Cas8 Dynamics and Regulation within a transposon-encoded Cascade-TniQ Complex.

Cascade is a class 1, type 1 CRISPR-Cas system with a variety of roles in prokaryote defense, specifically against DNA-based viruses. The Vibrio Cholerae transposon, Tn6677, encodes a variant of the type 1F Cascade known as type 1F-3. This Cascade variant complexes with a homodimer of the transposition protein TniQ and leverages the sequence specificity of Cascade to direct the integration activity of the heteromeric transposase tnsA/B, resulting in site-specific transposition of Tn6677. We desire to uncover the molecular details behind R Loop formation of 'Cascade-TniQ.' Due to the lack of a complete model of Cascade-TniQ available at atom-level resolution, we first build a complete model using AlphaFold V2.1. We then simulate this model via classical molecular dynamics and umbrella sampling to study an important regulatory component within Cascade-TniQ, known as the Cas8 'bundle.' Particularly, we show that this alpha helical bundle experiences a free energy barrier to its large-scale translatory motions and relative free energies of its states primarily dependent on a loop within a Cas7 subunit in Cascade-TniQ. Further, we comment on additional structural and dynamical regulatory points of Cascade-TniQ during R Loop formation, such as Cascade-TniQ backbone rigidity, and the potential role TniQ plays in regulating bundle dynamics. In summary, our outcomes provide the first all-atom dynamic representation of one of the largest CRISPR systems, with information that can contribute to understanding the mechanism of nucleic acid binding and, eventually, to transposase recruitment itself. Such information may prove informative to advance genome engineering efforts.

High-fidelity, hyper-accurate, and evolved mutants rewire atomic-level communication in CRISPR-Cas9.

The high-fidelity (HF1), hyper-accurate (Hypa), and evolved (Evo) variants of the CRISPR-associated protein 9 (Cas9) endonuclease are critical tools to mitigate off-target effects in the application of CRISPR-Cas9 technology. The mechanisms by which mutations in recognition subdomain 3 (Rec3) mediate specificity in these variants are poorly understood. Here, solution nuclear magnetic resonance and molecular dynamics simulations establish the structural and dynamic effects of high-specificity mutations in Rec3, and how they propagate the allosteric signal of Cas9. We reveal conserved structural changes and dynamic differences at regions of Rec3 that interface with the RNA:DNA hybrid, transducing chemical signals from Rec3 to the catalytic His-Asn-His (HNH) domain. The variants remodel the communication sourcing from the Rec3 α helix 37, previously shown to sense target DNA complementarity, either directly or allosterically. This mechanism increases communication between the DNA mismatch recognition helix and the HNH active site, shedding light on the structure and dynamics underlying Cas9 specificity and providing insight for future engineering principles.

Unveiling the RNA-mediated allosteric activation discloses functional hotspots in CRISPR-Cas13a.

Cas13a is a recent addition to the CRISPR-Cas toolkit that exclusively targets RNA, which makes it a promising tool for RNA detection. It utilizes a CRISPR RNA (crRNA) to target RNA sequences and trigger a composite active site formed by two 'Higher Eukaryotes and Prokaryotes Nucleotide' (HEPN) domains, cleaving any solvent-exposed RNA. In this system, an intriguing form of allosteric communication controls the RNA cleavage activity, yet its molecular details are unknown. Here, multiple-microsecond molecular dynamics simulations are combined with graph theory to decipher this intricate activation mechanism. We show that the binding of a target RNA acts as an allosteric effector, by amplifying the communication signals over the dynamical noise through interactions of the crRNA at the buried HEPN1-2 interface. By introducing a novel Signal-to-Noise Ratio (SNR) of communication efficiency, we reveal critical allosteric residues-R377, N378, and R973-that rearrange their interactions upon target RNA binding. Alanine mutation of these residues is shown to select target RNA over an extended complementary sequence beyond guide-target duplex for RNA cleavage, establishing the functional significance of these hotspots. Collectively our findings offer a fundamental understanding of the Cas13a mechanism of action and pave new avenues for the development of highly selective RNA-based cleavage and detection tools.

New design strategies for ultra-specific CRISPR-Cas13a-based RNA detection with single-nucleotide mismatch sensitivity.

