The C-Terminus of Panusin, a Lobster ß-Defensin, Is Crucial for Optimal Antimicrobial Activity and Serum Stability.

ß-defensins are one of the most abundant and studied families of antimicrobial peptides (AMPs). Because of their selective toxicity to bacterial membranes and a broad spectrum of microbicidal action, ß-defensins are regarded as potential therapeutic agents. This work focuses on a ß-defensin-like AMP from the spiny lobster Panulirus argus (hereafter referred to as panusin or PaD). This AMP is structurally related to mammalian defensins via the presence of an αß domain stabilized by disulfide bonds. Previous studies of PaD suggest that its C-terminus (Ct_PaD) contains the main structural determinants of antibacterial activity. To confirm this hypothesis, we made synthetic versions of PaD and Ct_PaD to determine the influence of the C-terminus on antimicrobial activity, cytotoxicity, proteolytic stability, and 3D structure. After successful solid-phase synthesis and folding, antibacterial assays of both peptides showed truncated Ct_PaD to be more active than native PaD, confirming the role of the C-terminus in activity and suggesting that cationic residues in that region enhance binding to negatively charged membranes. On the other hand, neither PaD nor Ct_PaD were hemolytic or cytotoxic in human cells. Proteolysis in human serum was also studied, showing high (>24 h) t1/2 values for PaD and lower but still considerable for Ct_PaD, indicating that the missing native disulfide bond in Ct_PaD alters protease resistance, albeit not decisively. NMR-2D experiments in water agree with the results obtained by circular dichroism (CD), where in SDS micelles, CD showed both peptides adopting an increasingly ordered structure in a hydrophobic environment, in tune with their ability to perturb bacterial membrane systems. In conclusion, while the ß-defensin features of PaD are confirmed as advantageous in terms of antimicrobial activity, toxicity, and protease stability, the results of the present work suggest that these same features are preserved, even enhanced, in the structurally simpler Ct_PaD, which must therefore be viewed as a valuable lead for the development of novel anti-infectives.

eIF4G1 N-terminal intrinsically disordered domain is a multi-docking station for RNA, Pab1, Pub1, and self-assembly.

Yeast eIF4G1 interacts with RNA binding proteins (RBPs) like Pab1 and Pub1 affecting its function in translation initiation and stress granules formation. We present an NMR and SAXS study of the N-terminal intrinsically disordered region of eIF4G1 (residues 1-249) and its interactions with Pub1, Pab1 and RNA. The conformational ensemble of eIF4G11-249 shows an α-helix within the BOX3 conserved element and a dynamic network of fuzzy π-π and π-cation interactions involving arginine and aromatic residues. The Pab1 RRM2 domain interacts with eIF4G1 BOX3, the canonical interaction site, but also with BOX2, a conserved element of unknown function to date. The RNA1 region interacts with RNA through a new RNA interaction motif and with the Pub1 RRM3 domain. This later also interacts with eIF4G1 BOX1 modulating its intrinsic self-assembly properties. The description of the biomolecular interactions involving eIF4G1 to the residue detail increases our knowledge about biological processes involving this key translation initiation factor.

Structure and function of the N-terminal extension of the formin INF2.

In INF2-a formin linked to inherited renal and neurological disease in humans-the DID is preceded by a short N-terminal extension of unknown structure and function. INF2 activation is achieved by Ca2+-dependent association of calmodulin (CaM). Here, we show that the N-terminal extension of INF2 is organized into two α-helices, the first of which is necessary to maintain the perinuclear F-actin ring and normal cytosolic F-actin content. Biochemical assays indicated that this helix interacts directly with CaM and contains the sole CaM-binding site (CaMBS) detected in INF2. The residues W11, L14 and L18 of INF2, arranged as a 1-4-8 motif, were identified as the most important residues for the binding, W11 being the most critical of the three. This motif is conserved in vertebrate INF2 and in the human population. NMR and biochemical analyses revealed that CaM interacts directly through its C-terminal lobe with the INF2 CaMBS. Unlike control cells, INF2 KO cells lacked the perinuclear F-actin ring, had little cytosolic F-actin content, did not respond to increased Ca2+ concentrations by making more F-actin, and maintained the transcriptional cofactor MRTF predominantly in the cytoplasm. Whereas expression of intact INF2 restored all these defects, INF2 with inactivated CaMBS did not. Our study reveals the structure of the N-terminal extension, its interaction with Ca2+/CaM, and its function in INF2 activation.

