Development of protease resistant and non-cytotoxic Jelleine analogs with enhanced broad spectrum antimicrobial efficacy.

Short systemic half- life of Antimicrobial Peptides (AMP) is one of the major bottlenecks that limits their successful commercialization as therapeutics. In this work, we have designed analogs of the natural AMP Jelleine, obtained from royal jelly of apis mellifera. Among the designed peptides, J3 and J4 were the most potent with broad spectrum activities against a varied class of ESKAPE pathogens and fungus C. albicans. All the developed peptides were more effective against Gram-negative bacteria in comparison to the Gram-positive pathogens, and were especially effective against P. aeruginosa and C. albicans.J3 and J4 were completely trypsin resistant and serum stable, while retaining the non-cytotoxicity of the parent Jelleine, Jc. The designed peptides were membranolytic in their mode of action. CD and MD simulations in the presence of bilayers, established that J3 and J4 were non-structured even upon membrane binding and suggested that biological properties of the AMPs were innocent of any specific secondary structural requirements. Enhancement of charge to increase the antimicrobial potency, controlling the hydrophobic-hydrophilic balance to maintain non-cytotoxicity and induction of unnatural amino acid residues to impart protease resistance, remains some of the fundamental principles in the design of more effective antimicrobial therapeutics of the future, which may help combat the quickly rising menace of antimicrobial resistance in the microbes.

Why Does the E1219V Mutation Expand T-Rich PAM Recognition in Cas9 from Streptococcus pyogenes?

Popular RNA-guided DNA endonuclease Cas9 from Streptococcus pyogenes (SpCas9) recognizes the canonical 5'-NGG-3' protospacer adjacent motif (PAM) and triggers double-stranded DNA cleavage activity. Mutations in SpCas9 were demonstrated to expand the PAM readability and hold promise for therapeutic and genome editing applications. However, the energetics of the PAM recognition and its relation to the atomic structure remain unknown. Using the X-ray structure (precatalytic SpCas9:sgRNA:dsDNA) as a template, we calculated the change in the PAM binding affinity in response to SpCas9 mutations using computer simulations. The E1219V mutation in SpCas9 fine-tunes the water accessibility in the PAM binding pocket and promotes new interactions in the SpCas9:noncanonical T-rich PAM, thus weakening the PAM stringency. The nucleotide-specific interaction of two arginine residues (i.e., R1333 and R1335 of SpCas9) ensured stringent 5'-NGG-3' PAM recognition. R1335A substitution (SpCas9R1335A) completely disrupts the direct interaction between SpCas9 and PAM sequences (canonical or noncanonical), accounting for the loss of editing activity. Interestingly, the double mutant (SpCas9R1335A,E1219V) boosts DNA binding affinity by favoring protein:PAM electrostatic contact in a desolvated pocket. The underlying thermodynamics explain the varied DNA cleavage activity of SpCas9 variants. A direct link between the energetics, structures, and activity is highlighted, which can aid in the rational design of improved SpCas9-based genome editing tools.

E-Protein Protonation Titration-Induced Single-Particle Chemical Force Spectroscopy for Microscopic Understanding and pI Estimation of Infectious DENV.

The ionization state of amino acids on the outer surface of a virus regulates its physicochemical properties toward the sorbent surface. Serologically different strains of the dengue virus (DENV) show different extents of infectivity depending upon their interactions with a receptor on the host cell. To understand the structural dependence of E-protein protonation over its sequence dependence, we have followed E-protein titration kinetics both experimentally and theoretically for two differentially infected dengue serotypes, namely, DENV-2 and DENV-4. We have performed E-protein protonation titration-induced single-particle chemical force spectroscopy using an atomic force microscope (AFM) to measure the surface chemistry of DENV in physiological aqueous solutions not only to understand the charge distribution dynamics on the virus surface but also to estimate the isoelectric point (pI) accurately for infectious dengue viruses. Cryo-EM structure-based theoretical pI calculations of the DENV-2 surface protein were shown to be consistent with the evaluated pI value from force spectroscopy measurements. We also highlighted here the role of the microenvironment around the titrable residues (in the 3D-folded structure of the protein) in altering the pKa. This is a comprehensive study to understand how the cumulative charge distribution on the outer surface of a specific serotype of DENV regulates a prominent role of infectivity over minute changes at the genetic level.

Mechanism of Protease Resistance of D-Amino Acid Residue Containing Cationic Antimicrobial Heptapeptides.

