Solution NMR studies of amphibian antimicrobial peptides: Linking structure to function?

The high-resolution three-dimensional structure of an antimicrobial peptide has implications for the mechanism of its antimicrobial activity, as the conformation of the peptide provides insights into the intermolecular interactions that govern the binding to its biological target. For many cationic antimicrobial peptides the negatively charged membranes surrounding the bacterial cell appear to be a main target. In contrast to what has been found for other classes of antimicrobial peptides, solution NMR studies have revealed that in spite of the wide diversity in the amino acid sequences of amphibian antimicrobial peptides (AAMPs), they all adopt amphipathic α-helical structures in the presence of membrane-mimetic micelles, bicelles or organic solvent mixtures. In some cases the amphipathic AAMP structures are directly membrane-perturbing (e.g. magainin, aurein and the rana-box peptides), in other instances the peptide spontaneously passes through the membrane and acts on intracellular targets (e.g. buforin). Armed with a high-resolution structure, it is possible to relate the peptide structure to other relevant biophysical and biological data to elucidate a mechanism of action. While many linear AAMPs have significant antimicrobial activity of their own, mixtures of peptides sometimes have vastly improved antibiotic effects. Thus, synergy among antimicrobial peptides is an avenue of research that has recently attracted considerable attention. While synergistic relationships between AAMPs are well described, it is becoming increasingly evident that analyzing the intermolecular interactions between these peptides will be essential for understanding the increased antimicrobial effect. NMR structure determination of hybrid peptides composed of known antimicrobial peptides can shed light on these intricate synergistic relationships. In this work, we present the first NMR solution structure of a hybrid peptide composed of magainin 2 and PGLa bound to SDS and DPC micelles. The hybrid peptide adopts a largely helical conformation and some information regarding the inter-helix organization of this molecule is reported. The solution structure of the micelle associated MG2-PGLa hybrid peptide highlights the importance of examining structural contributions to the synergistic relationships but it also demonstrates the limitations in the resolution of the currently used solution NMR techniques for probing such interactions. Future studies of antimicrobial peptide synergy will likely require stable isotope-labeling strategies, similar to those used in NMR studies of proteins.     

Solution NMR structure of the helicase associated domain BVU_0683(627–691) from Bacteroides vulgatus provides first structural coverage for protein domain family PF03457 and indicates domain binding to DNA

A high-quality NMR structure of the helicase associated (HA) domain comprising residues 627–691 of the 753-residue protein BVU_0683 from Bacteroides vulgatus exhibits an all α-helical fold. The structure presented here is the first representative for the large protein domain family PF03457 (currently 742 members) of HA domains. Comparison with structurally similar proteins supports the hypothesis that HA domains bind to DNA and that binding specificity varies greatly within the family of HA domains constituting PF03457.     

Redox activation of Fe(III)–thiosemicarbazones and Fe(III)–bleomycin by thioredoxin reductase: specificity of enzymatic redox centers and analysis of reactive species formation by ESR spin trapping

Thiosemicarbazones such as Triapine (Tp) and Dp44mT are tridentate iron (Fe) chelators that have well-documented antineoplastic activity. Although Fe–thiosemicarbazones can undergo redox cycling to generate reactive species that may have important roles in their cytotoxicity, there is only limited insight into specific cellular agents that can rapidly reduce Fe(III)–thiosemicarbazones and thereby promote their redox activity. Here we report that thioredoxin reductase-1 (TrxR1) and glutathione reductase (GR) have this activity and that there is considerable specificity to the interactions between specific redox centers in these enzymes and various Fe(III) complexes. Site-directed variants of TrxR1 demonstrate that the selenocysteine (Sec) of the enzyme is not required, whereas the C59 residue and the flavin have important roles. Although TrxR1 and GR have analogous C59/flavin motifs, TrxR is considerably faster than GR. For both enzymes, Fe(III)(Tp)₂ is reduced faster than Fe(III)(Dp44mT)₂. This reduction promotes redox cycling and the generation of hydroxyl radical (HO) in a peroxide-dependent manner, even with low-micromolar levels of Fe(Tp)₂. TrxR also reduces Fe(III)–bleomycin and this activity is Sec-dependent. TrxR cannot reduce Fe(III)–EDTA at significant rates. Our findings are the first to demonstrate pro-oxidant reductive activation of Fe(III)-based antitumor thiosemicarbazones by interactions with specific enzyme species. The marked elevation of TrxR1 in many tumors could contribute to the selective tumor toxicity of these drugs by enhancing the redox activation of Fe(III)–thiosemicarbazones and the generation of reactive oxygen species such as HO.     

