A Versatile Virus-Mimetic Engineering Approach for Concurrent Protein Nanocage Surface-Functionalization and Cargo Encapsulation.

Naturally occurring protein nanocages like ferritin are self-assembled from multiple subunits. Because of their unique cage-like structure and biocompatibility, there is a growing interest in their biomedical use. A multipurpose and straightforward engineering approach does not exist for using nanocages to make drug-delivery systems by encapsulating hydrophilic or hydrophobic drugs and developing vaccines by surface functionalization with a protein like an antigen. Here, a versatile engineering approach is described by mimicking the HIV-1 Gap polyprotein precursor. Various PREcursors of nanoCages (PREC) are designed and created by linking two ferritin subunits via a flexible linker peptide containing a protease cleavage site. These precursors can have additional proteins at their N-terminus, and their protease cleavage generates ferritin-like nanocages named protease-induced nanocages (PINCs). It is demonstrated that PINC formation allows concurrent surface decoration with a protein and hydrophilic or hydrophobic drug encapsulation up to fourfold more than the amount achieved using other methods. The PINCs/Drug complex is stable and efficiently kills cancer cells. This work provides insight into the precursors' design rules and the mechanism of PINCs formation. The engineering approach and mechanistic insight described here will facilitate nanocages' applications in drug delivery or as a platform for making multifunctional therapeutics like mosaic vaccines.

LARP4A recognizes polyA RNA via a novel binding mechanism mediated by disordered regions and involving the PAM2w motif, revealing interplay between PABP, LARP4A and mRNA.

LARP4A belongs to the ancient RNA-binding protein superfamily of La-related proteins (LARPs). In humans, it acts mainly by stabilizing mRNAs, enhancing translation and controlling polyA lengths of heterologous mRNAs. These activities are known to implicate its association with mRNA, protein partners and translating ribosomes, albeit molecular details are missing. Here, we characterize the direct interaction between LARP4A, oligoA RNA and the MLLE domain of the PolyA-binding protein (PABP). Our study shows that LARP4A-oligoA association entails novel RNA recognition features involving the N-terminal region of the protein that exists in a semi-disordered state and lacks any recognizable RNA-binding motif. Against expectations, we show that the La module, the conserved RNA-binding unit across LARPs, is not the principal determinant for oligoA interaction, only contributing to binding to a limited degree. Furthermore, the variant PABP-interacting motif 2 (PAM2w) featured in the N-terminal region of LARP4A was found to be important for both RNA and PABP recognition, revealing a new role for this protein-protein binding motif. Our analysis demonstrates the mutual exclusive nature of the PAM2w-mediated interactions, thereby unveiling a tantalizing interplay between LARP4A, polyA and PABP.

Binding Interactions of Peptide Aptamers.

Peptide aptamers are short amino acid chains that are capable of binding specifically to ligands in the same way as their much larger counterparts, antibodies. Ligands of therapeutic interest that can be targeted are other peptide chains or loops located on the surface of protein receptors (e.g., GCPR), which take part in cell-to-cell communications either directly or via the intermediary of hormones or signalling molecules. To confer on aptamers the same sort of conformational rigidity that characterises an antibody binding site, aptamers are often constructed in the form of cyclic peptides, on the assumption that this will encourage stronger binding interactions than would occur if the aptamers were simply linear chains. However, no formal studies have been conducted to confirm the hypothesis that linear peptides will engage in stronger binding interactions with cyclic peptides than with other linear peptides. In this study, the interaction of a model cyclic decamer with a series of linear peptide constructs was compared with that of a linear peptide with the same sequence, showing that the cyclic configuration does confer benefits by increasing the strength of binding.

Isothermal Titration Calorimetry Enables Rapid Characterization of Enzyme Kinetics and Inhibition for the Human Soluble Epoxide Hydrolase.

