Quantitative Binding Studies Research Articles

Structural elucidation of the mesothelin-mucin-16/CA125 interaction

Mesothelin (MSLN) is a cell-surface glycoprotein expressed at low levels on normal mesothelium but overexpressed in many cancers. Mesothelin has been implicated to play role/s in cell adhesion and multiple signaling pathways. Mucin-16/CA125 is an enormous cell-surface glycoprotein, also normally expressed on mesothelium and implicated in the progression and metastasis of several cancers, and directly binds mesothelin. However, the precise biological function/s of mesothelin and mucin-16/CA125 remain mysterious. We report protein engineering and recombinant production, qualitative and quantitative binding studies, and a crystal structure determination elucidating the molecular-level details governing recognition of mesothelin by mucin-16/CA125. The interface is small, consistent with the ∼micromolar binding constant and is free of glycan-mediated interactions. Sequence comparisons and modeling suggest that multiple mucin-16/CA125 modules can interact with mesothelin through comparable interactions, potentially generating a high degree of avidity at the cell surface to overcome the weak affinity, with implications for functioning and therapeutic interventions.

Structural basis for CEP192-mediated regulation of centrosomal AURKA

Aurora kinase A (AURKA) performs critical functions in mitosis. Thus, the activity and subcellular localization of AURKA are tightly regulated and depend on diverse factors including interactions with the multiple binding cofactors. How these different cofactors regulate AURKA to elicit different levels of activity at distinct subcellular locations and times is poorly understood. Here, we identified a conserved region of CEP192, the major cofactor of AURKA, that mediates the interaction with AURKA. Quantitative binding studies were performed to map the interactions of a conserved helix (Helix-1) within CEP192. The crystal structure of Helix-1 bound to AURKA revealed a distinct binding site that is different from other cofactor proteins such as TPX2. Inhibiting the interaction between Helix-1 and AURKA in cells led to the mitotic defects, demonstrating the importance of the interaction. Collectively, we revealed a structural basis for the CEP192-mediated AURKA regulation at the centrosome, which is distinct from TPX2-mediated regulation on the spindle microtubule.

How Choice of Model Membrane Affects Protein-Glycosphingolipid Interactions: Insights from Native Mass Spectrometry.

Interactions between glycan-binding proteins (GBPs) and glycosphingolipids (GSLs) are involved in numerous physiological and pathophysiological processes. Many model membrane systems are available for studying GBP-GSL interactions, but a systematic investigation has not been carried out on how the nature of the model membrane affects binding. In this work, we use electrospray ionization mass spectrometry (ESI-MS), both direct and competitive assays, to measure the binding of cholera toxin B subunit homopentamer (CTB5) to GM1 ganglioside in liposomes, bilayer islands [styrene maleic acid lipid particles (SMALPs), nanodiscs (NDs), and picodiscs (PDs)], and micelles. We find that direct ESI-MS analysis of CTB5 binding to GM1 is unreliable due to non-uniform response factors, incomplete extraction of bound GM1 in the gas phase, and nonspecific CTB5-GM1 interactions. Conversely, indirect proxy ligand ESI-MS measurements show that the intrinsic (per binding site) association constants of CTB5 for PDs, NDs, and SMALPs are similar and comparable to the affinity of soluble GM1 pentasaccharide (GM1os). The observed affinity decreases with increasing GM1 content due to molecular crowding stemming from GM1 clustering. Unlike the smaller model membranes, the observed affinity of CTB5 toward GM1 liposomes is ∼10-fold weaker than GM1os and relatively insensitive to the GM1 content. GM1 glycomicelles exhibit the lowest affinity, ∼35-fold weaker than GM1os. Together, the results highlight experimental design considerations for quantitative GBP-GSL binding studies involving multisubunit GBPs and factors to consider when comparing results obtained with different membrane systems. Notably, they suggest that bilayer islands with a low percentage of GSL, wherein clustering is minimized, are ideal for assessing intrinsic strength of GBP-GSL interactions in a membrane environment, while binding to liposomes, which is sub-optimal due to extensive clustering, may be more representative of authentic cellular environments.

