The discovery of protein inhibitors of CRISPR-Cas systems, called anti-CRISPRs (Acrs), has enabled the development of highly controllable and precise CRISPR-Cas tools. Anti-CRISPRs share very little ...structural or sequential resemblance to each other or to other proteins, which raises intriguing questions regarding their modes of action. Many structure-function studies have shed light on the mechanism(s) of Acrs, which can act as orthosteric or allosteric inhibitors of CRISPR-Cas machinery, as well as enzymes that irreversibly modify CRISPR-Cas components. Only recently has the breadth of diversity of Acr structures and functions come to light, and this remains a rapidly evolving field. Here, we draw attention to a plethora of Acr mechanisms, with particular focus on how their action toward Cas proteins modulates conformation, dynamic (allosteric) signaling, nucleic acid binding, and cleavage ability.
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.
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•A novel X-ray crystal structure of GeoHNH was solved to 1.9 Å.•HNH domains of GeoCas9 and SpCas9 are structurally homologous despite a dissimilar overall fold.•Thermophilic GeoHNH is ...highly dynamic on the ps-ns timescale while signaling in mesophilic SpHNH occurson the μs-ms timescale.•This shift in dynamic profile occurs despite sequentialand structural conservation in the core of HNH.
CRISPR-Cas9 is a widely used biochemical tool with applications in molecular biology and precision medicine. The RNA-guided Cas9 protein uses its HNH endonuclease domain to cleave the DNA strand complementary to its endogenous guide RNA. In this study, novel constructs of HNH from two divergent organisms, G. stearothermophilus (GeoHNH) and S. pyogenes (SpHNH) were engineered from their respective full-length Cas9 proteins. Despite low sequence similarity, the X-ray crystal structures of these constructs reveal that the core of HNH surrounding the active site is conserved. Structure prediction of the full-length GeoCas9 protein using Phyre2 and AlphaFold2 also showed that the crystallographic construct of GeoHNH represents the structure of the domain within the full-length GeoCas9 protein. However, significant differences are observed in the solution dynamics of structurally conserved regions of GeoHNH and SpHNH, the latter of which was shown to use such molecular motions to propagate the DNA cleavage signal. Indeed, molecular simulations show that the intradomain signaling pathways, which drive SpHNH function, are non-specific and poorly formed in GeoHNH. Taken together, these outcomes suggest mechanistic differences between mesophilic and thermophilic Cas9 species.
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.
HNH is one of two endonuclease domains of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein Cas9 that perform site-specific cleavage of double-stranded DNA. We ...engineered a novel construct of this critical nuclease from
Streptococcus pyogenes
Cas9 that not only maintains the wild-type amino acid sequence and fold, but displays enhanced thermostability when compared to the full-length Cas9 enzyme. Here, we report backbone and side chain assignments of the HNH nuclease as a foundational step toward the characterization of protein dynamics and allostery in CRISPR-Cas9.
Phage L encodes a trimeric 43 kDa decoration protein (Dec) that noncovalently binds and stabilizes the capsids of the homologous phages L and P22 in vitro. At physiological pH Dec was unsuitable for ...NMR. We were able to obtain samples amenable for NMR spectroscopy by unfolding Dec to pH 2 and refolding it to pH 4. Our unfolding/refolding protocol converted trimeric Dec to a folded 14.4 kDa monomer. We verified that the acid-unfolding protocol did not perturb the secondary structure, or the capsid-binding function of refolded Dec. We were able to obtain complete
1
H,
15
N, and
13
C assignments for the Dec monomer, as well as information on its secondary structure and dynamics based on chemical shift assignments. The assigned NMR spectrum is being used to determine the three-dimensional structure of Dec, which is important for understanding how the trimer binds phage capsids and for the use of the protein as a platform for phage-display nanotechnology.
HNH is one of two endonuclease domains of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein Cas9 that perform site-specific cleavage of double-stranded DNA. We ...engineered a novel construct of this critical nuclease from Streptococcus pyogenes Cas9 that not only maintains the wild-type amino acid sequence and fold, but displays enhanced thermostability when compared to the full-length Cas9 enzyme. Here, we report backbone and side chain assignments of the HNH nuclease as a foundational step toward the characterization of protein dynamics and allostery in CRISPR-Cas9.
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