Pyrrole ßC-B-N Fused Porphyrins: Molecular Structures and Opto-Electrochemical Studies.

Herein, we report the design, synthesis, structure, and electrochemical study of doubly ßC-B-N fused Ni(II) porphyrins (1-trans, 1-cis, 2-trans, and 2-cis). These compounds have been synthesized from A2B2 type dipyridyl Ni(II) porphyrins (Ar=Ph for 1 a; Ar=C6F5 for 2 a) via Lewis base-directed electrophilic aromatic borylation reactions. The solution state structures of these compounds have been established using 1H NMR, 11B NMR, 1H-1H COSY, 1H-13C HSQC, and 19F-13C HSQC NMR techniques. Single crystal X-ray analysis have revealed that 1-trans, 1-cis, and 2-trans adopt ruffled conformations, with alternate meso-carbons on the opposite sides of the mean porphyrin plane. The Soret bands in the absorption spectra of the B-N fused molecules are ~40 nm redshifted compared to unfused Ni(II) porphyrin precursors. The B-N fusion have diminished the redox potential of fused porphyrins. Although 1-trans and 1-cis, show four oxidation processes, 2-trans and 2-cis show only three oxidation processes. DFT studies have revealed that the tetrahedral geometry of the boron has induced a twist in the π-conjugation, which destabilizes the HOMO and stabilizes the LUMO in 1-trans, 1-cis, 2-trans, and 2-cis.

Structural, Functional, and Mutational Studies of a Potent Subtilisin Inhibitor from Budgett's Frog, Lepidobatrachus laevis.

Subtilases play a significant role in microbial pathogen infections by degrading the host proteins. Subtilisin inhibitors are crucial in fighting against these harmful microorganisms. LL-TIL, from skin secretions of Lepidobatrachus laevis, is a cysteine-rich peptide belonging to the I8 family of inhibitors. Protease inhibitory assays demonstrated that LL-TIL acts as a slow-tight binding inhibitor of subtilisin Carlsberg and proteinase K with inhibition constants of 91 pM and 2.4 nM, respectively. The solution structures of LL-TIL and a mutant peptide reveal that they adopt a typical TIL-type fold with a canonical conformation of a reactive site loop (RSL). The structure of the LL-TIL-subtilisin complex and molecular dynamics (MD) simulations provided an in-depth view of the structural basis of inhibition. NMR relaxation data and molecular dynamics simulations indicated a rigid conformation of RSL, which does not alter significantly upon subtilisin binding. The energy calculation for subtilisin inhibition predicted Ile31 as the highest contributor to the binding energy, which was confirmed experimentally by site-directed mutagenesis. A chimeric mutant of LL-TIL broadened the inhibitory profile and attenuated subtilisin inhibition by 2 orders of magnitude. These results provide a template to engineer more specific and potent TIL-type subtilisin inhibitors.

Insights into the solution structure and transcriptional regulation of the MazE9 antitoxin in Mycobacterium tuberculosis.

The present study endeavors to decode the details of the transcriptional autoregulation effected by the MazE9 antitoxin of the Mycobacterium tuberculosis MazEF9 toxin-antitoxin system. Regulation of this bicistronic operon at the level of transcription is a critical biochemical process that is key for the organism's stress adaptation and virulence. Here, we have reported the solution structure of the DNA binding domain of MazE9 and scrutinized the thermodynamic and kinetic parameters operational in its interaction with the promoter/operator region, specific to the mazEF9 operon. A HADDOCK model of MazE9 bound to its operator DNA has been calculated based on the information on interacting residues obtained from these studies. The thermodynamics and kinetics of the interaction of MazE9 with the functionally related mazEF6 operon indicate that the potential for intracellular cross-regulation is unlikely. An interesting feature of MazE9 is the cis ⇌ trans conformational isomerization of proline residues in the intrinsically disordered C-terminal domain of this antitoxin.

Structure and allosteric activity of a single-disulfide conopeptide from Conus zonatus at human α3ß4 and α7 nicotinic acetylcholine receptors.