Isothermal titration calorimetry (ITC) is conventionally used to acquire thermodynamic data for biological interactions. In recent years, ITC has emerged as a powerful tool to characterize enzyme kinetics. In this study, we have adapted a single-injection method (SIM) to study the kinetics of human soluble epoxide hydrolase (hsEH), an enzyme involved in cardiovascular homeostasis, hypertension, nociception, and insulin sensitivity through the metabolism of epoxy-fatty acids (EpFAs). In the SIM method, the rate of reaction is determined by monitoring the thermal power, while the substrate is being depleted, overcoming the need for synthetic substrates and reducing postreaction processing. Our results show that ITC enables the detailed, rapid, and reproducible characterization of the hsEH-mediated hydrolysis of several natural EpFA substrates. Furthermore, we have applied a variant of the single-injection ITC method for the detailed description of enzyme inhibition, proving the power of this approach in the rapid screening and discovery of new hsEH inhibitors using the enzyme's physiological substrates. The methods described herein will enable further studies on EpFAs' metabolism and biology, as well as drug discovery investigations to identify and characterize hsEH inhibitors. This also promises to provide a general approach for the characterization of lipid catalysis, given the challenges that lipid metabolism studies pose to traditional spectroscopic techniques.

Zinc-induced oligomerization of zinc α2 glycoprotein reveals multiple fatty acid-binding sites.

Zinc α2 glycoprotein (ZAG) is an adipokine with a class I MHC protein fold and is associated with obesity and diabetes. Although its intrinsic ligand remains unknown, ZAG binds the dansylated C11 fatty acid 11-(dansylamino)undecanoic acid (DAUDA) in the groove between the α1 and α2 domains. The surface of ZAG has approximately 15 weak zinc-binding sites deemed responsible for precipitation from human plasma. In the present study the functional significance of these metal sites was investigated. Analytical ultracentrifugation (AUC) and CD showed that zinc, but not other divalent metals, causes ZAG to oligomerize in solution. Thus ZAG dimers and trimers were observed in the presence of 1 and 2 mM zinc. Molecular modelling of X-ray scattering curves and sedimentation coefficients indicated a progressive stacking of ZAG monomers, suggesting that the ZAG groove may be occluded in these. Using fluorescence-detected sedimentation velocity, these ZAG-zinc oligomers were again observed in the presence of the fluorescent boron dipyrromethene fatty acid C16-BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid). Fluorescence spectroscopy confirmed that ZAG binds C16-BODIPY. ZAG binding to C16-BODIPY, but not to DAUDA, was reduced by increased zinc concentrations. We conclude that the lipid-binding groove in ZAG contains at least two distinct fatty acid-binding sites for DAUDA and C16-BODIPY, similar to the multiple lipid binding seen in the structurally related immune protein CD1c. In addition, because high concentrations of zinc occur in the pancreas, the perturbation of these multiple lipid-binding sites by zinc may be significant in Type 2 diabetes where dysregulation of ZAG and zinc homoeostasis occurs.

Synergic interplay of the La motif, RRM1 and the interdomain linker of LARP6 in the recognition of collagen mRNA expands the RNA binding repertoire of the La module.

The La-related proteins (LARPs) form a diverse group of RNA-binding proteins characterized by the possession of a composite RNA binding unit, the La module. The La module comprises two domains, the La motif (LaM) and the RRM1, which together recognize and bind to a wide array of RNA substrates. Structural information regarding the La module is at present restricted to the prototypic La protein, which acts as an RNA chaperone binding to 3' UUUOH sequences of nascent RNA polymerase III transcripts. In contrast, LARP6 is implicated in the regulation of collagen synthesis and interacts with a specific stem-loop within the 5' UTR of the collagen mRNA. Here, we present the structure of the LaM and RRM1 of human LARP6 uncovering in both cases considerable structural variation in comparison to the equivalent domains in La and revealing an unprecedented fold for the RRM1. A mutagenic study guided by the structures revealed that RNA recognition requires synergy between the LaM and RRM1 as well as the participation of the interdomain linker, probably in realizing tandem domain configurations and dynamics required for substrate selectivity. Our study highlights a considerable complexity and plasticity in the architecture of the La module within LARPs.

Analysis of the interaction with the hepatitis C virus mRNA reveals an alternative mode of RNA recognition by the human La protein.