An increasingly pressing need for clinical diagnostics has required the development of novel nucleic acid-based detection technologies that are sensitive, fast, and inexpensive, and that can be deployed at point-of-care. Recently, the RNA-guided ribonuclease CRISPR-Cas13 has been successfully harnessed for such purposes. However, developing assays for detection of genetic variability, for example single-nucleotide polymorphisms, is still challenging and previously described design strategies are not always generalizable. Here, we expanded our characterization of LbuCas13a RNA-detection specificity by performing a combination of experimental RNA mismatch tolerance profiling, molecular dynamics simulations, protein, and crRNA engineering. We found certain positions in the crRNA-target-RNA duplex that are particularly sensitive to mismatches and establish the effect of RNA concentration in mismatch tolerance. Additionally, we determined that shortening the crRNA spacer or modifying the direct repeat of the crRNA leads to stricter specificities. Furthermore, we harnessed our understanding of LbuCas13a allosteric activation pathways through molecular dynamics and structure-guided engineering to develop novel Cas13a variants that display increased sensitivities to single-nucleotide mismatches. We deployed these Cas13a variants and crRNA design strategies to achieve superior discrimination of SARS-CoV-2 strains compared to wild-type LbuCas13a. Together, our work provides new design criteria and Cas13a variants to use in future easier-to-implement Cas13-based RNA detection applications.

High-Fidelity, Hyper-Accurate, and Evolved Mutants Rewire Atomic Level Communication in CRISPR-Cas9.

The Cas9-HF1, HypaCas9, and evoCas9 variants of the Cas9 endonuclease are critical tools to mitigate off-target effects in the application of CRISPR-Cas9 technology. The mechanisms by which mutations in the Rec3 domain mediate specificity in these variants are poorly understood. Here, solution NMR and molecular dynamics simulations establish the structural and dynamic effects of high-specificity mutations in Rec3, and how they propagate the allosteric signal of Cas9. We reveal conserved structural changes and peculiar dynamic differences at regions of Rec3 that interface with the RNA:DNA hybrid, transducing chemical signals from Rec3 to the catalytic HNH domain. The variants remodel the communication sourcing from the Rec3 α-helix 37, previously shown to sense target DNA complementarity, either directly or allosterically. This mechanism increases communication between the DNA mismatch recognition helix and the HNH active site, shedding light on the structure and dynamics underlying Cas9 specificity and providing insight for future engineering principles.

RNA-mediated Allosteric Activation in CRISPR-Cas13a.

Cas13a is a recent addition to the CRISPR-Cas toolkit that exclusively targets RNA, which makes it a promising tool for RNA detection. The protein uses a CRISPR RNA (crRNA) to target RNA sequences, which are cleaved by a composite active site formed by two 'Higher Eukaryotes and Prokaryotes Nucleotide' (HEPN) catalytic domains. In this system, an intriguing form of allosteric communication controls RNA cleavage activity, yet its molecular details are unknown. Here, multiple-microsecond molecular dynamics simulations are combined with graph theory and RNA cleavage assays to decipher this activation mechanism. We show that the binding of a target RNA acts as an allosteric effector of the spatially distant HEPN catalytic cleft, by amplifying the allosteric signals over the dynamical noise, that passes through the buried HEPN interface. Critical residues in this region - N378, R973, and R377 - rearrange their interactions upon target RNA binding, and alter allosteric signalling. Alanine mutation of these residues is experimentally shown to select target RNA over an extended complementary sequence beyond guide-target duplex, for RNA cleavage. Altogether, our findings offer a fundamental understanding of the Cas13a mechanism of action and pave new avenues for the development of more selective RNA-based cleavage and detection tools.

New design strategies for ultra-specific CRISPR-Cas13a-based RNA-diagnostic tools with single-nucleotide mismatch sensitivity.

The pressing need for clinical diagnostics has required the development of novel nucleic acid-based detection technologies that are sensitive, fast, and inexpensive, and that can be deployed at point-of-care. Recently, the RNA-guided ribonuclease CRISPR-Cas13 has been successfully harnessed for such purposes. However, developing assays for detection of genetic variability, for example single-nucleotide polymorphisms, is still challenging and previously described design strategies are not always generalizable. Here, we expanded our characterization of LbuCas13a RNA-detection specificity by performing a combination of experimental RNA mismatch tolerance profiling, molecular dynamics simulations, protein, and crRNA engineering. We found certain positions in the crRNA-target-RNA duplex that are particularly sensitive to mismatches and establish the effect of RNA concentration in mismatch tolerance. Additionally, we determined that shortening the crRNA spacer or modifying the direct repeat of the crRNA leads to stricter specificities. Furthermore, we harnessed our understanding of LbuCas13a allosteric activation pathways through molecular dynamics and structure-guided engineering to develop novel Cas13a variants that display increased sensitivities to single-nucleotide mismatches. We deployed these Cas13a variants and crRNA design strategies to achieve superior discrimination of SARS-CoV-2 strains compared to wild-type LbuCas13a. Together, our work provides new design criteria and new Cas13a variants for easier-to-implement Cas13-based diagnostics. KEY POINTS: Certain positions in the Cas13a crRNA-target-RNA duplex are particularly sensitive to mismatches.Understanding Cas13a's allosteric activation pathway allowed us to develop novel high-fidelity Cas13a variants.These Cas13a variants and crRNA design strategies achieve superior discrimination of SARS-CoV-2 strains.