Conopeptides are neurotoxic peptides in the venom of marine cone snails and have broad therapeutic potential for managing pain and other conditions. Here, we identified the single-disulfide peptides Czon1107 and Cca1669 from the venoms of Conus zonatus and Conus caracteristicus, respectively. We observed that Czon1107 strongly inhibits the human α3ß4 (IC50 15.7 ± 3.0 µm) and α7 (IC50 77.1 ± 0.05 µm) nicotinic acetylcholine receptor (nAChR) subtypes, but the activity of Cca1669 remains to be identified. Czon1107 acted at a site distinct from the orthosteric receptor site. Solution NMR experiments revealed that Czon1107 exists in equilibrium between conformational states that are the result of a key Ser4-Pro5cis-trans isomerization. Moreover, we found that the X-Pro amide bonds in the inter-cysteine loop are rigidly constrained to cis conformations. Structure-activity experiments of Czon1107 and its variants at positions P5 and P7 revealed that the conformation around the X-Pro bonds (cis-trans) plays an important role in receptor subtype selectivity. The cis conformation at the Cys6-Pro7 peptide bond was essential for α3ß4 nAChR subtype allosteric selectivity. In summary, we have identified a unique single-disulfide conopeptide with a noncompetitive, potentially allosteric inhibitory mechanism at the nAChRs. The small size and rigidity of the Czon1107 peptide could provide a scaffold for rational drug design strategies for allosteric nAChR modulation. This new paradigm in the "conotoxinomic" structure-function space provides an impetus to screen venom from other Conus species for similar, short bioactive peptides that allosterically modulate ligand-gated receptor function.

Aromatic Interactions Drive the Coupled Folding and Binding of the Intrinsically Disordered Sesbania mosaic Virus VPg Protein.

The plant Sesbania mosaic virus [a (+)-ssRNA sobemovirus] VPg protein is intrinsically disordered in solution. For the virus life cycle, the VPg protein is essential for replication and for polyprotein processing that is carried out by a virus-encoded protease. The nuclear magnetic resonance (NMR)-derived tertiary structure of the protease-bound VPg shows it to have a novel tertiary structure with an α-ß-ß-ß topology. The quaternary structure of the high-affinity protease-VPg complex (≈27 kDa) has been determined using HADDOCK protocols with NMR (residual dipolar coupling, dihedral angle, and nuclear Overhauser enhancement) restraints and mutagenesis data as inputs. The geometry of the complex is in excellent agreement with long-range orientational restraints such as residual dipolar couplings and ring-current shifts. A "vein" of aromatic residues on the protease surface is pivotal for the folding of VPg via intermolecular edge-to-face π···π stacking between Trp271 and Trp368 of the protease and VPg, respectively, and for the CH···π interactions between Leu361 of VPg and Trp271 of the protease. The structure of the protease-VPg complex provides a molecular framework for predicting sites of important posttranslational modifications such as RNA linkage and phosphorylation and a better understanding of the coupled folding upon binding of intrinsically disordered proteins. The structural data presented here augment the limited structural data available on viral proteins, given their propensity for structural disorder.

CcdB at pH 4 Forms a Partially Unfolded State with a Dry Core.

pH is an important factor that affects the protein structure, stability, and activity. Here, we probe the nature of the low-pH structural form of the homodimeric CcdB (controller of cell death B) protein. Characterization of CcdB protein at pH 4 and 300 K using circular dichroism spectroscopy, 8-anilino-1-naphthalene-sulphonate binding, and Trp solvation studies suggests that it forms a partially unfolded state with a dry core at equilibrium under these conditions. CcdB remains dimeric at pH 4 as shown by multiple techniques, such as size-exclusion chromatography coupled to multiangle light scattering, analytical ultracentrifugation, and electron paramagnetic resonance. Comparative analysis using two-dimensional 15N-1H heteronuclear single-quantum coherence NMR spectra of CcdB at pH 4 and 7 suggests that the pH 4 and native state have similar but nonidentical structures. Hydrogen-exchange-mass-spectrometry studies demonstrate that the pH 4 state has substantial but anisotropic changes in local stability with core regions close to the dimer interface showing lower protection but some other regions showing higher protection relative to pH 7.

Crystallographic Structures of IlvN·Val/Ile Complexes: Conformational Selectivity for Feedback Inhibition of Aceto Hydroxy Acid Synthases.