Heterodinuclear Cu(II)Zn(II) complexes: F NMR as a versatile tool to control the synthesis

The dinucleating ligands 2,6-bis[(bis(2-pyridylmethyl)amino)methyl]-4-methylphenol (H-BPMP) and 2,6-bis[(bis(2-pyridylmethyl)amino)methyl]-4-fluorophenol (H-BPFP) were used to synthesize heterodinuclear (μ-phenoxo)(μ-hydroxo) Cu(II)Zn(II) complexes. The labeled ligand with a fluorine atom allows the use of ¹⁹F NMR spectroscopy, which turned to be a rapid and powerful tool to tune the synthesis of the heterodinuclear paramagnetic complex [CuZnBPFP(μOH)](ClO₄)₂ and avoid mixing of complexes with statistical distribution. When applied to the non-fluorinated ligand, this experimental procedure leads to prepare and isolate easily the complex [CuZnBPMP(μOH)](ClO₄)₂. The X-ray structure is described.     

Mechanism of zinc(II)-promoted amyloid formation: zinc(II) binding facilitates the transition from the partially α-helical conformer to aggregates of amyloid β protein(1–28)

The amyloidoses are a group of disorders characterized by aberrant protein folding and assembly, leading to the deposition of insoluble protein fibrils (amyloid), which provokes cell dysfunction and later cell death. One of the physiologically relevant environmental factors able to affect the conformation and hence the aggregation properties of amyloidogenic proteins/peptides is metal ions. Zn(II) promotes aggregation of most amyloidogenic peptides/proteins in vitro, including amyloid β protein (Aβ), but the underlying mechanism is not known. To better understand this mechanism the present study focused on the partially α-helical conformer, supposed to be an intermediate in Aβ aggregation. This partially α-helical conformer is stabilized by 10–20% 2,2,2-trifluoroethanol (TFE): therefore, the influence of Zn binding on the aggregation of the amylidogenic model peptide Aβ(1–28) (Aβ28) was investigated at different TFE concentrations. The results showed a synergistic effect of Zn(II) and 10% TFE, i.e., that either Zn or 10% TFE accelerated Aβ28 aggregation on its own, but with them together an at least 10 times promotion of Aβ28 aggregation was observed. Further studies by thioflavin T fluorescence spectroscopy, transmission electron microscopy, and circular dichroism (CD) spectroscopy suggested that the aggregates of Zn-Aβ28 formed in 10%TFE contain a β-sheet secondary structure and are more of the amyloid type. CD spectroscopy indicated that Zn binding disrupted partially the α-helical structure of Aβ28 in TFE. Thus, we propose that the promotion of Aβ28 aggregation by Zn is based on the transformation of the partially α-helical conformer (intermediate) towards the β-sheet amyloid structure by a destabilization of the α-helix in the intermediate. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Cationic membrane peptides: atomic-level insight of structure–activity relationships from solid-state NMR

Many membrane-active peptides, such as cationic cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs), conduct their biological functions by interacting with the cell membrane. The interactions of charged residues with lipids and water facilitate membrane insertion, translocation or disruption of these highly hydrophobic species. In this review, we will summarize high-resolution structural and dynamic findings towards the understanding of the structure–activity relationship of lipid membrane-bound CPPs and AMPs, as examples of the current development of solid-state NMR (SSNMR) techniques for studying membrane peptides. We will present the most recent atomic-resolution structure of the guanidinium-phosphate complex, as constrained from experimentally measured site-specific distances. These SSNMR results will be valuable specifically for understanding the intracellular translocation pathway of CPPs and antimicrobial mechanism of AMPs, and more generally broaden our insight into how cationic macromolecules interact with and cross the lipid membrane.     

Nickel(II)—bleomycin: spectral investigation of the metal binding site

Bleomycin (blm) solutions containing the nickel(II) ion have been investigated through ¹H nmr, ligand field and circular dichroism spectroscopies. It has been found the blm binds the metal ion in a pH dependent fashion. The spectral data are consistent with the presence of at least two species. It is suggested that in the low pH region blm binds to nickel(II) through the β-aminoalanino residue, whereas in the high pH region, the 4-amino-pyrimidine, imidazole, and amido group of β-hydroxy-histidine are also involved in coordination.     

Structure–activity relationships (SAR) and structure–kinetic relationships (SKR) of bicyclic heteroaromatic acetic acids as potent CRTh2 antagonists II: Lead optimization

Extensive structure–activity relationship (SAR) and structure–kinetic relationship (SKR) studies in the bicyclic heteroaromatic series of CRTh2 antagonists led to the identification of several molecules that possessed both excellent binding and cellular potencies along with long receptor residence times. A small substituent in the bicyclic core provided an order of magnitude jump in dissociation half-lives. Selected optimized compounds demonstrated suitable pharmacokinetic profiles.     