Characterization of the Interaction of Nerve Agent Mimics with Selected Synthetic Receptors

Qualitative NMR spectroscopic and quantitative calorimetric binding studies were performed to characterize the interaction of nontoxic mimics of the V-type nerve agent VX (O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate) and the Novichok nerve agent A-234 (ethyl (1-(diethylamino)ethylidene)phosphoramidofluoridate) with a series of receptors in 100 mM aqueous phosphate buffer at pH 7.4 and 37 °C. These investigations provided information about the preferred geometry with which the nerve agent mimics are included into the receptor cavities and about the stability of the complexes formed. According to the results, the positively charged VX mimic prefers to bind to cation receptors such as sulfonated calixarenes and an acyclic cucurbituril but does not noticeably interact with cyclodextrins. While binding to the acyclic cucurbituril is stronger than that to calixarenes, the mode of inclusion into the sulfonatocalix[4]arene cavity is better suited for the development of scavengers that bind and detoxify V-type nerve agents. The neutral Novichok mimic, on the other hand, only interacts with the acyclic cucurbituril with a strength required for scavenger development. These binding studies thus provided guidelines for the further development of nerve agent scavengers.

Inter-Leaflet Phospholipid Exchange Impacts the Ligand Density Available for Protein Binding at Supported Lipid Bilayers.

Phospholipid bilayers formed at solid-liquid interfaces have garnered interest as mimics of cell membranes to model association reactions of proteins with lipid bilayer-tethered ligands. Despite the importance of understanding how ligand density in a lipid bilayer impacts the protein-ligand association response, relating the ligand-modified lipid fraction to the absolute density of solution-accessible ligands in a lipid bilayer remains a challenge in interfacial quantitative analysis. In this work, confocal Raman microscopy is employed to quantify the association of anti-biotin IgG with a small fraction of biotinylated lipids dispersed in either gel-phase or liquid-crystalline supported lipid bilayers deposited on the interior surfaces of wide-pore silica surfaces. We examine the question of whether inter-leaflet lipid translocation contributes to the population of solution-accessible biotin ligands on the distal leaflet of a supported lipid bilayer by comparing their protein accumulation response with ligands dispersed in lipid monolayers on nitrile-derivatized silica surfaces. The binding of the antibody to biotin ligands dispersed in gel-phase bilayers exhibited an equivalent biotin coverage response as the accumulation of IgG onto gel-phase monolayers, indicating that gel-phase bilayer symmetry was preserved. This result contrasts with the ∼60% greater anti-biotin capture observed at fluid-phase bilayers compared to fluid-phase monolayers prepared at equivalent biotin fractions. This enhanced protein capture is attributed to biotin-capped lipids being transferred from the surface-associated proximal leaflet of the bilayer to the solution-exposed distal leaflet by the inter-leaflet exchange or lipid flip-flop, a facile process in fluid-phase supported lipid bilayers. The results suggest caution in interpreting the results of quantitative studies of protein binding to lipid-tethered ligands dispersed in fluid-phase phospholipid bilayers.

A tunable LIC1-adaptor interaction modulates dynein activity in a cargo-specific manner

Cytoplasmic dynein-1 (dynein) is the motor responsible for most retrograde transport of cargoes along microtubules in eukaryotic cells, including organelles, mRNA and viruses. Cargo selectivity and activation of processive motility depend on a group of so-called “activating adaptors” that link dynein to its general cofactor, dynactin, and cargoes. The mechanism by which these adaptors regulate dynein transport is poorly understood. Here, based on crystal structures, quantitative binding studies, and in vitro motility assays, we show that BICD2, CRACR2a, and HOOK3, representing three subfamilies of unrelated adaptors, interact with the same amphipathic helix of the dynein light intermediate chain-1 (LIC1). While the hydrophobic character of the interaction is conserved, the three adaptor subfamilies use different folds (coiled-coil, EF-hand, HOOK domain) and different surface contacts to bind the LIC1 helix with affinities ranging from 1.5 to 15.0 μM. We propose that a tunable LIC1-adaptor interaction modulates dynein’s motility in a cargo-specific manner.