Antimicrobial peptides (AMPs) have been an alternate promising class of therapeutics in combating global antibiotic resistance threat. However, the short half-life of AMPs, owing to protease degradability, is one of the major bottlenecks in its commercial success. In this study, we have developed all-D-amino acid containing small cationic peptides P4C and P5C, which are completely protease-resistant, noncytotoxic, nonhemolytic, and potent against the ESKAPE pathogens in comparison to their L analogues. MD simulations suggested marginal improvement in the peptide-binding affinity to the membrane-mimetic SDS micelle (∼ 1 kcal/mol) in response to L → D conversion, corroborating the marginal improvement in the antimicrobial activity. However, L → D chirality conversion severely compromised the peptide:protease (trypsin) binding affinity (≥10 kcal/mol). The relative distance between the scissile peptide carbonyl and the catalytic triad of the protease (H57, D102, and S195) was found to be significantly altered in the D-peptide:protease complex (inactive conformation) relative to the active L-peptide:protease complex. Thus, the poor binding affinity between D-peptides and the protease, resulting in the inactive complex formation, explained their experimentally observed proteolytic stability. This mechanistic insight might be extended to the proteolytic stability of the D-peptides in general and stimulate the rational design of protease-resistant AMPs.

Ambidexterity and Left-Handedness Induced by Geminally Disubstituted γ Amino Acid Residues in Chiral 310 Helices.

Chirality is an omnipresent feature in nature's architecture starting from simple molecules like amino acids to complex higher-order structures viz. proteins, DNA, and RNA. The L configuration of proteinogenic amino acids gives rise to right-handed helices. Ambidexterity is as rare in organisms as in molecules. There are only a few reports of ambidexterity in single-peptide molecules composed of either mixed L and D or achiral residues. Here, we report, for the first time, the ambidextrous and left-handed helical conformations in the chiral nonapeptides P1-P3 (Boc-LUVUγx,xULUV-OMe where U = Aib, x,x = 2,2/3,3/4,4), containing chiral L α amino acid residues, in addition to the usually observed right-handed helical conformation. The centrally located achiral γ residue, capable of adopting both left and right-handed helical conformations, induces its handedness on the neighboring chiral and achiral residues, leading to the observation of both left and right-handed helices in P2 and P3. The presence of a single water molecule proximal to the γ residue induces the reversal of helix handedness by forming distinct and stable water-mediated hydrogen bonds. This gives rise to ambidextrous helices as major conformers in P1 and P2. The absence of the observation of ambidexterity in P3 might be due to the inability of γ4,4 in the recruitment of a water molecule. Experiments (NMR, X-ray, and CD) and density functional theory (DFT) calculations suggest that the position of geminal disubstitution is crucial for determining the population of the amenable helical conformations (ambidextrous, left and right-handed) in these chiral peptides.

Effect of Spacer Length Modification of the Cationic Side Chain on the Energetics of Antimicrobial Peptide Binding to Membrane-Mimetic Bilayers.

Understanding the mechanism of action of the antimicrobial peptide (AMP) in terms of its structure and energetics is the key to designing new potent and selective AMPs. Recently, we reported a membranolytic 14-residue-long lysine-rich cationic antimicrobial peptide (LL-14: NH3+-LKWLKKLLKWLKKL-CONH2) against Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus, which is limited by cytotoxicity and expected to undergo facile protease degradation. Aliphatic side-chain-length modification of the cationic amino-acid residues (Lys and Arg) is a popular strategy for designing protease-resistant AMPs. However, the effect of the peptide side-chain length modifications on the membrane binding affinity and its relation to the atomic structure remain an unsolved problem. We report computer simulations that quantitatively calculated the difference in peptide binding affinity to membrane-mimetic-bilayer models (bacterial: 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)/1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) bilayer and mammalian: 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayer) upon decreasing or increasing the spacer length of the cationic lysine residues of LL-14 (as well as their arginine analogues). We show that the peptide/bilayer interaction energetics varies drastically in response to spacer length modification. The strength of peptide discrimination depends strongly on the nature of the bilayer (bacterial or mammalian mimetic model). An increase in the lysine spacer length by one carbon (i.e., homolysine analogue of LL-14) is weakly/strongly disfavored by the bacterial/mammalian-membrane-mimetic bilayer. Recently, we have demonstrated an excellent correlation between the antimicrobial activity of the membranolytic cationic peptides and their binding affinity to membrane-mimetic-bilayer models. Thus, the homolysine analogue of LL-14 is a promising noncytotoxic AMP with conserved activity. On the other hand, homoarginine analogue (arginine spacer length increment by a single carbon) was preferred by both the bacteria and the mammalian mimetic bilayers and displayed the strongest affinity for the former among the peptides studied in this work. Thus, the promising most potent homoarginine analogue is likely to be cytotoxic. Shortening the Lys/Arg side chain to a three-carbon spacer (Dab/Agb) improves the binding affinity to bacterial and mammalian-membrane-mimetic bilayers. Arginine and arginine-derivative peptides exhibited stronger binding affinity to the bilayers relative to the lysine analogue. The results provide a plausible explanation to the previous experimental observations, viz., superior antimicrobial activity of the arginine peptides relative to Lys peptides and the improvement of antimicrobial activity upon substitution of Lys with Dab in the cationic peptides. The simulations revealed that the small change in the peptide hydrophobicity by Lys/Arg spacer length modification could drastically alter the energetics of peptide/bilayer binding by fine-tuning the electrostatic interactions. The energetics underlying the peptide selectivity by simple membrane-mimetic bilayer models may be beneficial for designing new selective and protease-resistant AMPs.