Inter-hairpin linker sequences determine the structure of the ßß-solenoid fold: a "bottom-up" study of pneumococcal LytA choline-binding module.

The ßß-solenoid structures are part of many proteins involved in the recognition of bacterial cell wall. They are elongated polypeptides consisting of repeated ß-hairpins connected by linker sequences and disposed around a superhelical axis stabilised by short-range interactions. Among the most studied ßß-solenoids are those belonging to the family of choline-binding modules (CBMs) from the respiratory pathogen Streptococcus pneumoniae (pneumococcus) and its bacteriophages, and their properties have been employed to develop several biotechnological and biomedical tools. We have carried out a theoretical, spectroscopic and thermodynamic study of the ßß-solenoid structure of the CBM from the pneumococcal LytA autolysin using peptides of increasing length containing 1-3 repeats of this structure. Our results show that hints of native-like tertiary structure are only observed with a minimum of three ß-hairpins, corresponding to one turn of the solenoid superhelix, and identify the linker sequences between hairpins as the major directors of the solenoid folding. This study paves the way for the rational structural engineering of ßß-solenoids aimed to find novel applications.

DD04107-Derived neuronal exocytosis inhibitor peptides: Evidences for synaptotagmin-1 as a putative target.

The analgesic peptide DD04107 (Pal-EEMQRR-NH2) and its acetylated analogue inhibit α-calcitonin gene-related peptide (α-CGRP) exocytotic release from primary sensory neurons. Examining the crystal structure of the SNARE-Synaptotagmin-1(Syt1) complex, we hypothesized that these peptides could inhibit neuronal exocytosis by binding to Syt1, hampering at least partially its interaction with the SNARE complex. To address this hypothesis, we first interrogate the role of individual side-chains on the inhibition of α-CGRP release, finding that E1, M3, Q4 and R6 residues were crucial for activity. CD and NMR conformational analysis showed that linear peptides have tendency to adopt α-helical conformations, but the results with cyclic analogues indicated that this secondary structure is not needed for activity. Isothermal titration calorimetry (ITC) measurements demonstrate a direct interaction of some of these peptides with Syt1-C2B domain, but not with Syt7-C2B region, indicating selectivity. As expected for a compound able to inhibit α-CGRP release, cyclic peptide derivative Pal-E-cyclo[EMQK]R-NH2 showed potent in vivo analgesic activity, in a model of inflammatory pain. Molecular dynamics simulations provided a model consistent with KD values for the interaction of peptides with Syt1-C2B domain, and with their biological activity. Altogether, these results identify Syt1 as a potential new analgesic target.

Insights Into the Micelle-Induced ß-Hairpin-to-α-Helix Transition of a LytA-Derived Peptide by Photo-CIDNP Spectroscopy.

A choline-binding module from pneumococcal LytA autolysin, LytA239-252, was reported to have a highly stable nativelike ß-hairpin in aqueous solution, which turns into a stable amphipathic α-helix in the presence of micelles. Here, we aim to obtain insights into this DPC-micelle triggered ß-hairpin-to-α-helix conformational transition using photo-CIDNP NMR experiments. Our results illustrate the dependency between photo-CIDNP phenomena and the light intensity in the sample volume, showing that the use of smaller-diameter (2.5 mm) NMR tubes instead of the conventional 5 mm ones enables more efficient illumination for our laser-diode light setup. Photo-CIDNP experiments reveal different solvent accessibility for the two tyrosine residues, Y249 and Y250, the latter being less accessible to the solvent. The cross-polarization effects of these two tyrosine residues of LytA239-252 allow for deeper insights and evidence their different behavior, showing that the Y250 aromatic side chain is involved in a stronger interaction with DPC micelles than Y249 is. These results can be interpreted in terms of the DPC micelle disrupting the aromatic stacking between W241 and Y250 present in the nativelike ß-hairpin, hence initiating conversion towards the α-helix structure. Our photo-CIDNP methodology represents a powerful tool for observing residue-level information in switch peptides that is difficult to obtain by other spectroscopic techniques.