Conformational factors that predicate selectivity for valine or isoleucine binding to IlvN leading to the regulation of aceto hydroxy acid synthase I (AHAS I) of Escherichia coli have been determined for the first time from high-resolution (1.9-2.43 Å) crystal structures of IlvN·Val and IlvN·Ile complexes. The valine and isoleucine ligand binding pockets are located at the dimer interface. In the IlvN·Ile complex, among residues in the binding pocket, the side chain of Cys43 is 2-fold disordered (χ1 angles of gauche- and trans). Only one conformation can be observed for the identical residue in the IlvN·Val complexes. In a reversal, the side chain of His53, located at the surface of the protein, exhibits two conformations in the IlvN·Val complex. The concerted conformational switch in the side chains of Cys43 and His53 may play an important role in the regulation of the AHAS I holoenzyme activity. A significant result is the establishment of the subunit composition in the AHAS I holoenzyme by analytical ultracentrifugation. Solution nuclear magnetic resonance and analytical ultracentrifugation experiments have also provided important insights into the hydrodynamic properties of IlvN in the ligand-free and -bound states. The structural and biophysical data unequivocally establish the molecular basis for differential binding of the ligands to IlvN and a rationale for the resistance of IlvM to feedback inhibition by the branched-chain amino acids.

Engineering Aromatic-Aromatic Interactions To Nucleate Folding in Intrinsically Disordered Regions of Proteins.

Aromatic interactions are an important force in protein folding as they combine the stability of a hydrophobic interaction with the selectivity of a hydrogen bond. Much of our understanding of aromatic interactions comes from "bioinformatics" based analyses of protein structures and from the contribution of these interactions to stabilizing secondary structure motifs in model peptides. In this study, the structural consequences of aromatic interactions on protein folding have been explored in engineered mutants of the molten globule protein apo-cytochrome b5. Structural changes from disorder to order due to aromatic interactions in two variants of the protein, viz., WF-cytb5 and FF-cytb5, result in significant long-range secondary and tertiary structure. The results show that 54 and 52% of the residues in WF-cytb5 and FF-cytb5, respectively, occupy ordered regions versus 26% in apo-cytochrome b5. The interactions between the aromatic groups are offset-stacked and edge-to-face for the Trp-Phe and Phe-Phe mutants, respectively. Urea denaturation studies indicate that both mutants have a Cm higher than that of apo-cytochrome b5 and are more stable to chaotropic agents than apo-cytochrome b5. The introduction of these aromatic residues also results in "trimer" interactions with existing aromatic groups, reaffirming the selectivity of the aromatic interactions. These studies provide insights into the aromatic interactions that drive disorder-to-order transitions in intrinsically disordered regions of proteins and will aid in de novo protein design beyond small peptide scaffolds.

Influenza hemagglutinin stem-fragment immunogen elicits broadly neutralizing antibodies and confers heterologous protection.

Influenza hemagglutinin (HA) is the primary target of the humoral response during infection/vaccination. Current influenza vaccines typically fail to elicit/boost broadly neutralizing antibodies (bnAbs), thereby limiting their efficacy. Although several bnAbs bind to the conserved stem domain of HA, focusing the immune response to this conserved stem in the presence of the immunodominant, variable head domain of HA is challenging. We report the design of a thermotolerant, disulfide-free, and trimeric HA stem-fragment immunogen which mimics the native, prefusion conformation of HA and binds conformation specific bnAbs with high affinity. The immunogen elicited bnAbs that neutralized highly divergent group 1 (H1 and H5 subtypes) and 2 (H3 subtype) influenza virus strains in vitro. Stem immunogens designed from unmatched, highly drifted influenza strains conferred robust protection against a lethal heterologous A/Puerto Rico/8/34 virus challenge in vivo. Soluble, bacterial expression of such designed immunogens allows for rapid scale-up during pandemic outbreaks.

Nuclear Magnetic Resonance Structure of a Major Lens Protein, Human γC-Crystallin: Role of the Dipole Moment in Protein Solubility.