Bridging Thermodynamics, Antimicrobial Activity, and pH Sensitivity of Cationic Membranolytic Heptapeptides-A Computational and Experimental Study.

Understanding the thermodynamics of peptide:membrane binding and the factors that alter the stability is the key to designing potent and selective small antimicrobial peptides. Here, we report the thermodynamics, antimicrobial activity, and mechanism of a de novo designed seven residue long cationic antimicrobial peptide (P4: NH3+-LKWLKKL-CONH2, Charge +4) and its analogs (P5: Lysine's → Arginine's; P6: Lysine's → Uncharged-Histidine's; P7: Tryptophan → Leucine) by combining computation and experiments. Computer simulations predicted the order of decreasing peptide binding affinity to the membrane-mimetic systems (micelle/bilayer) as P5 > P4 > P7 ≫ P6. Antimicrobial assays of these peptides against P. aeruginosa and E. coli at physiological pH 7.4 confirmed P5 as the most potent peptide (followed by P4), whereas P6 showed inferior activity. P7 was found to be inactive against E. coli. Substitution of the uncharged-histidine (P6) by the charged-histidine (P6*) significantly favored micelle/bilayer binding. Thus, P6 was predicted to be an effective antimicrobial peptide only at low pH. Noticeable improvement in the antimicrobial activity of the histidine-peptide (P6) against E. coli (an acid-resistant bacteria) upon lowering the pH was demonstrated and validated the computational claim. The peptides displayed a membranolytic mode of action. The link between the structure and calculated energetics (ΔΔG) has been established, and the correlation between the calculated energetics and the antimicrobial activity has been highlighted. The histidine-peptide (P6) is reported to be active against acid-resistant bacteria, thus, a promising membranolytic pH-sensitive AMP.

Insights into the Mechanism of CRISPR/Cas9-Based Genome Editing from Molecular Dynamics Simulations.

The CRISPR/Cas9 system is a popular genome-editing tool with immense therapeutic potential. It is a simple two-component system (Cas9 protein and RNA) that recognizes the DNA sequence on the basis of RNA:DNA complementarity, and the Cas9 protein catalyzes the double-stranded break in the DNA. In the past decade, near-atomic resolution structures at various stages of the CRISPR/Cas9 DNA editing pathway have been reported along with numerous experimental and computational studies. Such studies have boosted knowledge of the genome-editing mechanism. Despite such advancements, the application of CRISPR/Cas9 in therapeutics is still limited, primarily due to off-target effects. Several studies aim at engineering high-fidelity Cas9 to minimize the off-target effects. Molecular Dynamics (MD) simulations have been an excellent complement to the experimental studies for investigating the mechanism of CRISPR/Cas9 editing in terms of structure, thermodynamics, and kinetics. MD-based studies have uncovered several important molecular aspects of Cas9, such as nucleotide binding, catalytic mechanism, and off-target effects. In this Review, the contribution of MD simulation to understand the CRISPR/Cas9 mechanism has been discussed, preceded by an overview of the history, mechanism, and structural aspects of the CRISPR/Cas9 system. These studies are important for the rational design of highly specific Cas9 and will also be extremely promising for achieving more accurate genome editing in the future.

Effect of a monovalent salt on the energetics of an antimicrobial-peptide: micelle dissociation.

