Identification of Family-Specific Features in Cas9 and Cas12 Proteins: A Machine Learning Approach Using Complete Protein Feature Spectrum.

The recent development of CRISPR-Cas technology holds promise to correct gene-level defects for genetic diseases. The key element of the CRISPR-Cas system is the Cas protein, a nuclease that can edit the gene of interest assisted by guide RNA. However, these Cas proteins suffer from inherent limitations such as large size, low cleavage efficiency, and off-target effects, hindering their widespread application as a gene editing tool. Therefore, there is a need to identify novel Cas proteins with improved editing properties, for which it is necessary to understand the underlying features governing the Cas families. In this study, we aim to elucidate the unique protein features associated with Cas9 and Cas12 families and identify the features distinguishing each family from non-Cas proteins. Here, we built Random Forest (RF) binary classifiers to distinguish Cas12 and Cas9 proteins from non-Cas proteins, respectively, using the complete protein feature spectrum (13,494 features) encoding various physiochemical, topological, constitutional, and coevolutionary information on Cas proteins. Furthermore, we built multiclass RF classifiers differentiating Cas9, Cas12, and non-Cas proteins. All the models were evaluated rigorously on the test and independent data sets. The Cas12 and Cas9 binary models achieved a high overall accuracy of 92% and 95% on their respective independent data sets, while the multiclass classifier achieved an F1 score of close to 0.98. We observed that Quasi-Sequence-Order (QSO) descriptors like Schneider.lag and Composition descriptors like charge, volume, and polarizability are predominant in the Cas12 family. Conversely Amino Acid Composition descriptors, especially Tripeptide Composition (TPC), predominate the Cas9 family. Four of the top 10 descriptors identified in Cas9 classification are tripeptides PWN, PYY, HHA, and DHI, which are seen to be conserved across all Cas9 proteins and located within different catalytically important domains of the Streptococcus pyogenes Cas9 (SpCas9) structure. Among these, DHI and HHA are well-known to be involved in the DNA cleavage activity of the SpCas9 protein. Mutation studies have highlighted the significance of the PWN tripeptide in PAM recognition and DNA cleavage activity of SpCas9, while Y450 from the PYY tripeptide plays a crucial role in reducing off-target effects and improving the specificity in SpCas9. Leveraging our machine learning (ML) pipeline, we identified numerous Cas9 and Cas12 family-specific features. These features offer valuable insights for future experimental and computational studies aiming at designing Cas systems with enhanced gene-editing properties. These features suggest plausible structural modifications that can effectively guide the development of Cas proteins with improved editing capabilities.

Identification of Family-Specific Features in Cas9 and Cas12 Proteins: A Machine Learning Approach Using Complete Protein Feature Spectrum.

The recent development of CRISPR-Cas technology holds promise to correct gene-level defects for genetic diseases. The key element of the CRISPR-Cas system is the Cas protein, a nuclease that can edit the gene of interest assisted by guide RNA. However, these Cas proteins suffer from inherent limitations like large size, low cleavage efficiency, and off-target effects, hindering their widespread application as a gene editing tool. Therefore, there is a need to identify novel Cas proteins with improved editing properties, for which it is necessary to understand the underlying features governing the Cas families. In the current study, we aim to elucidate the unique protein attributes associated with Cas9 and Cas12 families and identify the features that distinguish each family from the other. Here, we built Random Forest (RF) binary classifiers to distinguish Cas12 and Cas9 proteins from non-Cas proteins, respectively, using the complete protein feature spectrum (13,495 features) encoding various physiochemical, topological, constitutional, and coevolutionary information of Cas proteins. Furthermore, we built multiclass RF classifiers differentiating Cas9, Cas12, and Non-Cas proteins. All the models were evaluated rigorously on the test and independent datasets. The Cas12 and Cas9 binary models achieved a high overall accuracy of 95% and 97% on their respective independent datasets, while the multiclass classifier achieved a high F1 score of 0.97. We observed that Quasi-sequence-order descriptors like Schneider-lag descriptors and Composition descriptors like charge, volume, and polarizability are essential for the Cas12 family. More interestingly, we discovered that Amino Acid Composition descriptors, especially the Tripeptide Composition (TPC) descriptors, are important for the Cas9 family. Four of the identified important descriptors of Cas9 classification are tripeptides PWN, PYY, HHA, and DHI, which are seen to be conserved across all the Cas9 proteins and were located within different catalytically important domains of the Cas9 protein structure. Among these four tripeptides, tripeptides DHI and HHA are well-known to be involved in the DNA cleavage activity of the Cas9 protein. We therefore propose the the other two tripeptides, PWN and PYY, may also be essential for the Cas9 family. Our identified important descriptors enhanced the understanding of the catalytic mechanisms of Cas9 and Cas12 proteins and provide valuable insights into design of novel Cas systems to achieve enhanced gene-editing properties.