Novel pyrano 1,3 oxazine based ligand inhibits the epigenetic reader hBRD2 in glioblastoma.

Glioblastoma (GBM) is the most common primary brain malignancy, rarely amenable to treatment with a high recurrence rate. GBM are prone to develop resistance to the current repertoire of drugs, including the first-line chemotherapeutic agents with frequent recurrence, limiting therapeutic success. Recent clinical data has evidenced the BRD2 and BRD4 of the BET family proteins as the new druggable targets against GBM. In this relevance, we have discovered a compound (pyrano 1,3 oxazine derivative; NSC 328111; NS5) as an inhibitor of hBRD2 by the rational structure-based approach. The crystal structure of the complex, refined to 1.5 Å resolution, revealed that the NS5 ligand significantly binds to the N-terminal bromodomain (BD1) of BRD2 at the acetylated (Kac) histone binding site. The quantitative binding studies, by SPR and MST assay, indicate that NS5 binds to BD1 of BRD2 with a KD value of ∼1.3 µM. The cell-based assay, in the U87MG glioma cells, confirmed that the discovered compound NS5 significantly attenuated proliferation and migration. Furthermore, evaluation at the translational level established significant inhibition of BRD2 upon treatment with NS5. Hence, we propose that the novel lead compound NS5 has an inhibitory effect on BRD2 in glioblastoma.

The C-terminal tails of the mitochondrial transcription factors Mtf1 and TFB2M are part of an autoinhibitory mechanism that regulates DNA binding

The structurally homologous Mtf1 and TFB2M proteins serve as transcription initiation factors of mitochondrial RNA polymerases in Saccharomyces cerevisiae and humans, respectively. These transcription factors directly interact with the nontemplate strand of the transcription bubble to drive promoter melting. Given the key roles of Mtf1 and TFB2M in promoter-specific transcription initiation, it can be expected that the DNA binding activity of the mitochondrial transcription factors is regulated to prevent DNA binding at inappropriate times. However, little information is available on how mitochondrial DNA transcription is regulated. While studying C-terminal (C-tail) deletion mutants of Mtf1 and TFB2M, we stumbled upon a finding that suggested that the flexible C-tail region of these factors autoregulates their DNA binding activity. Quantitative DNA binding studies with fluorescence anisotropy-based titrations revealed that Mtf1 with an intact C-tail has no affinity for DNA but deletion of the C-tail greatly increases Mtf1's DNA binding affinity. Similar observations were made with TFB2M, although autoinhibition by the C-tail of TFB2M was not as complete as in Mtf1. Analysis of available TFB2M structures disclosed that the C-tail engages in intramolecular interactions with the DNA binding groove in the free factor, which, we propose, inhibits its DNA binding activity. Further experiments showed that RNA polymerase relieves this autoinhibition by interacting with the C-tail and engaging it in complex formation. In conclusion, our biochemical and structural analyses reveal autoinhibitory and activation mechanisms of mitochondrial transcription factors that regulate their DNA binding activities and aid in specific assembly of transcription initiation complexes.