Antimicrobial peptides (AMPs) are promising antimicrobial and therapeutic agents. Recently, we synthesized a cationic 14 residue AMP (LL-14: LKWLKKLLKWLKKL), which showed high broad-spectrum antimicrobial activity. However, the antimicrobial activity of LL-14 was compromised in the presence of NaCl. Salt sensitivity of antimicrobial potency is one of the fundamental limitations of AMP therapeutics. Thus, understanding the thermodynamics of AMP binding to simple membrane-mimetic systems and the effect of NaCl that contributes to their stability is crucial for designing promising AMPs against microbial infection. In this work, we reported computational analysis of LL-14 binding to SDS micelles (the simplest bacterial membrane mimic) at various NaCl concentrations (0.0%, 0.5%, 1.0% w/v). The thermodynamics of LL-14 dissociation from the SDS micelles was estimated by employing steered molecular dynamics (SMD) simulation followed by umbrella sampling. The results indicated that the increase in NaCl concentration systematically disfavoured the LL-14:SDS binding, primarily by stabilizing the dissociative state (i.e., free LL-14 and free micelles in water). We proposed a kinetic scheme in which the salt-induced selective stabilization of the dissociative state increased the activation barrier for the peptide:micelle binding event resulting in reduced affinity. Center-of-mass pulling indicated that the interactions involving the N-terminal of the LL-14 (residues 1-6) and SDS micelle were crucial for the stability of the LL-14:SDS complex, and LL-14 underwent a conformational change (helix → unstructured) before dissociating from the SDS micelle. The observed structural features from the peptide:micelle dissociation pathway corroborate our previous simulations as well as circular dichroism (CD), and fluorescence experiments.

Structure-based thermodynamics of ion selectivity (Mg2+versus Ca2+ and K+versus Na+) in the active site of the eukaryotic lariat group II intron from algae Pylaiella littoralis.

Group II introns are metalloenzymes that can catalyze self-splicing. Recently, the crystal structures of the eukaryotic group IIB lariat intron from the brown algae Pylaiella littoralis have been reported for two intermediate states (pre-hydrolytic (2s) and post-hydrolytic) along the self-splicing pathway. Three characteristic metal-ion binding sites (M1 and M2 sites for catalytic Mg2+ ions, and K1 site for K+) in the catalytic pocket of the lariat intron have been identified and proposed to be crucial for self-splicing. Using the X-ray structures as a template, we quantitatively estimated the energetics of divalent (Mg2+versus Ca2+) and monovalent (K+versus Na+) ion selectivity and established a direct link between the energetics and structures of this lariat intron (bound to cognate and near-cognate metal ions). Molecular dynamics (MD) free energy simulations showed that the lariat intron was strongly selective between divalent metal ions. The strength of divalent metal-ion selectivity was noticeably high in the post-hydrolytic state (ΔΔG ≈ 20 kcal mol-1) relative to its pre-hydrolytic (2s) state (ΔΔG ≈ 13 kcal mol-1). Quantum chemical calculations ensured that the sign of the estimated divalent metal-ion selectivity was correct. The M1-binding pocket was less solvent-exposed in the case of the post-hydrolytic state relative to the pre-hydrolytic (2s) state, which boosted the metal-ion selectivity of the former. Surprisingly, in contrast to the bacterial linear group II intron, the lariat intron was found to be non-selective between monovalent ions (K+versus Na+). The interaction network in the first coordination shell of Ca2+ in the M1-binding pocket was different relative to Mg2+. Mg2+ substitution by Ca2+ resulted in the substitution of a single M1-RNA interaction by the M1-water interaction. In the pre-hydrolytic (2s) state, Ca2+ substitution completely disrupted the M1⋯5'-exon interaction; thus, the nature of the divalent metal ion is critical for catalysis. The interaction network in the M2 site was independent of the nature of the divalent metal ions (Mg2+ or Ca2+). The monovalent ion was loosely bound in the wet binding pocket (K1 site) of the lariat intron; thus, the substitution of K+ by Na+ could not significantly alter the free energy of the complex. The metal ion selectivity was dependent on the solvent accessibility of the metal-ion-binding-pocket, dry pocket enhanced the selectivity.

Effect of Leu/Val Mutation on the Energetics of Antimicrobial Peptide:Micelle Binding.