Human La protein is an essential factor in the biology of both coding and non-coding RNAs. In the nucleus, La binds primarily to 3' oligoU containing RNAs, while in the cytoplasm La interacts with an array of different mRNAs lacking a 3' UUU(OH) trailer. An example of the latter is the binding of La to the IRES domain IV of the hepatitis C virus (HCV) RNA, which is associated with viral translation stimulation. By systematic biophysical investigations, we have found that La binds to domain IV using an RNA recognition that is quite distinct from its mode of binding to RNAs with a 3' UUU(OH) trailer: although the La motif and first RNA recognition motif (RRM1) are sufficient for high-affinity binding to 3' oligoU, recognition of HCV domain IV requires the La motif and RRM1 to work in concert with the atypical RRM2 which has not previously been shown to have a significant role in RNA binding. This new mode of binding does not appear sequence specific, but recognizes structural features of the RNA, in particular a double-stranded stem flanked by single-stranded extensions. These findings pave the way for a better understanding of the role of La in viral translation initiation.

Conformational flexibility determines selectivity and antibacterial, antiplasmodial, and anticancer potency of cationic α-helical peptides.

We used a combination of fluorescence, circular dichroism (CD), and NMR spectroscopies in conjunction with size exclusion chromatography to help rationalize the relative antibacterial, antiplasmodial, and cytotoxic activities of a series of proline-free and proline-containing model antimicrobial peptides (AMPs) in terms of their structural properties. When compared with proline-free analogs, proline-containing peptides had greater activity against Gram-negative bacteria, two mammalian cancer cell lines, and intraerythrocytic Plasmodium falciparum, which they were capable of killing without causing hemolysis. In contrast, incorporation of proline did not have a consistent effect on peptide activity against Mycobacterium tuberculosis. In membrane-mimicking environments, structures with high α-helix content were adopted by both proline-free and proline-containing peptides. In solution, AMPs generally adopted disordered structures unless their sequences comprised more hydrophobic amino acids or until coordinating phosphate ions were added. Proline-containing peptides resisted ordering induced by either method. The roles of the angle subtended by positively charged amino acids and the positioning of the proline residues were also investigated. Careful positioning of proline residues in AMP sequences is required to enable the peptide to resist ordering and maintain optimal antibacterial activity, whereas varying the angle subtended by positively charged amino acids can attenuate hemolytic potential albeit with a modest reduction in potency. Maintaining conformational flexibility improves AMP potency and selectivity toward bacterial, plasmodial, and cancerous cells while enabling the targeting of intracellular pathogens.

Control of pH responsive peptide self-association during endocytosis is required for effective gene transfer.

Cationic amphipathic histidine rich peptides demonstrate differential nucleic acid binding capabilities at neutral and acidic pH and adopt conformations at acidic pH that enable interaction with endosomal membranes, their subsequent disordering and facilitate entry of cargo to the cell cytosol. To better understand the relative contributions of each stage in the process and consequently the structural requirements of pH responsive peptides for optimal nucleic acid transfer, we used biophysical methods to dissect the series of events that occur during endosomal acidification. Far-UV circular dichroism was used to characterise the solution conformation of a series of peptides, containing either four or six histidine residues, designed to respond at differing pH while a novel application of near-UV circular dichroism was used to determine the binding affinities of the peptides for both DNA and siRNA. The peptide induced disordering of neutral and anionic membranes was investigated using (2)H solid-state NMR. While each of these parameters models key stages in the nucleic acid delivery process and all were affected by increasing the histidine content of the peptide, the effect of a more acidic pH response on peptide self-association was most notable and identified as the most important barrier to further enhancing nucleic acid delivery. Further, the results indicate that Coulombic interactions between the histidine residues modulate protonation and subsequent conformational transitions required for peptide mediated gene transfer activity and are an important factor to consider in future peptide design.

Spectroscopic studies on nicotine and nornicotine in the UV region.