Machines on Genes through the Computational Microscope.

Macromolecular machines acting on genes are at the core of life's fundamental processes, including DNA replication and repair, gene transcription and regulation, chromatin packaging, RNA splicing, and genome editing. Here, we report the increasing role of computational biophysics in characterizing the mechanisms of "machines on genes", focusing on innovative applications of computational methods and their integration with structural and biophysical experiments. We showcase how state-of-the-art computational methods, including classical and ab initio molecular dynamics to enhanced sampling techniques, and coarse-grained approaches are used for understanding and exploring gene machines for real-world applications. As this review unfolds, advanced computational methods describe the biophysical function that is unseen through experimental techniques, accomplishing the power of the "computational microscope", an expression coined by Klaus Schulten to highlight the extraordinary capability of computer simulations. Pushing the frontiers of computational biophysics toward a pragmatic representation of large multimegadalton biomolecular complexes is instrumental in bridging the gap between experimentally obtained macroscopic observables and the molecular principles playing at the microscopic level. This understanding will help harness molecular machines for medical, pharmaceutical, and biotechnological purposes.

Disruption of electrostatic contacts in the HNH nuclease from a thermophilic Cas9 rewires allosteric motions and enhances high-temperature DNA cleavage.

Allosteric signaling within multidomain proteins is a driver of communication between spatially distant functional sites. Understanding the mechanism of allosteric coupling in large multidomain proteins is the most promising route to achieving spatial and temporal control of the system. The recent explosion of CRISPR-Cas9 applications in molecular biology and medicine has created a need to understand how the atomic level protein dynamics of Cas9, which are the driving force of its allosteric crosstalk, influence its biophysical characteristics. In this study, we used a synergistic approach of nuclear magnetic resonance (NMR) and computation to pinpoint an allosteric hotspot in the HNH domain of the thermostable GeoCas9. We show that mutation of K597 to alanine disrupts a salt-bridge network, which in turn alters the structure, the timescale of allosteric motions, and the thermostability of the GeoHNH domain. This homologous lysine-to-alanine mutation in the extensively studied mesophilic S. pyogenes Cas9 similarly alters the dynamics of the SpHNH domain. We have previously demonstrated that the alteration of allostery via mutations is a source for the specificity enhancement of SpCas9 (eSpCas9). Hence, this may also be true in GeoCas9.

Twisting and swiveling domain motions in Cas9 to recognize target DNA duplexes, make double-strand breaks, and release cleaved duplexes.

The CRISPR-associated protein 9 (Cas9) has been engineered as a precise gene editing tool to make double-strand breaks. CRISPR-associated protein 9 binds the folded guide RNA (gRNA) that serves as a binding scaffold to guide it to the target DNA duplex via a RecA-like strand-displacement mechanism but without ATP binding or hydrolysis. The target search begins with the protospacer adjacent motif or PAM-interacting domain, recognizing it at the major groove of the duplex and melting its downstream duplex where an RNA-DNA heteroduplex is formed at nanomolar affinity. The rate-limiting step is the formation of an R-loop structure where the HNH domain inserts between the target heteroduplex and the displaced non-target DNA strand. Once the R-loop structure is formed, the non-target strand is rapidly cleaved by RuvC and ejected from the active site. This event is immediately followed by cleavage of the target DNA strand by the HNH domain and product release. Within CRISPR-associated protein 9, the HNH domain is inserted into the RuvC domain near the RuvC active site via two linker loops that provide allosteric communication between the two active sites. Due to the high flexibility of these loops and active sites, biophysical techniques have been instrumental in characterizing the dynamics and mechanism of the CRISPR-associated protein 9 nucleases, aiding structural studies in the visualization of the complete active sites and relevant linker structures. Here, we review biochemical, structural, and biophysical studies on the underlying mechanism with emphasis on how CRISPR-associated protein 9 selects the target DNA duplex and rejects non-target sequences.

Shifting Polar Residues Across Primary Sequence Frames of Transmembrane Domains Calibrates Membrane Permeation Thermodynamics.