Leveraging QM/MM and Molecular Dynamics Simulations to Decipher the Reaction Mechanism of the Cas9 HNH Domain to Investigate Off-Target Effects.

The clustered regularly interspaced short palindromic repeats (CRISPR) technology is an RNA-guided targeted genome-editing tool using Cas family proteins. Two magnesium-dependent nuclease domains of the Cas9 enzyme, termed HNH and RuvC, are responsible for cleaving the target DNA (t-DNA) and nontarget DNA strands, respectively. The HNH domain is believed to determine the DNA cleavage activity of both endonuclease domains and is sensitive to complementary RNA-DNA base pairing. However, the underlying molecular mechanisms of CRISPR-Cas9, by which it rebukes or accepts mismatches, are poorly understood. Thus, investigation of the structure and dynamics of the catalytic state of Cas9 with either matched or mismatched t-DNA can provide insights into improving its specificity by reducing off-target cleavages. Here, we focus on a recently discovered catalytic-active form of the Streptococcus pyogenes Cas9 (SpCas9) and employ classical molecular dynamics and coupled quantum mechanics/molecular mechanics simulations to study two possible mechanisms of t-DNA cleavage reaction catalyzed by the HNH domain. Moreover, by designing a mismatched t-DNA structure called MM5 (C to G at the fifth position from the protospacer adjacent motif region), the impact of single-guide RNA (sgRNA) and t-DNA complementarity on the catalysis process was investigated. Based on these simulations, our calculated binding affinities, minimum energy paths, and analysis of catalytically important residues provide atomic-level details of the differences between matched and mismatched cleavage reactions. In addition, several residues exhibit significant differences in their catalytic roles for the two studied systems, including K253, K263, R820, K896, and K913.

Application of molecular dynamic simulations in modeling the excited state behavior of confined molecules.

Relative to isotropic organic solvent medium, the structure and conformation of a reactant molecule in an organized and confining medium are often different. In addition, because of the rigidity of the immediate environment, the reacting molecule have a little freedom to undergo large changes even upon gaining energy or modifications in the electronic structure. These alterations give rise to differences in the photochemistry of a molecular and supramolecular species. In this study, one such example is presented. α-Alkyl dibenzylketones upon excitation in isotropic solvents give products via Norrish type I and type II reactions that are independent of the chain length of the alkyl substituent. On the other hand, when these molecules are enclosed within an organic capsule of volume ~ 550 Å3, they give products that are strikingly dependent on the length of the α-alkyl substitution. These previously reported experimental observations are rationalized based on the structures generated by molecular modeling (docking and molecular dynamics (MD) simulations). It is shown that MD simulations that are utilized extensively in biologically important macromolecules can also be useful to understand the excited state behavior of reactive molecules that are part of supramolecular assemblies. These simulations can provide structural information of the reactant molecule and the surroundings complementing that with the one obtained from 1 and 2D NMR experiments. MD simulated structures of seven α-alkyl dibenzylketones encapsulated within the octa acid capsule provide a clear understanding of their unique behavior in this restricted medium. Because of the rigidity of the medium, these structures although generated in the ground state can rationalize the photochemical behavior of the molecules in the excited state.

Distinct chemical factors in hydrolytic reactions catalyzed by metalloenzymes and metal complexes.