The UV absorption and electronic circular dichroism (ECD) spectra of (R)- and (S)-nicotine and (S)-nornicotine in aqueous solution were measured to a significantly lower wavelength range than previously reported, allowing the identification of four previously unobserved electronic transitions. The ECD spectra of the two enantiomers of nicotine were equal in magnitude and opposite in sign, while the UV absorption spectra were coincidental. In line with previous observations, (S)-nicotine exhibited a negative cotton effect centered on 263 nm with vibronic structure (π-π1 * transition) and a broad, positive ECD signal at around 240 nm associated with the n-π1 * transition. As expected this band disappeared when the pyridyl aromatic moiety was protonated. Four further electronic transitions are reported between 215 and 180 nm; it is proposed the negative maxima around 206 nm is either an n-σ* transition or a charge transfer band resulting from the movement of charge from the pyrrolidyl N lone pair to the pyridyl π* orbital. The pyridyl π-π2* transition may be contained within the negative ECD signal envelope at around 200 nm. Another negative maximum at 188 nm is thought to be the pyridyl π-π3 * transition, while the lowest wavelength end-absorption and positive ECD may be associated with the π-π4 * transition. The UV absorption spectra of (S)-nornicotine was similar to that of (S)-nicotine in the range 280-220 nm and acidification of the aqueous solution enhanced the absorption. The ECD signals of (S)-nornicotine were considerably less intense compared to (S)-nicotine and declined further on acidification; in the far UV region the ECD spectra diverge considerably.

Synergy between Winter Flounder antimicrobial peptides.

Some antimicrobial peptides (AMPs) have potent bactericidal activity and are being considered as potential alternatives to classical antibiotics. In response to an infection, such AMPs are often produced in animals alongside other peptides with low or no perceivable antimicrobial activity, whose role is unclear. Here we show that six AMPs from the Winter Flounder (WF) act in synergy against a range of bacterial pathogens and provide mechanistic insights into how this increases the cooperativity of the dose-dependent bactericidal activity and potency that enable therapy. Only two WF AMPs have potent antimicrobial activity when used alone but we find a series of two-way combinations, involving peptides which otherwise have low or no activity, yield potent antimicrobial activity. Weakly active WF AMPs modulate the membrane interactions of the more potent WF AMPs and enable therapy in a model of Acinetobacter baumannii burn wound infection. The observed synergy and emergent behaviour may explain the evolutionary benefits of producing a family of related peptides and are attractive properties to consider when developing AMPs towards clinical applications.

Solution model of the intrinsically disordered polyglutamine tract-binding protein-1.

Polyglutamine tract-binding protein-1 (PQBP-1) is a 265-residue nuclear protein that is involved in transcriptional regulation. In addition to its role in the molecular pathology of the polyglutamine expansion diseases, mutations of the protein are associated with X-linked mental retardation. PQBP-1 binds specifically to glutamine repeat sequences and proline-rich regions, and interacts with RNA polymerase II and the spliceosomal protein U5-15kD. In this work, we obtained a biophysical characterization of this protein by employing complementary structural methods. PQBP-1 is shown to be a moderately compact but largely disordered molecule with an elongated shape, having a Stokes radius of 3.7 nm and a maximum molecular dimension of 13 nm. The protein is monomeric in solution, has residual ß-structure, and is in a premolten globule state that is unaffected by natural osmolytes. Using small-angle x-ray scattering data, we were able to generate a low-resolution, three-dimensional model of PQBP-1.

Heterodimerization of the human RNase P/MRP subunits Rpp20 and Rpp25 is a prerequisite for interaction with the P3 arm of RNase MRP RNA.

Rpp20 and Rpp25 are two key subunits of the human endoribonucleases RNase P and MRP. Formation of an Rpp20-Rpp25 complex is critical for enzyme function and sub-cellular localization. We present the first detailed in vitro analysis of their conformational properties, and a biochemical and biophysical characterization of their mutual interaction and RNA recognition. This study specifically examines the role of the Rpp20/Rpp25 association in the formation of the ribonucleoprotein complex. The interaction of the individual subunits with the P3 arm of the RNase MRP RNA is revealed to be negligible whereas the 1:1 Rpp20:Rpp25 complex binds to the same target with an affinity of the order of nM. These results unambiguously demonstrate that Rpp20 and Rpp25 interact with the P3 RNA as a heterodimer, which is formed prior to RNA binding. This creates a platform for the design of future experiments aimed at a better understanding of the function and organization of RNase P and MRP. Finally, analyses of interactions with deletion mutant proteins constructed with successively shorter N- and C-terminal sequences indicate that the Alba-type core domain of both Rpp20 and Rpp25 contains most of the determinants for mutual association and P3 RNA recognition.

Observation of a single-stranded DNA/pyrrolobenzodiazepine adduct.