Permeation of the mitochondrial outer membrane (MOM) using the transmembrane domains (TMDs) is the key step of the Bcl-2 family of proteins to control apoptosis. The primary sequences of the TMDs of the family members like Bcl-xL, Bcl-2, Bak, etc. indicate the presence of charged residues at the C-terminal tip to be essential for drilling the membrane. However, Bax, a variant of the same family, is an exception, as the charged residues are shifted away from the tip by two positional frames in the primary sequence, but does it matter really? The free energy landscapes of membrane permeation, computed from a total of ∼13.3 µs of conformational sampling, show how such shifting of the amino acid frames in the primary sequence is correlated with the energy landscape that ensures the balance between membrane permeation and cytosolic population. Shifting the charged residues back to the terminal, in suitable mutants of Bax, proves the necessity of terminal charged residues by improving the insertion free energy but adds a high energy barrier unless some other polar residues are adjusted further. The difference in the TMDs of Bcl-xL and Bax is also reflected in their mechanism to drill the MOM-like anionic membrane; only Bax-TMD requires surface crowding to favorably shape the permeation landscape by weakening the bilayer integrity. So, this investigation suggests that such proteins can calibrate the free energy landscape of membrane permeation by adjusting the positions of the charged or polar residues in the primary sequence frames, a strategy analogous to the game of the "sliding tile puzzle" but played with primary sequence frames.

HKT1;5 Transporter Gene Expression and Association of Amino Acid Substitutions With Salt Tolerance Across Rice Genotypes.

Plants need to maintain a low Na+/K+ ratio for their survival and growth when there is high sodium concentration in soil. Under these circumstances, the high affinity K+ transporter (HKT) and its homologs are known to perform a critical role with HKT1;5 as a major player in maintaining Na+ concentration. Preferential expression of HKT1;5 in roots compared to shoots was observed in rice and rice-like genotypes from real time PCR, microarray, and RNAseq experiments and data. Its expression trend was generally higher under increasing salt stress in sensitive IR29, tolerant Pokkali, both glycophytes; as well as the distant wild rice halophyte, Porteresia coarctata, indicative of its importance during salt stress. These results were supported by a low Na+/K+ ratio in Pokkali, but a much lower one in P. coarctata. HKT1;5 has functional variability among salt sensitive and tolerant varieties and multiple sequence alignment of sequences of HKT1;5 from Oryza species and P. coarctata showed 4 major amino acid substitutions (140 P/A/T/I, 184 H/R, D332H, V395L), with similarity amongst the tolerant genotypes and the halophyte but in variance with sensitive ones. The best predicted 3D structure of HKT1;5 was generated using Ktrab potassium transporter as template. Among the four substitutions, conserved presence of aspartate (332) and valine (395) in opposite faces of the membrane along the Na+/K+ channel was observed only for the tolerant and halophytic genotypes. A model based on above, as well as molecular dynamics simulation study showed that valine is unable to generate strong hydrophobic network with its surroundings in comparison to leucine due to reduced side chain length. The resultant alteration in pore rigidity increases the likelihood of Na+ transport from xylem sap to parenchyma and further to soil. The model also proposes that the presence of aspartate at the 332 position possibly leads to frequent polar interactions with the extracellular loop polar residues which may shift the loop away from the opening of the constriction at the pore and therefore permit easy efflux of the Na+. These two substitutions of the HKT1;5 transporter probably help tolerant varieties maintain better Na+/K+ ratio for survival under salt stress.

Cholesterol Modulates Membrane Properties and the Interaction of gp41 Fusion Peptide To Promote Membrane Fusion.

An envelope glycoprotein, gp41, is crucial for the entry of human immunodeficiency virus (HIV) into the host cell. The 20-23 N-terminal amino acid sequence of gp41 plays an important role in promoting fusion between viral and host cells. Interestingly, the structure and function of the fusion peptide are extremely sensitive to the characteristics of the lipid environment. In this present work, we have extensively utilized steady-state and time-resolved fluorescence spectroscopy in tandem with molecular dynamics simulation to elucidate peptide binding and peptide-induced perturbation to the membrane. We have used two depth-dependent fluorescence probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and its trimethylammonium derivative (TMA-DPH), to monitor the effect of peptide binding along the bilayer normal and have reconciled the experimental observation with the insights from the simulated molecular events. We have further monitored the effect of membrane cholesterol on peptide-induced membrane perturbation. The molecular dynamics simulation data show that the peptide alters the membrane properties in the vicinity of the peptide and it penetrates to a larger extent into the bilayer when the membrane contains cholesterol. Our results clearly elucidate that cholesterol alters the membrane physical properties in favor of membrane fusion and interaction pattern of the fusion peptide with the membrane in a concentration-dependent fashion. The role of cholesterol is specifically important as the host eukaryotic cells contain a decent amount of cholesterol that might be critical for the entry of HIV into the host cells.