The selective hydrolysis of the extremely stable phosphoester, peptide and ester bonds of molecules by bio-inspired metal-based catalysts (metallohydrolases) is required in a wide range of biological, biotechnological and industrial applications. Despite the impressive advances made in the field, the ultimate goal of designing efficient enzyme mimics for these reactions is still elusive. Its realization will require a deeper understanding of the diverse chemical factors that influence the activities of both natural and synthetic catalysts. They include catalyst-substrate complexation, non-covalent interactions and the electronic nature of the metal ion, ligand environment and nucleophile. Based on our computational studies, their roles are discussed for several mono- and binuclear metallohydrolases and their synthetic analogues. Hydrolysis by natural metallohydrolases is found to be promoted by a ligand environment with low basicity, a metal bound water and a heterobinuclear metal center (in binuclear enzymes). Additionally, peptide and phosphoester hydrolysis is dominated by two competing effects, i.e. nucleophilicity and Lewis acid activation, respectively. In synthetic analogues, hydrolysis is facilitated by the inclusion of a second metal center, hydrophobic effects, a biological metal (Zn, Cu and Co) and a terminal hydroxyl nucleophile. Due to the absence of the protein environment, hydrolysis by these small molecules is exclusively influenced by nucleophile activation. The results gleaned from these studies will enhance the understanding of fundamental principles of multiple hydrolytic reactions. They will also advance the development of computational methods as a predictive tool to design more efficient catalysts for hydrolysis, Diels-Alder reaction, Michael addition, epoxide opening and aldol condensation.

Deconvoluting binding sites in amyloid nanofibrils using time-resolved spectroscopy.

Steady-state fluorescence spectroscopy has a central role not only for sensing applications, but also in biophysics and imaging. Light switching probes, such as ruthenium dipyridophenazine complexes, have been used to study complex systems such as DNA, RNA, and amyloid fibrils. Nonetheless, steady-state spectroscopy is limited in the kind of information it can provide. In this paper, we use time-resolved spectroscopy for studying binding interactions between amyloid-ß fibrillar structures and photoluminescent ligands. Using time-resolved spectroscopy, we demonstrate that ruthenium complexes with a pyrazino phenanthroline derivative can bind to two distinct binding sites on the surface of fibrillar amyloid-ß, in contrast with previous studies using steady-state photoluminescence spectroscopy, which only identified one binding site for similar compounds. The second elusive binding site is revealed when deconvoluting the signals from the time-resolved decay traces, allowing the determination of dissociation constants of 3 and 2.2 µM. Molecular dynamic simulations agree with two binding sites on the surface of amyloid-ß fibrils. Time-resolved spectroscopy was also used to monitor the aggregation of amyloid-ß in real-time. In addition, we show that common polypyridine complexes can bind to amyloid-ß also at two different binding sites. Information on how molecules bind to amyloid proteins is important to understand their toxicity and to design potential drugs that bind and quench their deleterious effects. The additional information contained in time-resolved spectroscopy provides a powerful tool not only for studying excited state dynamics but also for sensing and revealing important information about the system including hidden binding sites.

Promiscuous Catalytic Activity of a Binuclear Metallohydrolase: Peptide and Phosphoester Hydrolyses.

In this study, chemical promiscuity of a binuclear metallohydrolase Streptomyces griseus aminopeptidase (SgAP) has been investigated using DFT calculations. SgAP catalyzes two diverse reactions, peptide and phosphoester hydrolyses, using its binuclear (Zn-Zn) core. On the basis of the experimental information, mechanisms of these reactions have been investigated utilizing leucine p-nitro aniline (Leu-pNA) and bis(4-nitrophenyl) phosphate (BNPP) as the substrates. The computed barriers of 16.5 and 16.8 kcal/mol for the most plausible mechanisms proposed by the DFT calculations are in good agreement with the measured values of 13.9 and 18.3 kcal/mol for the Leu-pNA and BNPP hydrolyses, respectively. The former was found to occur through the transfer of two protons, while the latter with only one proton transfer. They are in line with the experimental observations. The cleavage of the peptide bond was the rate-determining process for the Leu-pNA hydrolysis. However, the creation of the nucleophile and its attack on the electrophile phosphorus atom was the rate-determining step for the BNPP hydrolysis. These calculations showed that the chemical nature of the substrate and its binding mode influence the nucleophilicity of the metal bound hydroxyl nucleophile. Additionally, the nucleophilicity was found to be critical for the Leu-pNA hydrolysis, whereas double Lewis acid activation was needed for the BNPP hydrolysis. That could be one of the reasons why peptide hydrolysis can be catalyzed by both mononuclear and binuclear metal cofactors containing hydrolases, while phosphoester hydrolysis is almost exclusively by binuclear metallohydrolases. These results will be helpful in the development of versatile catalysts for chemically distinct hydrolytic reactions.