Recently, we had reported a synthetic positively charged leucine-rich 14-residue-long antimicrobial peptide (AMP, LL-14: NH3+-LKWLKKLLKWLKKL-CONH2), which was highly active and cytotoxic relative to its valine analogue (VV-14). However, the thermodynamics underlying this differential toxicity and antimicrobial activity was unclear. Understanding the energetics of peptide binding to micelles (simplest membrane mimic, viz., SDS as a bacterial membrane and DPC as a eukaryotic membrane) and the effect of Leu → Val peptide mutations on the stability of the peptide:micelle complexes are of great academic interest and relevant for the rational design of potent and selective AMPs for therapeutic use. Here, we have reported the molecular dynamics free energy simulations that allowed us to quantitatively estimate the strength of peptide discrimination (based on single- or multiple-site Leu/Val mutations in LL-14) by membrane mimetic micelles (SDS and DPC) and decipher the energetics underlying peptide selectivity by micelles. The Leu-containing peptide (LL-14) was found to be preferred for micelle (SDS and DPC) binding relative to its Val analogues (single or multiple Val mutants). The strength of the preference depended on the position of the Leu/Val mutation in the peptide. Surprisingly, the N-terminal LL-14 single mutation (Leu → Val: L1V) was found to fine-tune the electrostatic interactions, resulting in the highest peptide selectivity (ΔΔG ∼ 8 kcal/mol for both SDS and DPC). However, the mechanism of L1V peptide selectivity was distinctly different for SDS and DPC micelles. SDS ensured high selectivity by disrupting the peptide:micelle salt bridge, whereas DPC desolvated the broken-peptide-backbone hydrogen bond in the V1 peptide:micelle complex. Mutations (Leu → Val) in the middle positions of the LL-14 (4th, 7th, 8th, and 11th) were disfavored by the micelles primarily due to the loss of peptide:micelle hydrophobic interactions. Peptides differing at the C-terminal (i.e., L14V) were recognized by SDS micelles (ΔΔG ∼ 4 kcal/mol) by altering peptide:micelle interactions. L14V mutation, on the other hand, did not play any role in the peptide:DPC binding, as no direct interactions between the C-terminal and DPC micelle were observed due to obvious electrostatic reasons. The strength of selectivity favoring LL-14 binding against VV-14 was found to be much higher for DPC micelles (ΔΔG ∼ 25 kcal/mol) relative to SDS micelles (ΔΔG ∼ 19 kcal/mol). The loss of the peptide:micelle hydrophobic contact in response to LL-14 → VV-14 mutation was found to be significantly larger for DPC relative to SDS micelles, resulting in higher discriminatory power for the former. Peptide:SDS salt bridges seemed to prevent the loss of peptide:micelle hydrophobic contact to some extent, leading to weaker selectivity for SDS micelles. High selectivity of DPC micelles provided an efficient mechanism for VV-14 dissociation from DPC micelles, whereas low-selectivity of SDS micelles ensured binding of both LL-14 and VV-14. To the best of our knowledge, this is the first study in which the experimental observations (antimicrobial activity and toxicity) between leucine-rich and valine-rich peptides have been explained by establishing a direct link between the energetics and structures.

Delineating the Mechanism of Action of a Protease Resistant and Salt Tolerant Synthetic Antimicrobial Peptide against Pseudomonas aeruginosa.

Rapidly growing antimicrobial resistance (AMR) against antibiotics has propelled the development of synthetic antimicrobial peptides (AMPs) as potential antimicrobial agents. An antimicrobial peptide Nle-Dab-Trp-Nle-Dab-Dab-Nle-CONH2 (P36; Nle = norleucine, Dab = diaminobutyric acid, Trp = tryptophan) potent against Pseudomonas aeruginosa (P. aeruginosa) has been developed in the present study. Rational design strategy adopted in this study led to the improvisation of the therapeutic qualities such as activity, salt tolerance, cytotoxicity, and protease resistance of the template peptide P4, which was earlier reported from our group. P36 exhibited salt tolerant antimicrobial potency against P. aeruginosa, along with very low cytotoxicity against mammalian cell lines. P36 was found to be nonhemolytic and resistant toward protease degradation which qualified it as a potent antimicrobial agent. We have investigated the mechanism of action of this molecule in detail using several experimental techniques (spectroscopic, biophysical, and microscopic) and molecular dynamics simulations. P36 was a membrane active AMP with membrane destabilization and deformation abilities, leading to leakage of the intracellular materials and causing eventual cell death. The interaction between P36 and the microbial membrane/membrane mimics was primarily driven by electrostatics. P36 was unstructured in water and upon binding to the microbial membrane mimic SDS, suggesting no influence of secondary structure on its antimicrobial potency. Positive charge, optimum hydrophobic-hydrophilic balance, and chain length remained the most important concerns to be addressed while designing small cationic antimicrobial peptides.