Munc13 Recruits SNAP25 to Facilitate SNARE Complex Assembly

Synaptic vesicle exocytosis in neurons requires SNARE complex formation during which VAMP2 from vesicles zippers with t-SNAREs (SNAP25 and Syntaxin1a) on the plasma membrane. Munc13-1 acts as a chaperone for this process by both tethering vesicles to PIP2-microdomains on the plasma membrane containing Syntaxin1a and by activating Syntaxin1a. How membrane-anchored SNAP25 enters this reaction is unknown. Here we report a novel function of Munc13 in the recruitment of SNAP25 to initiate the SNARE priming process. Using biochemical analyses, including quantitative binding studies, we show the MUN domain of Munc13 directly binds the SNAP25 loop region (connecting the two helices). Indeed, replacing the SNAP25 loop with the loop region of closely-related SNAP23 or with a non-specific GGGGS repeat sequence abrogated the Munc13 interaction. Interestingly, the Munc13 mutant NF->AA (1128/1131) that has been shown to be incapable of activating Syntaxin1a, still bound SNAP25 suggesting the binding sites on Munc13 for Syntaxin1a and SNAP25 are different. Furthermore, total internal reflection fluorescence (TIRF) microscopy analysis revealed that Munc13 co-clusters with SNAP25 on a supported lipid bilayer setup and these clusters typically contain 5-6 molecules each of Munc13 and SNAP25. This co-clustering was absent in the GGGGS mutant. Overall, our data suggest Munc13 recruits SNAP25 to the site of vesicle docking via direct MUN-SNAP25 loop interaction and by virtue of increasing its local concentration, it facilitates the initiation of the SNARE complex formation.

Mechanism of IRSp53 inhibition by 14-3-3

Filopodia are precursors of dendritic spines and polarized cell migration. The I-BAR-domain protein IRSp53 is a key regulator of filopodia dynamics that couples Rho-GTPase signaling to cytoskeleton and membrane remodeling, playing essential roles in neuronal development and cell motility. Here, we describe the structural-functional basis for 14-3-3-dependent inhibition of IRSp53. Phosphoproteomics, quantitative binding and crystallographic studies demonstrate that 14-3-3 binds to two pairs of phosphorylation sites in IRSp53. Using bicistronic expression, we obtain an IRSp53 heterodimer in which only one subunit is phosphorylated, and show that each subunit of IRSp53 independently binds one 14-3-3 dimer. A FRET-sensor assay using natively phosphorylated IRSp53 reveals opposite conformational changes upon binding of activatory (Cdc42, Eps8) or inhibitory (14-3-3) inputs. Finally, we show that 14-3-3 inhibits IRSp53 binding to membranes. Collectively, our findings support a mechanism whereby phosphorylation-dependent inhibition of IRSp53 by 14-3-3 counters membrane binding and interactions with Cdc42 and downstream cytoskeletal effectors.

TiO2 Nanoparticles Catalyze Oxidation of Huntingtin Exon 1-Derived Peptides Impeding Aggregation: A Quantitative NMR Study of Binding and Kinetics.

Polyglutamine expansion within the N-terminal region of the huntingtin protein results in the formation of intracellular aggregates responsible for Huntington's disease, a fatal neurodegenerative condition. The interaction between TiO2 nanoparticles and huntingtin peptides comprising the N-terminal amphiphilic domain without (httNT) or with (httNTQ10) a ten-residue C-terminal polyglutamine tract, is investigated by NMR spectroscopy. TiO2 nanoparticles decrease aggregation of httNTQ10 by catalyzing the oxidation of Met7 to a sulfoxide, resulting in an aggregation-incompetent peptide. The oxidation agent is hydrogen peroxide generated on the surface of the TiO2 nanoparticles either by UV irradiation or at low steady-state levels in the dark. The binding kinetics of nonaggregating httNT to TiO2 nanoparticles is characterized by quantitative analysis of 15N dark state exchange saturation transfer and lifetime line broadening NMR data. Binding involves a sparsely populated intermediate that experiences hindered rotational diffusion relative to the free state. Catalysis of methionine oxidation within the N-terminal domain of the huntingtin protein may potentially provide a strategy for delaying the onset of Huntington's disease.

Quantitative studies of DNA binding with trans complexes of PtII and PdII featuring tetrazolylacetic acids and their derivatives as ligands

The interaction of calf thymus DNA (CT DNA) with trans-[PtCl2L2] and trans-[PdCl2L2] complexes (L–5-methyl-1H-tetrazol-1-ylacetic acid and its ethyl, butyl, and isobutyl esters; ethyl esters of 2-R-2H-tetrazol-5-ylacetic acids, R = But, CH2CH2OH) have been quantitatively studied by a spectrophotometric method. The binding constants (Kb) of the tetrazolecontaining coordination compounds with CT DNA at pH 7.2 and 298 K are in the range of (3.67–5.93)•105 mol L–1 for the PdII complexes and (7.82–13.30)•105 mol L–1 for and the PtII complexes, respectively. The Kb values, as well as negative values of ΔGb (–(31.7–34.9) kJ mol–1) indicate the effective binding between the studied complexes and CT DNA.