Dissecting the thermodynamic contributions of the charged residues in the membrane anchoring of Bcl-xl C-terminal domain.

The C-terminal helix of the Bcl-xl is known to initiate the membrane insertion of the protein by anchoring into the mitochondrial outer membrane. The C-terminal charged residues of that helix, R232 and K233, are reported to have an important structural role in the process of that insertion. The present work provides a quantitative understanding of the thermodynamic contribution of these residues on the membrane insertion energy-profile, calculated from the Adaptive Biasing Force based MD simulations of 2.67 µs altogether. Interestingly, the effect of the single neutralizing mutations at the C-terminus, i.e. K233A or R232A, is easily tolerated by the peptide without impacting the nature of insertion energy-profile, indicating the efficiency of one positively charged residue to drive the insertion. Whereas a double mutant, i.e. R232A and K233A, makes a significant impact on the energy-profile by destabilizing the membrane-associated states, as well as the membrane-embedded states. The finding provides molecular-level mechanistic insight. The water-mediated interaction formed by the peptide polar side chains within the bilayer core is found to modulate the membrane response during peptide insertion and that subsequently regulates the insertion mechanism. Mutation of the C-terminal residues eventually alters such a cascade of interactions that results in an insertion through energetically more expensive pathway. Since any one of the positively charged residues at the terminal is critical to ensure the membrane insertion, it appears that the natural selection of 'two' instead of 'one' charged residue is redundant in the context of membrane anchoring but may be important for other biochemical events.

BIM Binding Remotely Regulates BAX Activation: Insights from the Free Energy Landscapes.

Activation of the pro-apoptotic BAX protein, a BCL-2 family member, is known to trigger apoptosis by forming pores in the mitochondrial outer membrane (MOM). While in the cytosol, release of its transmembrane C-terminal helix (called α9 helix) from a well-characterized binding pocket (BC groove) and subsequent permeabilization of the MOM are understood to be the initiating events of the activation. Concerning what initiates BAX activation, so far one plausible suggestion has been that the transient attachment of BH3-only peptide at a distal site from the BC groove triggers the activation process. Yet how this pivotal step displaces α9 from the BC groove has remained poorly understood. Using a combination of standard molecular dynamics and enhanced sampling methods, the energy landscape of BIM (BH3-only peptide) induced BAX activation has been computed, and the molecular origin of those events is hereby reported in atomistic detail. The simulated transition pathway of α9 release reveals that BIM subdues the energetic cost of the process by reducing the activation energy barrier to some extent but mostly by minimizing the free energy difference between the active (α9-released) and inactive (α9-bound) states. Interestingly, the flexibility of the α9 helix itself plays a decisive role in this mechanism. The impact of BIM encounter at the distal site is found to propagate to the α9 (BC groove bound) mostly through conserved pathways of residue level interactions. Overall, the thermodynamic basis of the "hit-and-run" mechanism for activation of the BCL-2 family is presented reconciling the available biochemical observations.

C-terminal tail insertion of Bcl-xL in membrane occurs via partial unfolding and refolding cycle associating microsolvation.

Bcl-xL, a member of the Bcl-2 family of proteins, remains distributed over the cytosol and the mitochondrial membrane, maintaining a balance between apoptosis and the survival of the cell. Passage to the membrane is essential for its biological functions (e.g. to antagonize pro-apoptotic proteins of the Bcl2 family), which is known to be initiated by the insertion of the C-terminal segment into the membrane. This tail, composed of ∼24 residues, is reported to act as a pseudo-inhibitor of the protein itself, adapting a helical conformation. It gets released from the confinement when Bcl-xL approaches the membrane. This article reports the events associated with the insertion of the helical tail into an explicitly modeled all-atom membrane, which reveals a partial unfolding to refolding cycle of the peptide, correlating with the early insertion, to a fully inserted state. The polar interactions have been found to have a dominant role in steering the peptide towards the membrane at the desired orientation. The landscape of the potential of mean force (PMF) is consistent with the proposed mechanism. Molecular dynamics further brings the insight that the peptide insertion is associated with the encapsulation of a thin water layer around the peptide throughout the course of insertion, which motivates the protein to refold once the insertion is complete.