Self-Assembly of a Dipeptide with a Reduced Amount of Copper into Antifungal and Antibacterial Particles.

With the growing concern over the environmental impact and health risks associated with conventional pesticides, there is a great need for developing safer and more sustainable alternatives. This study demonstrates the self-assembly of antimicrobial and antifungal spherical particles by a dipeptide utilizing a reduced amount of copper salt compared to the commonly employed formulation. The particles can be sprayed on a surface and form an antimicrobial coating. The effectiveness of the coating against the bacteria Pectobacterium brasiliense, a common pathogen affecting potato crops, was demonstrated, as the coating reduced the bacterial load by 7.3 log. Moreover, a comprehensive field trial was conducted, where the formulation was applied to potato seeds. Remarkably, it exhibited good efficacy against three prevalent potato pathogens (P. brasiliense, Pythium spp., and Spongospora subterranea) while demonstrating no phytotoxic effects on the potatoes. These findings highlight the tremendous potential of this formulation as a nonphytotoxic alternative to replace hazardous pesticides currently available in the market.

Structural insights into the role of the WW2 domain on tandem WW-PPxY motif interactions of oxidoreductase WWOX.

Class I WW domains are present in many proteins of various functions and mediate protein interactions by binding to short linear PPxY motifs. Tandem WW domains often bind peptides with multiple PPxY motifs, but the interplay of WW-peptide interactions is not always intuitive. The WW domain-containing oxidoreductase (WWOX) harbors two WW domains: an unstable WW1 capable of PPxY binding and stable WW2 that cannot bind PPxY. The WW2 domain has been suggested to act as a WW1 domain chaperone, but the underlying mechanism of its chaperone activity remains to be revealed. Here, we combined NMR, isothermal calorimetry, and structural modeling to elucidate the roles of both WW domains in WWOX binding to its PPxY-containing substrate ErbB4. Using NMR, we identified an interaction surface between these two domains that supports a WWOX conformation compatible with peptide substrate binding. Isothermal calorimetry and NMR measurements also indicated that while binding affinity to a single PPxY motif is marginally increased in the presence of WW2, affinity to a dual-motif peptide increases 10-fold. Furthermore, we found WW2 can directly bind double-motif peptides using its canonical binding site. Finally, differential binding of peptides in mutagenesis experiments was consistent with a parallel N- to C-terminal PPxY tandem motif orientation in binding to the WW1-WW2 tandem domain, validating structural models of the interaction. Taken together, our results reveal the complex nature of tandem WW-domain organization and substrate binding, highlighting the contribution of WWOX WW2 to both protein stability and target binding.

Studying Peptide-Metal Ion Complex Structures by Solution-State NMR.

Metal chelation can provide structural stability and form reactive centers in metalloproteins. Approximately one third of known protein structures are metalloproteins, and metal binding, or the lack thereof, is often implicated in disease, making it necessary to be able to study these systems in detail. Peptide-metal complexes are both present in nature and can provide a means to focus on the binding region of a protein and control experimental variables to a high degree. Structural studies of peptide complexes with metal ions by nuclear magnetic resonance (NMR) were surveyed for all the essential metal complexes and many non-essential metal complexes. The various methods used to study each metal ion are presented together with examples of recent research. Many of these metal systems have been individually reviewed and this current overview of NMR studies of metallopeptide complexes aims to provide a basis for inspiration from structural studies and methodology applied in the field.

Determining the structure and binding mechanism of oxytocin-Cu2+ complex using paramagnetic relaxation enhancement NMR analysis.

Oxytocin is a neuropeptide that binds copper ions in nature. The structure of oxytocin in interaction with Cu2+ was determined here by NMR, showing which atoms of the peptide are involved in binding. Paramagnetic relaxation enhancement NMR analyses indicated a binding mechanism where the amino terminus was required for binding and subsequently Tyr2, Ile3 and Gln4 bound in that order. The aromatic ring of Tyr2 formed a π-cation interaction with Cu2+. Oxytocin copper complex structure revealed by paramagnetic relaxation enhancement NMR analyses.

The Bacterial Extracellular Matrix Protein TapA Is a Two-Domain Partially Disordered Protein.