A hallmark of the crystallin proteins is their exceptionally high solubility, which is vital for maintaining the high refractive index of the eye lens. Human γC-crystallin is a major γ-crystallin whose mutant forms are associated with congenital cataracts but whose three-dimensional structure is not known. An earlier study of a homology model concluded that human γC-crystallin has low intrinsic solubility, mainly because of the atypical magnitude and fluctuations of its dipole moment. On the contrary, the high-resolution tertiary structure of human γC-crystallin determined here shows unequivocally that it is a highly soluble, monomeric molecule in solution. Notable differences between the orientations and interactions of several side chains are observed upon comparison to those in the model. No evidence of the pivotal role ascribed to the effect of dipole moment on protein solubility was found. The nuclear magnetic resonance structure should facilitate a comprehensive understanding of the deleterious effects of cataract-associated mutations in human γC-crystallin.

The coil-to-helix transition in IlvN regulates the allosteric control of Escherichia coli acetohydroxyacid synthase I.

The solution structure of IlvN, the regulatory subunit of Escherichia coli acetohydroxyacid synthase I, in the valine-bound form has been determined using high-resolution multidimensional, multinuclear nuclear magnetic resonance (NMR) methods. IlvN in the presence or absence of the effector molecule is present as a 22.5 kDa dimeric molecule. The ensemble of 20 low-energy structures shows a backbone root-mean-square deviation of 0.73 ± 0.13 Å and a root-mean-square deviation of 1.16 ± 0.13 Å for all heavy atoms. Furthermore, more than 98% of the backbone φ and ψ dihedral angles occupy the allowed and additionally allowed regions of the Ramachandran map, which is indicative of the fact that the structures are of high stereochemical quality. Each protomer exhibits a ßαßßαßα topology that is a characteristic feature of the ACT domain seen in metabolic enzymes. In the valine-bound form, IlvN exists apparently as a single conformer. In the free form, IlvN exists as a mixture of conformational states that are in intermediate exchange on the NMR time scale. Thus, a large shift in the conformational equilibrium is observed upon going from the free form to the bound form. The structure of the valine-bound form of IlvN was found to be similar to that of the ACT domain of the unliganded form of IlvH. Comparisons of the structures of the unliganded forms of these proteins suggest significant differences. The structural and conformational properties of IlvN determined here have allowed a better understanding of the mechanism of regulation of branched chain amino acid biosynthesis.

Solution nuclear magnetic resonance structure of the GATase subunit and structural basis of the interaction between GATase and ATPPase subunits in a two-subunit-type GMPS from Methanocaldococcus jannaschii.

The solution structure of the monomeric glutamine amidotransferase (GATase) subunit of the Methanocaldococcus janaschii (Mj) guanosine monophosphate synthetase (GMPS) has been determined using high-resolution nuclear magnetic resonance methods. Gel filtration chromatography and ¹5N backbone relaxation studies have shown that the Mj GATase subunit is present in solution as a 21 kDa (188-residue) monomer. The ensemble of 20 lowest-energy structures showed root-mean-square deviations of 0.35 ± 0.06 Å for backbone atoms and 0.8 ± 0.06 Å for all heavy atoms. Furthermore, 99.4% of the backbone dihedral angles are present in the allowed region of the Ramachandran map, indicating the stereochemical quality of the structure. The core of the tertiary structure of the GATase is composed of a seven-stranded mixed ß-sheet that is fenced by five α-helices. The Mj GATase is similar in structure to the Pyrococcus horikoshi (Ph) GATase subunit. Nuclear magnetic resonance (NMR) chemical shift perturbations and changes in line width were monitored to identify residues on GATase that were responsible for interaction with magnesium and the ATPPase subunit, respectively. These interaction studies showed that a common surface exists for the metal ion binding as well as for the protein-protein interaction. The dissociation constant for the GATase-Mg(2+) interaction has been found to be ∼1 mM, which implies that interaction is very weak and falls in the fast chemical exchange regime. The GATase-ATPPase interaction, on the other hand, falls in the intermediate chemical exchange regime on the NMR time scale. The implication of this interaction in terms of the regulation of the GATase activity of holo GMPS is discussed.

The structure of the thioredoxin-triosephosphate isomerase complex provides insights into the reversible glutathione-mediated regulation of triosephosphate isomerase.