Organic Host Encapsulation Effects on Nitrosobenzene Monomer-Dimer Distribution and C-NO Bond Rotation in an Aqueous Solution.

The intermolecular (monomer-dimer equilibrium) and intramolecular (C-NO and C-NMe2 rotations) dynamics of 4-nitrosocumene (1a) and 4-(N,N-dimethylamino)nitrosobenzene (1b), respectively, were found to be controlled by the medium (water) and the host environment (organic capsules and cavitands). The ability of water to shift the equilibrium toward the dimer appears to result from dipolar stabilization of the polar dimer structure and has a resemblance to water's known ability to favor organic cycloaddition reactions. In an aqueous medium, a range of organic hosts selectively include only the nitrosocumene monomer 1a. Encapsulation in the octa acid duplex (OA2) selects two 1a monomers rather than a dimer structure. Octa acid encapsulation also results in more restricted intramolecular C-N rotations of the guest 1b.

Degradation of a Main Plastic Pollutant Polyethylene Terephthalate by Two Distinct Proteases (Neprilysin and Cutinase-like Enzyme).

In this DFT study, hydrolysis of polyethylene terephthalate (PET), a major cause of plastic pollution, by two distinct enzymes, neprilysin (NEP, a mononuclear metalloprotease) and cutinase-like enzyme (CLE, a serine protease), has been investigated. These enzymes utilize different mechanisms for the degradation of PET. NEP uses either the metal-bound hydroxide attack (MH) mechanism or reverse protonation (RP) mechanism, while CLE utilizes a general acid/base mechanism that includes acylation and deacylation processes. Additionally, the RP mechanism of NEP can proceed through three pathways, RP0, RP1, and RP2. The DFT calculations predict that, among all these mechanisms, the MH mechanism is the energetically most favorable one for the NEP enzyme. In comparison, CLE catalyzes this reaction with a significantly higher barrier. These results suggest that the Lewis acid and nucleophile activations provided by the Zn metal center of NEP are more effective than the hydrogen bonding interactions afforded by the catalytic Ser85-His180-Asp165 triad of CLE. They have provided intrinsic details regarding PET degradation and will pave the way for the design of efficient metal-based catalysts for this critical reaction.

Heteromeric three-stranded coiled coils designed using a Pb(II)(Cys)3 template mediated strategy.

Three-stranded coiled coils are peptide structures constructed from amphipathic heptad repeats. Here we show that it is possible to form pure heterotrimeric three-stranded coiled coils by combining three distinct characteristics: (1) a cysteine sulfur layer for metal coordination, (2) a thiophilic, trigonal pyramidal metalloid (Pb(II)) that binds to these sulfurs and (3) an adjacent layer of reduced steric bulk generating a cavity where water can hydrogen bond to the cysteine sulfur atoms. Cysteine substitution in an a site yields Pb(II)A2B heterotrimers, while d sites provide pure Pb(II)C2D or Pb(II)CD2 scaffolds. Altering the metal from Pb(II) to Hg(II) or shifting the relative position of the sterically less demanding layer removes heterotrimer specificity. Because only two of the eight or ten hydrophobic layers are perturbed, catalytic sites can be introduced at other regions of the scaffold. A Zn(II)(histidine)3(H2O) centre can be incorporated at a remote location without perturbing the heterotrimer selectivity, suggesting a unique strategy to prepare dissymmetric catalytic sites within self-assembling de novo-designed proteins.

Effects of the Metal Ion on the Mechanism of Phosphodiester Hydrolysis Catalyzed by Metal-Cyclen Complexes.