Biofilms are aggregates of microbial cells that form on surfaces and at interfaces, and are encased in an extracellular matrix. In biofilms made by the soil bacterium Bacillus subtilis, the protein TapA mediates the assembly of the functional amyloid protein TasA into extracellular fibers, and it anchors these fibers to the cell surface. We used circular dichroism and NMR spectroscopy to show that, unlike the structured TasA, TapA is disordered. In addition, TapA is composed of two weakly interacting domains: a disordered C-terminal domain and a more structured N-terminal domain. These two domains also exhibited different structural changes in response to changes in external conditions, such as increased temperatures and the presence of lipid vesicles. Although the two TapA domains weakly interacted in solution, their cooperative interaction with lipid vesicles prevented disruption of the vesicles. These findings therefore suggest that the two-domain composition of TapA is important in its interaction with single or multiple partners in the extracellular matrix in biofilms.

Substitution of an Internal Disulfide Bridge with a Diselenide Enhances both Foldability and Stability of Human Insulin.

Insulin analogues, mainstays in the modern treatment of diabetes mellitus, exemplify the utility of protein engineering in molecular pharmacology. Whereas chemical syntheses of the individual A and B chains were accomplished in the early 1960s, their combination to form native insulin remains inefficient because of competing disulfide pairing and aggregation. To overcome these limitations, we envisioned an alternative approach: pairwise substitution of cysteine residues with selenocysteine (Sec, U). To this end, CysA6 and CysA11 (which form the internal intrachain A6-A11 disulfide bridge) were each replaced with Sec. The A chain[C6U, C11U] variant was prepared by solid-phase peptide synthesis; while sulfitolysis of biosynthetic human insulin provided wild-type B chain-di-S-sulfonate. The presence of selenium atoms at these sites markedly enhanced the rate and fidelity of chain combination, thus solving a long-standing challenge in chemical insulin synthesis. The affinity of the Se-insulin analogue for the lectin-purified insulin receptor was indistinguishable from that of WT-insulin. Remarkably, the thermodynamic stability of the analogue at 25 °C, as inferred from guanidine denaturation studies, was augmented (ΔΔGu ≈0.8 kcal mol-1 ). In accordance with such enhanced stability, reductive unfolding of the Se-insulin analogue and resistance to enzymatic cleavage by Glu-C protease occurred four times more slowly than that of WT-insulin. 2D-NMR and X-ray crystallographic studies demonstrated a native-like three-dimensional structure in which the diselenide bridge was accommodated in the hydrophobic core without steric clash.

A tandem in situ peptide cyclization through trifluoroacetic acid cleavage.

We present a new approach for peptide cyclization during solid phase synthesis under highly acidic conditions. Our approach involves simultaneous in situ deprotection, cyclization and trifluoroacetic acid (TFA) cleavage of the peptide, which is achieved by forming an amide bond between a lysine side chain and a succinic acid linker at the peptide N-terminus. The reaction proceeds via a highly active succinimide intermediate, which was isolated and characterized. The structure of a model cyclic peptide was solved by NMR spectroscopy. Theoretical calculations support the proposed mechanism of cyclization. Our new methodology is applicable for the formation of macrocycles in solid-phase synthesis of peptides and organic molecules.

Peptide models of Cu(I) and Zn(II) metallochaperones: the effect of pH on coordination and mechanistic implications.

The first NMR structures of Cu(I) and Zn(II) peptide complexes as models of metallochaperones were derived with no predetermined binding mode. The cyclic peptide MDCSGCSRPG was reacted with Cu(I) and Zn(II) at low and moderate pH. This peptide features the conserved sequence of copper chaperones but with Asp at position 2 as appears in the zinc binding domain of ZntA. The structures were compared with those of the Cu(I) complexes of the wild-type sequence peptide MTCSGCSRPG. All analyses were conducted first with no metal-binding constraints to ensure accurate binding ligand assignment. Several structures included metal-Met binding, raising a possible role of Met in the metal transport mechanism. Both Cu(I) and Zn(II) gave different complexes when reacted with the peptide of the native-like sequence under different pH conditions, raising the possibility of pH-dependent transport mechanisms. Cu(I) bound the MTCSGCSRPG peptide through one Cys and the Met under acidic conditions and differently under basic conditions; Zn(II) bound the MDCSGCSRPG peptide through two Cys and the Met residues under acidic conditions and through one Cys and the Met under basic conditions, while Cu(I) bound the non-native Asp mutant peptide through the Asp and one Cys under both conditions, suggesting that Asp may inhibit pH-dependent binding for Cu(I). NOESY and ESI-HRMS supported the presence of an aqua ligand for Zn(II), which likely deprotonated under basic conditions to give a hydroxo group. Coordination similarities were detected among the model system and native proteins, which overall suggest that coordination flexibility is required for the function of metallochaperones.