Protein-protein interactions are crucial for many biological functions. The redox interactome encompasses numerous weak transient interactions in which thioredoxin plays a central role. Proteomic studies have shown that thioredoxin binds to numerous proteins belonging to various cellular processes, including energy metabolism. Thioredoxin has cross talk with other redox mechanisms involving glutathionylation and has functional overlap with glutaredoxin in deglutathionylation reactions. In this study, we have explored the structural and biochemical interactions of thioredoxin with the glycolytic enzyme, triosephosphate isomerase. Nuclear magnetic resonance chemical shift mapping methods and molecular dynamics-based docking have been applied in deriving a structural model of the thioredoxin-triosephosphate isomerase complex. The spatial proximity of active site cysteine residues of thioredoxin to reactive thiol groups on triosephosphate isomerase provides a direct link to the observed deglutathionylation of cysteine 217 in triosephosphate isomerase, thereby reversing the inhibitory effect of S-glutathionylation of triosephosphate isomerase.

Solution structure of BTK-2, a novel hK(v)1.1 inhibiting scorpion toxin, from the eastern Indian scorpion Mesobuthus tamulus.

The three dimensional structure of a 32 residue three disulfide scorpion toxin, BTK-2, from the Indian red scorpion Mesobuthus tamulus has been determined using isotope edited solution NMR methods. Samples for structural and electrophysiological studies were prepared using recombinant DNA methods. Electrophysiological studies show that the peptide is active against hK(v)1.1 channels. The structure of BTK-2 was determined using 373 distance restraints from NOE data, 66 dihedral angle restraints from NOE, chemical shift and scalar coupling data, 6 constraints based on disulfide linkages and 8 constraints based on hydrogen bonds. The root mean square deviation (r.m.s.d) about the averaged co-ordinates of the backbone (N, C(α), C') and all heavy atoms are 0.81 ± 0.23Å and 1.51 ± 0.29Å respectively. The backbone dihedral angles (ϕ and ψ) for all residues occupy the favorable and allowed regions of the Ramachandran map. The three dimensional structure of BTK-2 is composed of three well defined secondary structural regions that constitute the α-ß-ß structural motif. Comparisons between the structure of BTK-2 and other closely related scorpion toxins pointed towards distinct differences in surface properties that provide insights into the structure-function relationships among this important class of voltage-gated potassium channel inhibiting peptides.

Structural and mutational analysis of MazE6-operator DNA complex provide insights into autoregulation of toxin-antitoxin systems.

Of the 10 paralogs of MazEF Toxin-Antitoxin system in Mycobacterium tuberculosis, MazEF6 plays an important role in multidrug tolerance, virulence, stress adaptation and Non Replicative Persistant (NRP) state establishment. The solution structures of the DNA binding domain of MazE6 and of its complex with the cognate operator DNA show that transcriptional regulation occurs by binding of MazE6 to an 18 bp operator sequence bearing the TANNNT motif (-10 region). Kinetics and thermodynamics of association, as determined by NMR and ITC, indicate that the nMazE6-DNA complex is of high affinity. Residues in N-terminal region of MazE6 that are key for its homodimerization, DNA binding specificity, and the base pairs in the operator DNA essential for the protein-DNA interaction, have been identified. It provides a basis for design of chemotherapeutic agents that will act via disruption of TA autoregulation, leading to cell death.

Ammonia channeling in Plasmodium falciparum GMP synthetase: investigation by NMR spectroscopy and biochemical assays.

GMP synthetase, a class I amidotransferase, catalyzes the last step of the purine biosynthetic pathway, where ammonia from glutamine is incorporated into xanthosine 5'-monophospate to yield guanosine 5'-monnophosphate as the main product. Combined biochemical, structural, and computational studies of glutamine amidotransferases have revealed the existence of physically separate active sites connected by molecular tunnels that efficiently transfer ammonia from the glutaminase site to the synthetase site. Here, we have investigated aspects of ammonia channeling in P. falciparum GMP synthetase using biochemical assays in conjunction with 15N-edited proton NMR spectroscopy. Our results suggest that (1) ammonia released from glutamine is not equilibrated with the external medium, (2) saturating concentrations of glutamine do not obliterate the incorporation of external ammonia into GMP, and (3) ammonia in the external medium can access the thioester intermediate when the ATPPase domain is bound to substrates. Further, mutation of Cys-102 to alanine confirmed its identity as the catalytic residue in the glutaminase domain, and ammonia-dependent assays on the mutant indicated glutamine to be a partial uncompetitive inhibitor of the enzyme.