Pyrrolobenzodiazepine (PBD) antitumor agents have, to date, only been observed to bind to duplex DNA, apparently requiring a minor groove environment for covalent bond formation between their C11-position and the C2-NH(2) functionality of a guanine base. Using an HPLC/MS assay we have now observed and isolated for the first time PBD adducts with single-stranded DNA fragments. Surprisingly, these adducts could only be formed through dissociation of duplex DNA adducts and not by direct interaction of PBDs with single-stranded DNA. They were sufficiently stable for characterization by MALDI-TOF-MS and remained intact after storing at -20 °C for at least 20 days, although the PBD became detached from the DNA within 7 days if stored at room temperature. Furthermore, addition of a complementary strand allowed the duplex adduct to reform. The relative stability of single-stranded PBD/DNA adducts despite a complete loss of minor groove structure was further confirmed by CD spectroscopic analysis. The CD signal induced by the presence of a PBD molecule in the single-stranded adducts remained prominent despite heating for 2 h at 50-60 °C, thus indicating their relatively robust nature.

Mutants of the tumour suppressor p53 L1 loop as second-site suppressors for restoring DNA binding to oncogenic p53 mutations: structural and biochemical insights.

To assess the potential of mutations from the L1 loop of the tumour suppressor p53 as second-site suppressors, the effect of H115N and S116M on the p53 'hot spot' mutations has been investigated using the double-mutant approach. The effects of these two mutants on the p53 hot spots in terms of thermal stability and DNA binding were evaluated. The results show that: (i) the p53 mutants H115N and S116M are thermally more stable than wild-type p53; (ii) H115N but not S116M is capable of rescuing the DNA binding of one of the most frequent p53 mutants in cancer, R248Q, as shown by binding of R248Q/H115N to gadd45 (the promoter of a gene involved in cell-cycle arrest); (iii) the double mutant R248Q/H115N is more stable than wild-type p53; (iv) the effect of H115N as a second-site suppressor to restore DNA-binding activity is specific to R248Q, but not to R248W; (v) molecular-dynamics simulations indicate that R248Q/H115N has a conformation similar to wild-type p53, which is distinct from that of R248Q. These findings could be exploited in designing strategies for cancer therapy to identify molecules that could mimic the effect of H115N in restoring function to oncogenic p53 mutants.

Lipid domain formation and non-lamellar structures associated with varied lysylphosphatidylglycerol analogue content in a model Staphylococcal plasma membrane.

Dipalmitoyl-3-aza-dehydroxy-lysylphosphatidylglycerol (DP3adLPG), is a chemically stable synthetic analogue of the bacterial lipid lysylphosphatidylglycerol (LPG), designed as a substitute for the notoriously labile native lipid in biophysical investigations. In Staphylococcus aureus, LPG is known to play a role in resistance to antibiotics by altering membrane charge properties in response to environmental stress, but little is known about how LPG influences other bilayer physicochemical properties or lateral organisation, through the formation of complexes with lipids such as phosphatidylglycerol (PG). In this study we have investigated the different phases formed by biomimetic mixtures of 3adLPG and PG in different thermotropic states, using neutron diffraction and electron microscopy. In a DPPG/DP3adLPG 70:30 mol% mixture, two distinct lamellar phases were observed below the lipid melting transition: Lß' 1 and Lß' 2 with respective periodicities of 82 and 62 Å. Increasing the proportion of DP3adLPG to mimic the effects of environmental stress led to the disappearance of the Lß' 1 phase and the formation of an inverse hexagonal phase. The compositions of these different phases were identified by investigating the thermotropic properties of the two mixtures, and probing their interaction with the antimicrobial peptide magainin 2 F5W. We propose that the observed polymorphism results from the preferential formation of either triplet PG-3adLPG-PG, or paired PG-3adLPG complexes, dependent upon the mixing proportions of the two lipids. The relevance of these findings to the role native LPG in S. aureus, are discussed with respect to their influence on antibiotic resistance and lateral membrane organisation.

Ridge-tile-like chiral topology: synthesis, resolution, and complete chiroptical characterization of enantiomers of edge-sharing binuclear square planar complexes of Ni(II) bearing achiral ligands.

