scholarly journals Enhanced specificity mutations perturb allosteric signaling in CRISPR-Cas9

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Lukasz Nierzwicki ◽  
Kyle W East ◽  
Uriel N Morzan ◽  
Pablo R Arantes ◽  
Victor S Batista ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Dna Cleavage ◽  
Molecular Level ◽  
Signal Transmission ◽  
Dna Recognition ◽  
Solution Nmr ◽  
Allosteric Communication ◽  
Catalytic Sites ◽  
Biochemical Studies

CRISPR-Cas9 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.

scholarly journals Enhanced Specificity Mutations Perturb Allosteric Signaling in CRISPR-Cas9

2021 ◽  
Author(s):  
Łukasz Nierzwicki ◽  
Kyle W. East ◽  
Uriel N. Morzan ◽  
Pablo R. Arantes ◽  
Victor S. Batista ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Graph Theory ◽  
Genome Editing ◽  
Dna Cleavage ◽  
Molecular Level ◽  
Signal Transmission ◽  
Dna Recognition ◽  
Solution Nmr ◽  
Catalytic Sites

ABSTRACTCRISPR-Cas9 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, solution NMR is combined with multi-microsecond molecular dynamics and graph theory-derived models to probe the allosteric role of key enhancement specificity mutations. We show that the 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 HNH domain allosteric structure, exerting the highest perturbation on the signaling transfer, while K810A and K848A result in more moderate effects on the allosteric intercommunication. This differential perturbation of the allosteric signaling reflects the different capabilities of the single mutants to increase Cas9 specificity, with the mutation achieving the highest specificity also strongly perturbing the signaling transfer. These outcomes reveal that the allosteric regulation is critical for the specificity enhancement of the Cas9 enzyme, and are valuable to harness the signaling network to improve the system’s specificity.

scholarly journals Flexible pivoting of dynamin PH-domain catalyzes fission: Insights into molecular degrees of freedom

2019 ◽  
Author(s):  
K. K. Baratam ◽  
K. Jha ◽  
A. Srivastava
Keyword(s):  
Molecular Dynamics ◽  
Free Energy ◽  
Synaptic Vesicle ◽  
Molecular Level ◽  
Pleckstrin Homology Domain ◽  
Membrane Association ◽  
Membrane Interactions ◽  
Vesicle Recycling ◽  
Membrane Fission

ABSTRACTThe neuronal dynamin1 functions in the release of synaptic vesicles by orchestrating the process of GTPase-dependent membrane fission. Dynamin1 associates with the plasma membrane-localized phosphatidylinositol-4,5-bisphosphate (PIP2) with its centrally-located pleckstrin homology domain (PHD). The PHD is dispensable as fission can be managed, albeit at much slower rates, even when the PHD-PIP2 interaction is replaced by a generic polyhistidine- or polylysine-lipid interaction. However, even when the PHD is present, the length of the dynamin scaffold and in turn the membrane remodeling and fission rates are severely restricted with mutations such as I533A on membrane-interacting variable loop 1 (VL1) of PHD. These observations suggest that PIP2-containing membrane interactions of PHD could have evolved to expedite fission to fulfill the requirement of rapid kinetics of synaptic vesicle recycling. Here, we use a suite of multiscale modeling approaches that combine atomistic molecular dynamics simulations, mixed resolution membrane mimetic models, coarse-grained molecular simulations and advanced free-energy sampling methods (metadynamics and umbrella sampling) to explore PHD-membrane interactions. Our results reveal that: (a) the binding of PHD to PIP2-containing membranes modulates the lipids towards fission-favoring conformations and softens the membrane, (b) that PHD engages another loop (VL4) for membrane association, which acts as an auxiliary pivot and modulates the orientation flexibility of PHD on the membrane – a mechanism we believe may be important for high fidelity dynamin collar assembly on the membrane. (c) Through analyses of our trajectories data and free-energy calculations on membrane-bound WT and mutant systems, we also identify key residues on multiple VLs that stabilizes PHD membrane association. And we suggest experiments to explore the ability of PHD to associate with membrane in orientations that favors faster fission. Together, these insights provide a molecular-level understanding of the “catalytic” role of the PHD in dynamin-mediated membrane fission.SIGNIFICANCEDynamin, a large multi-domain GTPase, remodels the membrane by self-assembling onto the neck of a budding vesicle and induces fission by its energy driven conformational changes. In this work, we use multi-scale molecular simulations to probe the role of dynamin’s pleckstrin-homology domain (PHD), which facilitates membrane interactions. Notably, PHD is dispensable for fission as is the case with extant bacterial and mitochondrial dynamins. However, reconstitution experiments suggest that the functional role of PHD in neuronal-membrane goes beyond that of an adaptor domain as it possibly ‘expedites’ the fission reaction during synaptic vesicle recycling. We provide a molecular-dynamics picture of how PHDs make membranes more pliable for fission and suggest new insights into the molecular-level processes driving the expedited fission behavior.

scholarly journals Allosteric Motions of the CRISPR-Cas9 HNH Nuclease Probed by NMR and Molecular Dynamics

2019 ◽  
Author(s):  
Kyle W. East ◽  
Jocelyn C. Newton ◽  
Uriel N. Morzan ◽  
Atanu Acharya ◽  
Erin Skeens ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Genome Editing ◽  
Dna Cleavage ◽  
Simulation Method ◽  
Accelerated Molecular Dynamics ◽  
Allosteric Communication ◽  
Double Stranded Dna ◽  
Dna Cleavage Activity ◽  
Mechanistic Basis ◽  
Allosteric Pathway

ABSTRACTCRISPR-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 timescale 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.Abstract Figure

scholarly journals Recent advances in the nutritional biochemistry of trivalent chromium

2004 ◽  
Vol 63 (1) ◽  
pp. 41-47 ◽  
Author(s):  
John B. Vincent
Keyword(s):  
Kinase Activity ◽  
Molecular Level ◽  
Nutritional Supplement ◽  
Trivalent Chromium ◽  
The Other ◽  
Chromium Picolinate ◽  
Biologically Relevant ◽  
Carbohydrate And Lipid Metabolism ◽  
Biochemical Studies

The nutritional biochemistry of trivalent Cr has been a poorly understood field of study; investigations of the biochemistry of the other essential transition metals have not proven as problematic. Despite over four decades of endeavour, only recently has a picture of the role of Cr potentially started to be defined. The biologically-relevant form is the trivalent ion. Cr3+appears to be required for proper carbohydrate and lipid metabolism in mammals, although fortunately Cr deficiency is difficult to achieve. Conditions that increase circulating glucose and insulin concentrations increase urinary Cr output. Cr is probably excreted in the form of the oligopeptide chromodulin. Chromodulin may be the key to understanding the role of Cr at a molecular level, as the molecule has been found to bind to activated insulin receptor, stimulating its kinase activity. A mechanism for the action of chromodulin has recently been proposed; this mechanism can serve as a potential framework for further studies to test the role of Cr in metabolism. An examination of the nutritional supplement chromium picolinate illustrates some of the difficulties associated with these biochemical studies.

Sign in / Sign up

Export Citation Format

Share Document