Effect of Differential Backbone Di-substitution of Gamma Amino Acid Residues on the Conformation and Assembly of their Fmoc Derivatives in Solid and Solution States.

We studied the effect of variable backbone dimethyl-substitution of γ amino acid residues (γ2,2 , γ3,3 and γ4,4 ) on the conformation and assembly, in crystals and solution of their Fmoc derivatives. The crystal structure of γ2,2 and γ4,4 derivatives showed distinct conformations (open/close for γ2,2 /γ4,4 ) that differed in torsion angles, hydrogen-bonding and most importantly the π-π Fmoc-stacking interactions (relatively favourable for γ4,4 -close). Fmoc derivatives existed in an equilibrium between major-monomeric (low energy, non-hydrogen bonded) and minor-dimeric (high energy, hydrogen bonded) populations in solution. The rate of major/minor population exchange was dependent on the position of substitution, highest being for γ4,4 derivative. In solution, assembly of Fmoc derivatives was solvent dependent, but it was independent of the position of geminal substitution. Crystallization was primarily governed by the stabilization of high-energy dimer by favourable π-π stacking involving Fmoc moieties. High free-energy of the dimers (γ2,2 -close, γ3,3 -open/close) offset favourable stacking interactions and hindered crystallization.

Venetoclax: a promising repurposed drug against SARS-CoV-2 main protease.

Global health care emergency caused by a new coronavirus (severe acute respiratory syndrome coronavirus 2 or SARS-CoV-2) demands urgent need to repurpose the approved pharmaceutical drugs. Main protease, Mpro of SARS-CoV-2 draws significant attention as a drug target. Herein, we have screened FDA approved organosulfur drugs (till 2016) and our laboratory synthesized organosulfur and organoselenium compounds (L1-L306) against Mpro-apo using docking followed by classical MD simulations. Additionally, a series of compounds (L307-L364) were chosen from previous experimental studies, which were reported to exhibit inhibitory potentials towards Mpro. We found several organosulfur drugs, particularly Venetoclax (FDA approved organosulfur drug for Leukemia) to be a high-affinity binders to the Mpro of SARS-CoV-2. The results reveal that organosulfur compounds including Venetoclax preferentially bind (non-covalently) to the non-catalytic pocket of the protein located in the dimer interface. We found that the ligand binding is primarily favoured by ligand-protein van der Waals interaction and penalized by desolvation effect. Interestingly, Venetoclax binding alters the local flexibility of Mpro and exerts pronounced effect in the C-terminal as well as two loop regions (Loop-A and Loop-B) that play important roles in catalysis. These findings highlighted the importance of drug repurposing and explored the non-catalytic pockets of Mpro in combating COVID-19 infection in addition to the importance of catalytic binding pocket of the protein.Communicated by Ramaswamy H. Sarma.

Divalent-Metal-Ion Selectivity of the CRISPR-Cas System-Associated Cas1 Protein: Insights from Classical Molecular Dynamics Simulations and Electronic Structure Calculations.