In this study, mechanisms of phosphodiester hydrolysis catalyzed by six di- and tetravalent metal-cyclen (M-C) complexes (Zn-C, Cu-C, Co-C, Ce-C, Zr-C and Ti-C) have been investigated using DFT calculations. The activities of these complexes were studied using three distinct mechanisms: (1) direct attack ( DA ), (2) catalyst-assisted ( CA ), and (3) water-assisted ( WA ). All divalent metal complexes (Zn-C, Cu-C and Co-C) coordinated to the BNPP substrate in a monodentate fashion and activated its scissile phosphoester bond. However, all tetravalent metal complexes (Ce-C, Zr-C, and Ti-C) interacted with BNPP in a bidentate manner and strengthened this bond. The DA mechanism was energetically the most feasible for all divalent M-C complexes, while the WA mechanism was favored by the tetravalent complexes, except Ce-C. The divalent complexes were found to be more reactive than their tetravalent counterparts. Zn-C catalyzed the hydrolysis with the lowest barrier among all M-C complexes, while Ti-C was the most reactive tetravalent complex. The activities of Ce-C and Zr-C, except Ti-C, were improved with an increase in the coordination number of the metal ion. The structural and mechanistic information provided in this study will be very helpful in the development of more efficient metal complexes for this critical reaction.

Investigating coordination flexibility of glycerophosphodiesterase (GpdQ) through interactions with mono-, di-, and triphosphoester (NPP, BNPP, GPE, and paraoxon) substrates.

In this study, interactions of the catalytically active binuclear form of glycerophosphodiesterase (GpdQ) with four chemically diverse substrates, i.e. NPP (a phosphomonoester), BNPP and GPE (both phosphodiesters), and paraoxon (a phosphotriester) have been investigated using all-atom molecular dynamics (MD) simulations. The roles of metal ions and key amino acid residues, coordination flexibility, and dynamic transformations in all enzyme-substrate complexes have been elucidated. The roles of important first and second coordination shell residues in substrate binding and coordination flexibility of the enzyme suggested by simulations are supported by experimental data. The chemical nature of the substrate is found to influence the mode of binding, electrostatic surface potential, metal-metal distance, and reorganization of the active site. The experimentally proposed association between the substrate binding and coordination flexibility is analyzed using principal component analysis (PCA), movements of loops, and root-mean-square-fluctuations (RMSF) as parameters. The PCA of these substrates provides different energy basins, i.e. one, three, two and five for NPP, BNPP, GPE, and paraoxon, respectively. Additionally, the area of an irregular hexagon (268.3, 288.9, 350.8, and 362.5 Å2) formed by the residues on these loops illustrates their distinct motions. The substrate binding free energies of NPP, BNPP, and GPE are quite close (22.4-24.3 kcal mol-1), but paraoxon interacts with the smallest binding free energy (14.1 kcal mol-1). The metal binding energies in the presence of these substrates are substantially different, i.e. the lowest for NPP and the highest for paraoxon. These results thus provide deeper insight into the chemical promiscuity and coordination flexibility of this important enzyme.

Hydrolysis of chemically distinct sites of human serum albumin by polyoxometalate: A hybrid QM/MM (ONIOM) study.

In this study, mechanisms of hydrolysis of all four chemically diverse cleavage sites of human serum albumin (HSA) by [Zr(OH)(PW11 O39 )]4- (ZrK) have been investigated using the hybrid two-layer QM/MM (ONIOM) method. These reactions have been proposed to occur through the following two mechanisms: internal attack (IA) and water assisted (WA). In both mechanisms, the cleavage of the peptide bond in the Cys392-Glu393 site of HSA is predicted to occur in the rate-limiting step of the mechanism. With the barrier of 27.5 kcal/mol for the hydrolysis of this site, the IA mechanism is found to be energetically more favorable than the WA mechanism (barrier = 31.6 kcal/mol). The energetics for the IA mechanism are in line with the experimentally measured values for the cleavage of a wide range of dipeptides. These calculations also suggest an energetic preference (Cys392-Glu393, Ala257-Asp258, Lys313-Asp314, and Arg114-Leu115) for the hydrolysis of all four sites of HSA. © 2018 Wiley Periodicals, Inc.