Binuclear square planar Ni(II) complexes are described, formed by two tridentate ligands with two imine-nitrogens coordinating two nickel atoms. Such complexes are synthetically readily available with great structural variety and present new types of ridge-tile-like chiral compounds that are reasonably stable in the appropriate "bent" conformation. Enantiomerically pure samples of these compounds have been obtained for the first time using HPLC with a chiral stationary phase. Absolute configurations and chiroptical properties are fully characterized by ECD, VCD, ORD spectroscopy, and theoretical calculations. These new compounds with ridge-tile-like chiral topology are configurationally reasonably stable [DeltaG(double dagger) = 121.4 kJ mol(-1), t(1/2) = 14.9 h (78 degrees C, ethanol)], and therefore their chemistry, physical properties, and applications can be systematically studied.

Synthetic Antimicrobial Peptide Tuning Permits Membrane Disruption and Interpeptide Synergy.

The ribosomally produced antimicrobial peptides of bacteria (bacteriocins) represent an unexplored source of membrane-active antibiotics. We designed a library of linear peptides from a circular bacteriocin and show that pore-formation dynamics in bacterial membranes are tunable via selective amino acid substitution. We observed antibacterial interpeptide synergy indicating that fundamentally altering interactions with the membrane enables synergy. Our findings suggest an approach for engineering pore-formation through rational peptide design and increasing the utility of novel antimicrobial peptides by exploiting synergy.

A pleurocidin analogue with greater conformational flexibility, enhanced antimicrobial potency and in vivo therapeutic efficacy.

Antimicrobial peptides (AMPs) are a potential alternative to classical antibiotics that are yet to achieve a therapeutic breakthrough for treatment of systemic infections. The antibacterial potency of pleurocidin, an AMP from Winter Flounder, is linked to its ability to cross bacterial plasma membranes and seek intracellular targets while also causing membrane damage. Here we describe modification strategies that generate pleurocidin analogues with substantially improved, broad spectrum, antibacterial properties, which are effective in murine models of bacterial lung infection. Increasing peptide-lipid intermolecular hydrogen bonding capabilities enhances conformational flexibility, associated with membrane translocation, but also membrane damage and potency, most notably against Gram-positive bacteria. This negates their ability to metabolically adapt to the AMP threat. An analogue comprising D-amino acids was well tolerated at an intravenous dose of 15 mg/kg and similarly effective as vancomycin in reducing EMRSA-15 lung CFU. This highlights the therapeutic potential of systemically delivered, bactericidal AMPs.

Probing the steric barrier of nonionic surfactant vesicles with melittin.

The role of the surface polymer brush of nonionic surfactant vesicles (NSV) in inhibiting interactions with small membrane-perturbing molecules was investigated using the bee venom peptide melittin as a probe. The interaction between melittin and NSV was compared with that of distearoylphosphatidylcholine (DSPC) vesicles and sterically stabilised liposomes (SSL) containing 5 mol% pegylated distearoylphosphatidylethanolamine (DSPE.E44). The degree of melittin interaction with the various vesicles was determined by measuring peptide binding and folding, using intrinsic tryptophan fluorescence and circular dichroism respectively, in addition to monitoring the release of encapsulated carboxyfluorescein dye. NSV composed of 1,2-di-O-octadecyl-rac-glyceryl-3-(omega-dodecaethylene glycol) (2C18E12) showed a strong affinity for melittin, whilst exhibiting approximately 50% less bound peptide than SSL. 2C18E12:Chol vesicles showed reduced melittin interaction, in a manner consistent with Chol incorporation into DSPC vesicles. These results are discussed with respect to the effect of Chol on the in-plane order of 2C18E12 bilayers and consequent attenuation of hydrophobic interactions with the peptide. NSV formed from equimolar mixtures of polyoxyethylene-n-stearoyl ethers C18E2 and C18E20 showed a greater interaction with melittin than 2C18E12. However, replacing C18E20 with C18E10 was sufficient to achieve an attenuation of melittin interaction similar to that observed in 2C18E12:Chol vesicles. This indicates that the presence of surface polymer brush alone may confer resistance to melittin, provided hydrophobic interactions between the peptide and the vesicles can be minimised, through improved in-plane bilayer order.