CRISPR-associated protein 1 (Cas1) is a universally conserved essential metalloenzyme of the clustered regularly interspaced short palindromic repeat (CRISPR) immune system of prokaryotes (bacteria, archaea) that can cut and integrate a part of viral DNA to its host genome with the help of other proteins. The integrated DNA acts as a memory of viral infection, which can be transcribed to RNA and stop future infection by recognition (based on the RNA/DNA complementarity principle) followed by protein-mediated degradation of the viral DNA. It has been proposed that the presence of a single manganese (Mn2+) ion in a conserved divalent-metal-ion binding pocket (key residues: E190, H254, D265, D268) of Cas1 is crucial for its function. Cas1-mediated DNA degradation was proposed to be hindered by metal substitution, metal chelation, or mutation of the binding pocket residues. Cas1 is active toward dsDNA degradation with both Mn2+ and Mg2+. X-ray structures of Cas1 revealed an intricate atomic interaction network of the divalent-metal-ion binding pocket and opened up the possibility of modeling related metal ions (viz., Mg2+, Ca2+) in the binding pocket of wild-type (WT) and mutated Cas1 proteins for computational analysis, which includes (1) quantitative estimation of the energetics of the divalent-metal-ion preference and (2) exploring the structural and dynamical aspects of the protein in response to divalent-metal-ion substitution or amino acid mutation. Using the X-ray structure of the Cas1 protein from Pseudomonas aeruginosa as a template (PDB 3GOD), we performed (∼2.23 µs) classical molecular dynamics (MD) simulations to compare structural and dynamical differences between Mg2+- and Ca2+-bound binding pockets of wild-type (WT) and mutant (E190A, H254A, D265A, D268A) Cas1. Furthermore, reduced binding pocket models were generated from X-ray and molecular dynamics (MD) trajectories, and the resulting structures were subjected to quantum chemical calculations. Results suggest that Cas1 prefers Mg2+ binding relative to Ca2+ and the preference is the strongest for WT and the weakest for the D268A mutant. Quantum chemical calculations indicate that Mn2+ is the most preferred relative to both Mg2+ and Ca2+ in the wild-type and mutant Cas1. Substitution of Mg2+ by Ca2+ does not alter the interaction network between Cas1 and the divalent metal ion but increases the wetness of the binding pocket by introducing a single water molecule in the first coordination shell of the latter. The strength of metal-ion preference (Mg2+ versus Ca2+) seems to be dependent on the solvent accessibility of the divalent-metal-ion binding pocket, strongest for wild-type Cas1 (in which the metal-ion binding pocket is dry, which includes two water molecules) and the weakest for the D268A mutant (in which the metal-ion binding pocket is wet, which includes four water molecules).

Effect of Differential Geminal Substitution of γ Amino Acid Residues at the (i + 2) Position of αγ Turn Segments on the Conformation of Template ß-Hairpin Peptides.

The effect of insertion of three geminally dimethyl substituted γ amino acid residues [γ2,2 (4-amino-2,2-dimethylbutanoic acid), γ3,3 (4-amino-3,3-dimethylbutanoic acid), and γ4,4 (4-amino-4,4-dimethylbutanoic acid)] at the (i + 2) position of a two-residue αγ C12 turn segment in a model octapeptide sequence Leu-Phe-Val-Aib-Xxx-Leu-Phe-Val (where Xxx = γ amino acid residues) has been investigated in this study. Solution conformational studies (NMR, CD, and IR) and ab initio calculations indicated that γ3,3 and γ4,4 residues were well accommodated in the ß-hairpin nucleating αγ C12 turns, which gave rise to well-registered hairpins, in contrast to γ2,2, which was unable to form a tight C12 ß-hairpin nucleating turn and promote a well-registered ß-hairpin. Geminal disubstitution at the Cα carbon in γ2,2 led to unfavorable steric contacts, disabling its accommodation in the αγ C12 hairpin nucleating turn unlike the γ3,3 and γ4,4 residues. Geminal substitutions at different carbons along the backbone constrained backbone torsion angles for the three γ amino acid residues differently, generating diverse conformational preferences in them. Folded hairpins were energetically more stable (∼8 to 9 kcal/mol) than the unfolded peptides. Conformational preference of the peptides was independent of the N-terminal protecting group. Such fundamental understanding will instrumentalize the future directed design of foldamers.

Triazole based isatin derivatives as potential inhibitor of key cancer promoting kinases- insight from electronic structure, docking and molecular dynamics simulations.

Computer Aided Drug Design approaches have been applied to predict potential inhibitors for two different kinases, namely, cyclin-dependent kinase 2 (CDK2) and Epidermal Growth Factor Receptor (EGFR) which are known to play crucial role in cancer growth. We have designed alkyl and aryl substituted isatin-triazole ligands and performed molecular docking to rank and predict possible binding pockets in CDK2 and EGFR kinases. Best-scoring ligands in the kinase-binding pocket were selected from the docking study and subjected to molecular dynamics simulation. Absolute binding affinities were estimated from the MD trajectories using the MM/PBSA approach. The results suggest that aryl substituted isatin-triazole ligands are better binder to the kinases relative to its alkyl analogue. Furthermore, aryl substituted isatin-triazole ligands prefer binding to EGFR kinases relative to CDK2. The ligand binding pockets of the kinases are primarily hydrophobic in nature. Ligand-kinase binding is favoured by electrostatic and Van der Waals interactions, later being the major contributor. Large estimated negative binding affinities (~ -10 to -25 kcal/mol) indicate that the ligands might inhibit the kinases. Physicochemical property analysis suggests that the proposed ligands could be orally bio-available.