Cloning, overexpression, folding and purification of a biosynthetically derived three disulfide scorpion toxin (BTK-2) from Mesobuthus tamulus.

BTK-2, a 32 residue scorpion toxin initially identified in the venom of red Indian scorpion Mesobuthus tamulus was cloned, overexpressed and purified using Cytochrome b(5) fusion protein system developed in our laboratory. The synthetic gene coding for the peptide was designed taking into account optimal codon usage by Escherichia coli. High expression levels of the fusion protein enabled facile purification of this peptide. The presence of disulfide bonded isomers, occurring as distinctly populated states even in the fusion protein, were separated by gel filtration chromatography. The target peptide was liberated from the host protein by Tev protease cleavage and subsequent purification was achieved using RP-HPLC methods. Reverse phase HPLC clearly showed the presence of at least two isomeric forms of the peptide that were significantly populated. The oxidative folding of BTK-2 was achieved under ambient conditions during the course of purification. Structural characterization of the two forms, by solution homonuclear and heteronuclear NMR methods, has shown that these two forms exhibit significantly different structural properties, and represent the natively folded and a "misfolded" form of the peptide. The formation of properly folded BTK-2 as a major fraction without the use of in vitro oxidative refolding methods clearly indicate the versatility of the Cytochrome b(5) fusion protein system for the efficient production of peptides for high resolution NMR studies.

Ω76: A designed antimicrobial peptide to combat carbapenem- and tigecycline-resistant Acinetobacter baumannii.

Drug resistance is a public health concern that threatens to undermine decades of medical progress. ESKAPE pathogens cause most nosocomial infections, and are frequently resistant to carbapenem antibiotics, usually leaving tigecycline and colistin as the last treatment options. However, increasing tigecycline resistance and colistin's nephrotoxicity severely restrict use of these antibiotics. We have designed antimicrobial peptides using a maximum common subgraph approach. Our best peptide (Ω76) displayed high efficacy against carbapenem and tigecycline-resistant Acinetobacter baumannii in mice. Mice treated with repeated sublethal doses of Ω76 displayed no signs of chronic toxicity. Sublethal Ω76 doses co-administered alongside sublethal colistin doses displayed no additive toxicity. These results indicate that Ω76 can potentially supplement or replace colistin, especially where nephrotoxicity is a concern. To our knowledge, no other existing antibiotics occupy this clinical niche. Mechanistically, Ω76 adopts an α-helical structure in membranes, causing rapid membrane disruption, leakage, and bacterial death.

Antimicrobial activity of marine bacteria associated with sponges from the waters off the coast of South East India.

Seventy-five marine bacterial strains associated with four species of sponges (Echinodictyum sp., Spongia sp., Sigmadocia fibulatus and Mycale mannarensis) were isolated from the Tuticorin coast, Gulf of Mannar region. The agar-overlay method was used to screen for antibiotic production by these strains against four bacteria, viz., Bacillus subtilis, Escherichia coli, Vibrio parahaemolyticus, and Vibrio harveyi and one fungal pathogen, viz., Candida albicans. Twenty-one per cent of the bacterial strains were found to be antibiotic producers and their activities ranged from broad spectral to species specific. A strain coded SC3 was found to be highly potent and was mass cultured. The ethyl acetate extract of the culture broth was further fractionated by reverse phase HPLC and the active fraction identified. In addition, SC3 was subjected to morphological and physiological characterization. The results of the tests showed SC3 to be a Gram-positive rod, sporulating, motile, catalase and oxidase positive. Phylogenetic analysis based on comparative analysis of sequenced 16s rRNA of the active strains indicated a preponderance of bacteria belonging to Vibrio and Bacillus genera with 95-99% sequence similarities. To our knowledge this is the first report on phylogenetic identification of antibiotic producing bacteria associated with sponges from Indian waters.