Backbone cyclic helix mimetic of chemokine (C-C motif) receptor 2: a rational approach for inhibiting dimerization of G protein-coupled receptors.

The transmembrane helical bundle of G protein-coupled receptors (GPCRs) dimerize through helix-helix interactions in response to inflammatory stimulation. A strategy was developed to target the helical dimerization site of GPCRs by peptidomimetics with drug like properties. The concept was demonstrated by selecting a potent backbone cyclic helix mimetic from a library that derived from the dimerization region of chemokine (C-C motif) receptor 2 (CCR2) that is a key player in Multiple Sclerosis. We showed that CCR2 based backbone cyclic peptide having a stable helix structure inhibits specific CCR2-mediated chemotactic migration.

A comparative study of backbone versus side chain peptide cyclization: application for HIV-1 integrase inhibitors.

Peptide cyclization is an important tool for overcoming the limitations of linear peptides as drugs. Backbone cyclization (BC) has advantages over side chain (SC) cyclization because it combines N-alkylation for extra peptide stability. However, the appropriate building blocks for BC are not yet commercially available. This problem can be overcome by preparing SC cyclic peptide analogs of the most active BC peptide using commercially available building blocks. We have recently developed BC peptides that inhibit the HIV-1 integrase enzyme (IN) activity and HIV-1 replication in infected cells. Here we used this system as a model for systematically comparing the BC and SC cyclization modes using biophysical, biochemical and structural methods. The most potent SC cyclic peptide was active almost as the BC peptide and inhibited IN activity in vitro and blocked IN activity in cells even after 6 days. We conclude that both cyclization types have their respective advantages: The BC peptide is more active and stable, probably due to the N-alkylation, while SC cyclic peptides are easier to synthesize. Due to the high costs and efforts involved in preparing BC peptides, SC may be a more approachable method in many cases. We suggest that both methods are interchangeable.

The positively charged region of the myosin IIC non-helical tailpiece promotes filament assembly.

The motor protein, non-muscle myosin II (NMII), must undergo dynamic oligomerization into filaments to participate in cellular processes such as cell migration and cytokinesis. A small non-helical region at the tail of the long coiled-coil region (tailpiece) is a common feature of all dynamically assembling myosin II proteins. In this study, we investigated the role of the tailpiece in NMII-C self-assembly. We show that the tailpiece is natively unfolded, as seen by circular dichroism and NMR experiments, and is divided into two regions of opposite charge. The positively charged region (Tailpiece(1946-1967)) starts at residue 1946 and is extended by seven amino acids at its N terminus from the traditional coiled-coil ending proline (Tailpiece(1953-1967)). Pull-down and sedimentation assays showed that the positive Tailpiece(1946-1967) binds to assembly incompetent NMII-C fragments inducing filament assembly. The negative region, residues 1968-2000, is responsible for NMII paracrystal morphology as determined by chimeras in which the negative region was swapped between the NMII isoforms. Mixing the positive and negative peptides had no effect on the ability of the positive peptide to bind and induce filament assembly. This study provides molecular insight into the role of the structurally disordered tailpiece of NMII-C in shifting the oligomeric equilibrium of NMII-C toward filament assembly and determining its morphology.

Molecular basis of the interaction between the antiapoptotic Bcl-2 family proteins and the proapoptotic protein ASPP2.