A conserved interaction of the dynein light intermediate chain with dynein-dynactin effectors necessary for processivity

Cytoplasmic dynein is the major minus-end-directed microtubule-based motor in cells. Dynein processivity and cargo selectivity depend on cargo-specific effectors that, while generally unrelated, share the ability to interact with dynein and dynactin to form processive dynein–dynactin-effector complexes. How this is achieved is poorly understood. Here, we identify a conserved region of the dynein Light Intermediate Chain 1 (LIC1) that mediates interactions with unrelated dynein–dynactin effectors. Quantitative binding studies map these interactions to a conserved helix within LIC1 and to N-terminal fragments of Hook1, Hook3, BICD2, and Spindly. A structure of the LIC1 helix bound to the N-terminal Hook domain reveals a conformational change that creates a hydrophobic cleft for binding of the LIC1 helix. The LIC1 helix competitively inhibits processive dynein–dynactin-effector motility in vitro, whereas structure-inspired mutations in this helix impair lysosomal positioning in cells. The results reveal a conserved mechanism of effector interaction with dynein–dynactin necessary for processive motility.

Monoamide Derivatives of EDTA Incorporating Pendent Carboxylates or Pyridyls: Synthesis, Metal Binding, and Crystal Structure of a Dinuclear Ca2+ Complex Featuring Bridging Na+ Ions

AbstractEDTA is a powerful, cheap and widely‐used chelating unit for a large range of metal ions. To link it covalently to other molecules, the formation of an amide bond using one of the carboxylates is an attractive and simple approach, even though it may compromise metal ion binding as one of the four carboxylate donors is lost. Here we undertake a quantitative study of the metal ion binding of two new mono‐amide derivatives of EDTA, namely AmGly1 and AmPy1, featuring an additional coordinating carboxylate or pyridyl group in the amide, respectively. The compounds are conveniently synthesised through alkylation of the tris‐t‐butyl ester of ethylenediamine‐triacetic acid with the appropriate α‐chloroamide. The switch from carboxylate to amide is found to impact Fe3+ binding the most amongst the metal ions examined (Mg2+, Ca2+, Mn2+, Fe3+, Zn2+), with a drop in log K(ML) of around 10 units in aqueous solution. The affinities for the other metals are less severely affected. Nevertheless, at the physiologically relevant pH of 7.4, their ability to bind Fe3+ is only reduced by around 6 log units when pFe3+ is considered (where [L]:[Fe3+] = 10:1). AmGly1 forms a dinuclear Ca2+ complex which has been characterised crystallographically. It is a C2‐symmetric 2:2 complex featuring a dicapped octahedral coordination geometry around Ca2+. In the crystal, the Ca2(AmGly1)2 units are linked via hydrated Na+ ions coordinated by a number of the oxygen atoms, including the pendent carboxylate.

Characterization of [3H] oxymorphone binding sites in mouse brain: Quantitative autoradiography in opioid receptor knockout mice

Oxymorphone, one of oxycodone’s metabolic products, is a potent opioid receptor agonist which is thought to contribute to the analgesic effect of its parent compound and may have high potential abuse liability. Nonetheless, the in vivo pharmacological binding profile of this drug is still unclear. This study uses mice lacking mu (MOP), kappa (KOP) or delta (DOP) opioid receptors as well as mice lacking all three opioid receptors to provide full characterisation of oxymorphone binding sites in the brain. Saturation binding studies using [3H]oxymorphone revealed high affinity binding sites in mouse brain displaying Kd of 1.7nM and Bmax of 147fmol/mg. Furthermore, we performed quantitative autoradiography binding studies using [3H]oxymorphone in mouse brain. The distribution of [3H]oxymorphone binding sites was found to be similar to the selective MOP agonist [3H]DAMGO in the mouse brain. [3H]Oxymorphone binding was completely abolished across the majority of the brain regions in mice lacking MOP as well as in mice lacking all three opioid receptors. DOP and KOP knockout mice retained [3H]oxymorphone binding sites suggesting oxymorphone may not target DOP or KOP. These results confirm that the MOP, and not the DOP or the KOP is the main high affinity binding target for oxymorphone.