Water soluble (η-arene) ruthenium(II) complexes incorporating marine derived bioligand: Synthesis, spectral and structural studies

A series of water soluble compounds of general formula [{(η⁶-arene)Ru(HMP)Cl}], [η⁶-arene = η⁶-cymene (1), η⁶-HMB (2), η⁶-C₆H₆ (3); HMP = 5-hydroxy-2-(hydroxymethyl)-4-pyrone] have been prepared by the reaction of [{(η⁶-arene) RuCl₂}₂] with HMP. The complexes 1 and 2 react with NaN₃ to give in excellent yield tetra-azido complexes [{(η⁶-arene)Ru(μN₃)N₃}₂] (arene = cymene 4, HMB = 5) but similar reaction of complex 3 with NaN₃ yielded di-azdo complex [{(η⁶-C₆H₆)Ru(μN₃)Cl}₂] (6). Reaction of [{(η⁶-arene)Ru(μN₃)Cl}₂] with HMP in the presence of NaOMe resulted in the formation of azido complex [{(η⁶-arene)Ru(HMP)N₃}]. Mono and dinuclear complexes [{(η⁶-arene)Ru(HMP)(L₁)}]⁺ and [{(η⁶-arene)Ru(HMP)}₂(μL₂)]²⁺ were also prepared by the reaction of complexes 1 and 2 with the appropriate ligand, L₁ or L₂ in the presence of AgBF₄ (L₁ = PyCN, DMAP; L₂ = 4,4′-bipy, pyrazine). The complexes are characterized on the basis of spectroscopic data and molecular structures of three representative compounds have been determined by single crystal X-ray diffraction study.     

A new insulin-mimetic bis(allixinato)zinc(II) complex: structure–activity relationship of zinc(II) complexes

During the investigation of the development of insulin-mimetic zinc(II) complexes with a blood glucose-lowering effect in experimental diabetic animals, we found a potent bis(maltolato)zinc(II) complex, Zn(ma)₂, exhibiting significant insulin-mimetic effects in a type 2 diabetic animal model. By using this Zn(ma)₂ as the leading compound, we examined the in vitro and in vivo structure–activity relationships of Zn(ma)₂ and its related complexes. The in vitro insulin-mimetic activity of these complexes was determined by the inhibition of free fatty acid release and the enhancement of glucose uptake in isolated rat adipocytes treated with epinephrine. A new Zn(II) complex with allixin isolated from garlic, Zn(alx)₂, exhibited the highest insulin-mimetic activity among the complexes analyzed. The insulin-mimetic activity of the Zn(II) complexes examined strongly correlated (correlation coefficient=0.96) with the partition coefficient (logP) of the ligand, indicating that the activity of Zn(ma)₂-related complexes depends on the lipophilicity of the ligand. The blood glucose-lowering effects of Zn(alx)₂ and Zn(ma)₂ were then compared, and both complexes were found to normalize hyperglycemia in KK-A^y mice after a 14-day course of daily intraperitoneal injections. However, Zn(alx)₂ improved glucose tolerance in KK-A^y mice much more than did Zn(ma)₂, indicating that Zn(alx)₂ possesses greater in vivo anti-diabetic activity than Zn(ma)₂. In addition, Zn(alx)₂ improved leptin resistance and suppressed the progress of obesity in type 2 diabetic KK-A^y mice. On the basis of these observations, we conclude that the Zn(alx)₂ complex is a novel potent candidate for the treatment of type 2 diabetes mellitus.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0590-8     

In silico and in vitro studies of transition metal complexes derived from curcumin–isoniazid Schiff base

Abstract

A series of transition metal complexes have been synthesized from biologically active curcumin and isoniazid Schiff base. They are characterized by various spectral techniques like UV–Vis, Fourier transform infrared (FT-IR), nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR) and mass spectroscopies. Moreover, elemental analysis, magnetic susceptibility and molar conductivity measurements are also carried out. All these data evidence that the metal complexes acquire square planar except zinc(II) which adopts a tetrahedral geometry, and they are non-electrolytic in nature. Groove mode of binding between the calf thymus DNA (CT DNA) and metal complexes is confirmed by electronic absorption titration, viscosity and cyclic voltammetry studies. In addition to that, all the metal complexes are able to cleave pUC 19 DNA. Optimized geometry and ground-state electronic structure calculations of all the synthesized compounds are established out by density functional theory (DFT) using B3LYP method which theoretically reveals that copper(II) complex explores higher stability and higher biological accessibility. This is experimentally corroborated by antimicrobial studies. In silico Absorption, Distribution, Metabolism, Excretion (ADME) studies reveal the biological potential of all synthesized complexes, and also biological activity of the ligand is predicted by PASS online biological activity prediction software. Molecular docking studies are also carried out to confirm the groove mode of binding and receptor–complex interactions.     