Mimicking human Drp1 disease-causing mutations in yeast Dnm1 reveals altered mitochondrial dynamics.

The dynamin-related protein 1 (Drp1) and its homologs in various eukaryotes are essential to maintain mitochondrial morphology and regulate mitochondrial division. Several mutations in different domains of Drp1 have been reported, which result in debilitating conditions. Four such disease-causing mutations of the middle domain of Drp1 were mimicked in the yeast dynamin-related GTPase (Dnm1) and were characterized in this study. Mitochondrial morphology and protein function were observed to be altered to a variable extent in cells expressing the mutated variants of Dnm1. Several aspects related to the protein such as punctate formation, localization to mitochondria, dynamic behavior and structure were analyzed by microscopy, biochemical studies and molecular dynamics simulations. Significant effects on the protein structure and function were observed in cells expressing A430D and G397D mutations. Overall, our data provide insight into the molecular and cellular alterations resulting from middle domain mutations in Dnm1.

Effect of monovalent salt concentration and peptide secondary structure in peptide-micelle binding.

Recently, we reported a cationic 14 residue peptide LL-14 (LKWLKKLLKWLKKL) with salt-sensitive broad-spectrum antimicrobial potency. However, the mechanism of its salt (NaCl) sensitivity remained unclear. In this study, we have reported computational (∼14.2 µs of MD) and experimental (CD, fluorescence) investigations to examine the salt-sensitivity and the role of peptide secondary structure on LL-14 binding to simple membrane mimetic (SDS, DPC) systems. LL-14 was shown to adopt a random coil (Pc) conformation in water and α-helical conformation (Ph) in the peptide:SDS micelle complex, accompanied by tryptophan burial, using both simulations and experiments. Simulations successfully deconvoluted the LL-14:micelle binding event in terms of secondary structure (random coil Pc versus helix Ph) and gave atomic insight into the initial and final LL-14:SDS complexes. Electrostatics drove the N-terminus (L1 and K2) of LL-14 (Pc or Ph) to bind the SDS micellar surface, initiating complex formation. LL-14 in amphipathic Ph conformation bound faster and buried deeper into the SDS micelle relative to Pc. Increasing NaCl concentration incrementally delayed LL-14:micelle binding by shielding the overall charges of the interacting partners. LL-14 binding to the SDS micelle was significantly faster relative to that of the zwitterionic DPC micelle due to electrostatic reasons. Cationic α-helical amphipathic peptides (with positively charged N-terminus) with low salt-ion concentration seemed to be ideal for faster SDS binding.

dUMP/F-dUMP Binding to Thymidylate Synthase: Human Versus Mycobacterium tuberculosis.

Thymidylate synthase is an enzyme that catalyzes deoxythymidine monophosphate (dTMP) synthesis from substrate deoxyuridine monophosphate (dUMP). Thymidylate synthase of Mycobacterium tuberculosis (MtbThyX) is structurally distinct from its human analogue human thymidylate synthase (hThyA), thus drawing attention as an attractive drug target for combating tuberculosis. Fluorodeoxyuridylate (F-dUMP) is a successful inhibitor of both MtbThyX and hThyA, thus limited by poor selectivity. Understanding the dynamics and energetics associated with substrate/inhibitor binding to thymidylate synthase in atomic details remains a fundamental unsolved problem, which is necessary for a new selective inhibitor design. Structural studies of MtbThyX and hThyA bound substrate/inhibitor complexes not only revealed the extensive specific interaction network between protein and ligands but also opened up the possibility of directly computing the energetics of the substrate versus inhibitor recognition. Using experimentally determined structures as a template, we report extensive computer simulations (∼4.5 µs) that allow us to quantitatively estimate ligand selectivity (dUMP vs F-dUMP) by MtbThyX and hThyA. We show that MtbThyX prefers deprotonated dUMP (enolate form) as the substrate, whereas hThyA binds to the keto form of dUMP. Computed energetics clearly show that MtbThyX is less selective between dUMP and F-dUMP, favoring the latter, relative to hThyA. The simulations reveal the role of tyrosine at position 135 (Y135) of hThyA in amplifying the selectivity. The protonation state of the pyrimidine base of the ligand (i.e., keto or enolate) seems to have no role in MtbThyX ligand selectivity. A molecular gate (consists of Y108, K165, H203, and a water molecule) restricts water accessibility and offers a desolvated dry ligand-binding pocket for MtbThyX. The ligand-binding pocket of hThyA is relatively wet and exposed to bulk water.