Oxoanion binding to a cyclic pseudopeptide containing 1,4-disubstituted 1,2,3-triazole moieties.

A macrocyclic pseudopeptide 3 is described featuring three amide groups and three 1,4-disubstituted 1,2,3-triazole units along the ring. This pseudopeptide was designed such that the amide NH groups and the triazole CH groups converge toward the cavity, thus creating an environment well suited for anion recognition. Conformational studies in solution combined with X-ray crystallography confirmed this preorganisation. Solubility of 3 restricted binding studies to organic media such as 5 vol% DMSO/acetone or DMSO/water mixtures with a water content up to 5 vol%. These binding studies demonstrated that 3 binds to a variety of inorganic anions in DMSO/acetone including chloride, nitrate, sulfate, and dihydrogenphosphate anions. In the more competitive DMSO/water mixtures, only affinity to the more strongly coordinating oxoanions is retained. Quantitative binding studies showed that dihydrogen phosphate complexation in DMSO/water involves the dimer of the H2PO4- anion. By contrast, sulfate and hydrogenpyrophosphate complexation involves a stepwise process comprising formation of a 1 : 1 complex followed by a 2R : 1A complex in which two molecules of 3 (R) bind to a single anion (A). While the second binding equilibrium is associated with a much smaller stability constant in comparison with the first one in the case of sulfate complexation, the two binding constants are of similar magnitude in the case of the hydrogenpyrophosphate anion. Formation of the 2R : 1A complex was attributed to the fact that the cavity size and rigidity of 3 prevents saturation of all hydrogen acceptor sites on the anionic guests.

Multifaceted Histone H3 Methylation and Phosphorylation Readout by the Plant Homeodomain Finger of Human Nuclear Antigen Sp100C

The decoding of histone post-translational modifications by chromatin-binding modules ("readers") constitutes one major mechanism of epigenetic regulation. Nuclear antigen Sp100 (SPECKLED, 100 kDa), a constitutive component of the promyelocytic leukemia nuclear bodies, plays key roles in intrinsic immunity and transcriptional repression. Sp100C, a splicing isoform specifically up-regulated upon interferon stimulation, harbors a unique tandem plant homeodomain (PHD) finger and bromodomain at its C terminus. Combining structural, quantitative binding, and cellular co-localization studies, we characterized Sp100C PHD finger as an unmethylated histone H3 Lys(4) (H3K4me0) reader that tolerates histone H3 Thr(3) phosphorylation (H3T3ph), histone H3 Lys(9) trimethylation (H3K9me3), and histone H3 Ser(10) phosphorylation (H3S10ph), hallmarks associated with the mitotic chromosome. In contrast, whereas H3K4me0 reader activity is conserved in Sp140, an Sp100C paralog, the multivalent tolerance of H3T3ph, H3K9me3, and H3S10ph was lost for Sp140. The complex structure determined at 2.1 Å revealed a highly coordinated lysine ϵ-amine recognition sphere formed by an extended N-terminal motif for H3K4me0 readout. Interestingly, reader pocket rigidification by disulfide bond formation enhanced H3K4me0 binding by Sp100C. An additional complex structure solved at 2.7 Å revealed that H3T3ph is recognized by the arginine residue, Arg(713), that is unique to the PHD finger of Sp100C. Consistent with a restrictive cellular role of Sp100C, these results establish a direct chromatin targeting function of Sp100C that may regulate transcriptional gene silencing and promyelocytic leukemia nuclear body-mediated intrinsic immunity in response to interferon stimulation.