Solution NMR structure of CD1104B from pathogenic Clostridium difficile reveals a distinct α-helical architecture and provides first structural representative of protein domain family PF14203

A high-quality structure of the 68-residue protein CD1104B from Clostridium difficile strain 630 exhibits a distinct all α-helical fold. The structure presented here is the first representative of bacterial protein domain family PF14203 (currently 180 members) of unknown function (DUF4319) and reveals that the side-chains of the only two strictly conserved residues (Glu 8 and Lys 48) form a salt bridge. Moreover, these two residues are located in the vicinity of the largest surface cleft which is predicted to contribute to a surface area involved in protein–protein interactions. This, along with its coding in transposon CTn4, suggests that CD1104B (and very likely all members of Pfam 14203) functions by interacting with other proteins required for the transfer of transposons between different bacterial species.     

Modeling of the structure of protein–DNA complexes using the data from FRET and footprinting experiments

We discuss the question of constructing three-dimensional models of DNA in complex with proteins using computer modeling and indirect methods of studying the conformation of macromolecules. We consider the methods of interpreting the experimental data obtained by indirect methods of studying the three-dimensional structure of biomolecules. We discuss some aspects of integrating such data into the process of constructing the molecular models of DNA–protein complexes based on the geometric characteristics of DNA. We propose an algorithm for estimating conformations of such complexes based on the information about the local flexibility of DNA and on the experimental data obtained by Forster resonance energy transfer (FRET) and hydroxyl footprinting. Finally, we use this algorithm to predict the hypothetical configuration of DNA in a nucleosome bound with histone H1.     

Novel aminopeptidase N inhibitors derived from antineoplaston AS2–5 (Part II)

As our ongoing work, a series of peptidomimetic l-iso-glutamine derivatives derived from antineoplaston AS2–5 scaffold were prepared and their APN/CD13 and MMP-2 inhibitory activities were evaluated hereby. The results displayed that these compounds exhibited selective inhibition against APN as compared with MMP-2, with IC₅₀ values in micromole range. Compounds A1 and A2 showed comparable APN inhibitory activities than the positive control bestatin.     

Functional and structural characterization of α-(1->2) branching sucrase derived from DSR-E glucansucrase

ΔN₁₂₃-glucan-binding domain-catalytic domain 2 (ΔN₁₂₃-GBD-CD2) is a truncated form of the bifunctional glucansucrase DSR-E from Leuconostoc mesenteroides NRRL B-1299. It was constructed by rational truncation of GBD-CD2, which harbors the second catalytic domain of DSR-E. Like GBD-CD2, this variant displays α-(1→2) branching activity when incubated with sucrose as glucosyl donor and (oligo-)dextran as acceptor, transferring glucosyl residues to the acceptor via a ping-pong bi-bi mechanism. This allows the formation of prebiotic molecules containing controlled amounts of α-(1→2) linkages. The crystal structure of the apo α-(1→2) branching sucrase ΔN₁₂₃-GBD-CD2 was solved at 1.90 Å resolution. The protein adopts the unusual U-shape fold organized in five distinct domains, also found in GTF180-ΔN and GTF-SI glucansucrases of glycoside hydrolase family 70. Residues forming subsite −1, involved in binding the glucosyl residue of sucrose and catalysis, are strictly conserved in both GTF180-ΔN and ΔN₁₂₃-GBD-CD2. Subsite +1 analysis revealed three residues (Ala-2249, Gly-2250, and Phe-2214) that are specific to ΔN₁₂₃-GBD-CD2. Mutation of these residues to the corresponding residues found in GTF180-ΔN showed that Ala-2249 and Gly-2250 are not directly involved in substrate binding and regiospecificity. In contrast, mutant F2214N had lost its ability to branch dextran, although it was still active on sucrose alone. Furthermore, three loops belonging to domains A and B at the upper part of the catalytic gorge are also specific to ΔN₁₂₃-GBD-CD2. These distinguishing features are also proposed to be involved in the correct positioning of dextran acceptor molecules allowing the formation of α-(1→2) branches.