We have characterized the molecular basis of the interaction between ASPP2 and Bcl-2, which are key proteins in the apoptotic pathway. The C-terminal ankyrin repeats and SH3 domain of ASPP2 (ASPP2(Ank-SH3)) mediate its interactions with the antiapoptotic protein Bcl-2. We used biophysical and computational methods to identify the interaction sites of Bcl-2 and its homologues with ASPP2. Using peptide array screening, we found that ASPP2(Ank-SH3) binds two homologous sites in all three Bcl proteins tested: (i) the conserved BH4 motif, and (ii) a binding site for proapoptotic regulators. Quantitative binding studies revealed that binding of ASPP2(Ank-SH3) to the Bcl-2 family members is selective at two levels: (i) interaction with Bcl-2-derived peptides is the tightest compared to peptides from the other family members, and (ii) within Bcl-2, binding of ASPP2(Ank-SH3) to the BH4 domain is tightest. Sequence alignment of the ASPP2-binding peptides combined with binding studies of mutated peptides revealed that two nonconserved positions where only Bcl-2 contains positively charged residues account for its tighter binding. The experimental binding results served as a basis for docking analysis, by which we modeled the complexes of ASPP2(Ank-SH3) with the full-length Bcl proteins. Using peptide arrays and quantitative binding studies, we found that Bcl-2 binds three loops in ASPP2(Ank-SH3) with similar affinity, in agreement with our predicted model. Based on our results, we propose a mechanism in which ASPP2 induces apoptosis by inhibiting functional sites of the antiapoptotic Bcl-2 proteins.

Diselenide crosslinks for enhanced and simplified oxidative protein folding.

The in vitro oxidative folding of proteins has been studied for over sixty years, providing critical insight into protein folding mechanisms. Hirudin, the most potent natural inhibitor of thrombin, is a 65-residue protein with three disulfide bonds, and is viewed as a folding model for a wide range of disulfide-rich proteins. Hirudin's folding pathway is notorious for its highly heterogeneous intermediates and scrambled isomers, limiting its folding rate and yield in vitro. Aiming to overcome these limitations, we undertake systematic investigation of diselenide bridges at native and non-native positions and investigate their effect on hirudin's folding, structure and activity. Our studies demonstrate that, regardless of the specific positions of these substitutions, the diselenide crosslinks enhanced the folding rate and yield of the corresponding hirudin analogues, while reducing the complexity and heterogeneity of the process. Moreover, crystal structure analysis confirms that the diselenide substitutions maintained the overall three-dimensional structure of the protein and left its function virtually unchanged. The choice of hirudin as a study model has implications beyond its specific folding mechanism, demonstrating the high potential of diselenide substitutions in the design, preparation and characterization of disulfide-rich proteins.

The butyrylcholinesterase K variant confers structurally derived risks for Alzheimer pathology.

The K variant of butyrylcholinesterase (BChE-K, 20% incidence) is a long debated risk factor for Alzheimer disease (AD). The A539T substitution in BChE-K is located at the C terminus, which is essential both for BChE tetramerization and for its capacity to attenuate beta-amyloid (Abeta) fibril formation. Here, we report that BChE-K is inherently unstable as compared with the "usual" BChE (BChE-U), resulting in reduced hydrolytic activity and predicting prolonged acetylcholine maintenance and protection from AD. A synthetic peptide derived from the C terminus of BChE-K (BSP-K), which displayed impaired intermolecular interactions, was less potent in suppressing Abeta oligomerization than its BSP-U counterpart. Correspondingly, highly purified recombinant human rBChE-U monomers suppressed beta-amyloid fibril formation less effectively than dimers, which also protected cultured neuroblastoma cells from Abeta neurotoxicity. Dual activity structurally derived changes due to the A539T substitution can thus account for both neuroprotective characteristics caused by sustained acetylcholine levels and elevated AD risk due to inefficient interference with amyloidogenic processes.

Mechanism of action of the HIV-1 integrase inhibitory peptide LEDGF 361-370.

The HIV-1 integrase protein (IN) mediates integration of the viral cDNA into the host genome and is a target for anti-HIV drugs. We have recently described a peptide derived from residues 361-370 of the IN cellular partner protein LEDGF/p75, which inhibited IN catalytic activity in vitro and HIV-1 replication in cells. Here we performed a comprehensive study of the LEDGF 361-370 mechanism of action in vitro, in cells and in vivo. Alanine scan, fluorescence anisotropy binding studies, homology modeling and NMR studies demonstrated that all residues in LEDGF 361-370 contribute to IN binding and inhibition. Kinetic studies in cells showed that LEDGF 361-370 specifically inhibited integration of viral cDNA. Thus, the full peptide was chosen for in vivo studies, in which it inhibited the production of HIV-1 RNA in mouse model. We conclude that the full LEDGF 361-370 peptide is a potent HIV-1 inhibitor and may be used for further development as an anti-HIV lead compound.