Binding of Substrates to the Central Pore of the Vps4 ATPase Is Autoinhibited by the Microtubule Interacting and Trafficking (MIT) Domain and Activated by MIT Interacting Motifs (MIMs)

The endosomal sorting complexes required for transport (ESCRT) pathway drives reverse topology membrane fission events within multiple cellular pathways, including cytokinesis, multivesicular body biogenesis, repair of the plasma membrane, nuclear membrane vesicle formation, and HIV budding. The AAA ATPase Vps4 is recruited to membrane necks shortly before fission, where it catalyzes disassembly of the ESCRT-III lattice. The N-terminal Vps4 microtubule-interacting and trafficking (MIT) domains initially bind the C-terminal MIT-interacting motifs (MIMs) of ESCRT-III subunits, but it is unclear how the enzyme then remodels these substrates in response to ATP hydrolysis. Here, we report quantitative binding studies that demonstrate that residues from helix 5 of the Vps2p subunit of ESCRT-III bind to the central pore of an asymmetric Vps4p hexamer in a manner that is dependent upon the presence of flexible nucleotide analogs that can mimic multiple states in the ATP hydrolysis cycle. We also find that substrate engagement is autoinhibited by the Vps4p MIT domain and that this inhibition is relieved by binding of either Type 1 or Type 2 MIM elements, which bind the Vps4p MIT domain through different interfaces. These observations support the model that Vps4 substrates are initially recruited by an MIM-MIT interaction that activates the Vps4 central pore to engage substrates and generate force, thereby triggering ESCRT-III disassembly.

Solvent effects upon guest binding and dynamics of a Fe(II)4L4 cage.

Solvent-dependent host-guest chemistry and favoring of otherwise disfavored conformations of large guests has been achieved with an adaptive, self-assembled Fe(II)4L4 coordination cage. Depending on the counterion, this face-capped tetrahedral capsule is soluble either in water or in acetonitrile and shows a solvent-dependent preference for encapsulation of certain classes of guest molecules. Quantitative binding studies were undertaken, revealing that both aromatic and aliphatic guests bind in water, whereas only aliphatic guests bind in acetonitrile. The flexibility of its subcomponent building blocks allows this cage to expand or contract upon guest binding, as studied by VT-NMR, thereby ensuring strong binding of both small and large guests. Upon encapsulation, large guest molecules can adopt conformations which are not thermodynamically favored in the free state. In addition, the chirotopic inner phase of the cage renders enantiotopic guest proton signals diastereotopic in specific cases.

Delineation of interfaces on human alpha-defensins critical for human adenovirus and human papillomavirus inhibition.

Human α-defensins are potent anti-microbial peptides with the ability to neutralize bacterial and viral targets. Single alanine mutagenesis has been used to identify determinants of anti-bacterial activity and binding to bacterial proteins such as anthrax lethal factor. Similar analyses of α-defensin interactions with non-enveloped viruses are limited. We used a comprehensive set of human α-defensin 5 (HD5) and human neutrophil peptide 1 (HNP1) alanine scan mutants in a combination of binding and neutralization assays with human adenovirus (AdV) and human papillomavirus (HPV). We have identified a core of critical hydrophobic residues that are common determinants for all of the virus-defensin interactions that were analyzed, while specificity in viral recognition is conferred by specific surface-exposed charged residues. The hydrophobic residues serve multiple roles in maintaining the tertiary and quaternary structure of the defensins as well as forming an interface for virus binding. Many of the important solvent-exposed residues of HD5 group together to form a critical surface. However, a single discrete binding face was not identified for HNP1. In lieu of whole AdV, we used a recombinant capsid subunit comprised of penton base and fiber in quantitative binding studies and determined that the anti-viral potency of HD5 was a function of stoichiometry rather than affinity. Our studies support a mechanism in which α-defensins depend on hydrophobic and charge-charge interactions to bind at high copy number to these non-enveloped viruses to neutralize infection and provide insight into properties that guide α-defensin anti-viral activity.