Cyclic peptide inhibitors of HIV-1 integrase derived from the LEDGF/p75 protein.

Restricting linear peptides to their bioactive conformation is an attractive way of improving their stability and activity. We used a cyclic peptide library with conformational diversity for selecting an active and stable peptide that mimics the structure and activity of the HIV-1 integrase (IN) binding loop from its cellular cofactor LEDGF/p75 (residues 361-370). All peptides in the library had the same primary sequence, and differed only in their conformation. Library screening revealed that the ring size and linker structure had a huge effect on the conformation, binding and activity of the peptides. One of the cyclic peptides, c(MZ 4-1), was a potent and stable inhibitor of IN activity in vitro and in cells even after 8 days. The NMR structure of c(MZ 4-1) showed that it obtains a bioactive conformation that is similar to the parent site in LEDGF/p75.

A Rapid and Efficient Building Block Approach for Click Cyclization of Peptoids.

Cyclic peptide-peptoid hybrids possess improved stability and selectivity over linear peptides and are thus better drug candidates. However, their synthesis is far from trivial and is usually difficult to automate. Here we describe a new rapid and efficient approach for the synthesis of click-based cyclic peptide-peptoid hybrids. Our methodology is based on a combination between easily synthesized building blocks, automated microwave assisted solid phase synthesis and bioorthogonal click cyclization. We proved the concept of this method using the INS peptide, which we have previously shown to activate the HIV-1 integrase enzyme. This strategy enabled the rapid synthesis and biophysical evaluation of a library of cyclic peptide-peptoid hybrids derived from HIV-1 integrase in high yield and purity. The new cyclic hybrids showed improved biological activity and were significantly more stable than the original linear INS peptide.

Cyclizing Painkillers: Development of Backbone-Cyclic TAPS Analogs.

Painkillers are commonly used medications. Native peptide painkillers suffer from various pharmacological disadvantages, while small molecule painkillers like morphine are highly addictive. We present a general approach aimed to use backbone-cyclization to develop a peptidomimetic painkiller. Backbone-cyclization was applied to transform the linear peptide Tyr-Arg-Phe-Sar (TAPS) into an active backbone-cyclic peptide with improved drug properties. We designed and synthesized a focused backbone-cyclic TAPS library with conformational diversity, in which the members of the library have the generic name TAPS c(n-m) where n and m represent the lengths of the alkyl chains on the nitrogens of Gly and Arg, respectively. We used a combined screening approach to evaluate the pharmacological properties and the potency of the TAPS c(n-m) library. We focused on an in vivo active compound, TAPS c(2-6), which is metabolically stable and has the potential to become a peripheral painkiller being a full µ opioid receptor functional agonist. To prepare a large quantity of TAPS c(2-6), we optimized the conditions of the on-resin reductive alkylation step to increase the efficiency of its SPPS. NMR was used to determine the solution conformation of the peptide lead TAPS c(2-6).

Mass spectrometry reveals the chemistry of formaldehyde cross-linking in structured proteins.

Whole-cell cross-linking coupled to mass spectrometry is one of the few tools that can probe protein-protein interactions in intact cells. A very attractive reagent for this purpose is formaldehyde, a small molecule which is known to rapidly penetrate into all cellular compartments and to preserve the protein structure. In light of these benefits, it is surprising that identification of formaldehyde cross-links by mass spectrometry has so far been unsuccessful. Here we report mass spectrometry data that reveal formaldehyde cross-links to be the dimerization product of two formaldehyde-induced amino acid modifications. By integrating the revised mechanism into a customized search algorithm, we identify hundreds of cross-links from in situ formaldehyde fixation of human cells. Interestingly, many of the cross-links could not be mapped onto known atomic structures, and thus provide new structural insights. These findings enhance the use of formaldehyde cross-linking and mass spectrometry for structural studies.