Structural Insights into ß-arrestin/CB1 Receptor Interaction: NMR and CD Studies on Model Peptides.

Activation of the cannabinoid CB1 receptor induces different cellular signaling cascades through coupling to different effector proteins (G-proteins and ß-arrestins), triggering numerous therapeutic effects. Conformational changes and rearrangements at the intracellular domain of this GPCR receptor that accompany ligand binding dictate the signaling pathways. The GPCR-binding interface for G proteins has been extensively studied, whereas ß-arrestin/GPCR complexes are still poorly understood. To gain knowledge in this direction, we designed peptides that mimic the motifs involved in the putative interacting region: ß-arrestin1 finger loop and the transmembrane helix 7-helix 8 (TMH7-H8) elbow located at the intracellular side of the CB1 receptor. According to circular dichroism and NMR data, these peptides form a native-like, helical conformation and interact with each other in aqueous solution, in the presence of trifluoroethanol, and using zwitterionic detergent micelles as membrane mimics. These results increase our understanding of the binding mode of ß-arrestin and CB1 receptor and validate minimalist approaches to structurally comprehend complex protein systems.

Influence of the Ovine Genital Tract Microbiota on the Species Artificial Insemination Outcome. A Pilot Study in Commercial Sheep Farms.

To date, there is a lack of research into the vaginal and sperm microbiome and its bearing on artificial insemination (AI) success in the ovine species. Using hypervariable regions V3-V4 of the 16S rRNA, we describe, for the first time, the combined effect of the ovine microbiome of both females (50 ewes belonging to five herds) and males (five AI rams from an AI center) on AI outcome. Differences in microbiota abundance between pregnant and non-pregnant ewes and between ewes carrying progesterone-releasing intravaginal devices (PRID) with or without antibiotic were tested at different taxonomic levels. The antibiotic treatment applied with the PRID only altered Streptobacillus genus abundance, which was significantly lower in ewes carrying PRID with antibiotic. Mageebacillus, Histophilus, Actinobacilllus and Sneathia genera were significantly less abundant in pregnant ewes. In addition, these genera were more abundant in two farms with higher AI failure. Species of these genera such as Actinobacillus seminis and Histophilus somni have been associated with reproductive disorders in the ovine species. These genera were not present in the sperm samples of AI rams, but were found in the foreskin samples of rams belonging to herd 2 (with high AI failure rate) indicating that their presence in ewes' vagina could be due to prior transmission by natural mating with rams reared in the herd.

Peptides Mimicking the ß7/ß8 Loop of HIV-1 Reverse Transcriptase p51 as "Hotspot-Targeted" Dimerization Inhibitors.

A conformationally constrained short peptide designed to target a protein-protein interaction hotspot in HIV-1 reverse transcriptase (RT) disrupts p66-p51 interactions and paves the way to the development of novel RT dimerization inhibitors.

Effect of Phosphorylation on the Structural Behaviour of Peptides Derived from the Intrinsically Disordered C-Terminal Domain of Histone H1.0.

To investigate the structural impact of phosphorylation on the human histone H1.0 C-terminal domain, we performed NMR structural studies of model peptides containing a single phosphorylation site: T118 -H1.0 (T118 PKK motif) and T140 -H1.0 (T140 PVK motif). Both model peptides are mainly disordered in aqueous solution in their non-phosphorylated and phosphorylated forms, but become structured in the presence of trifluoroethanol. The peptides T118 -H1.0 and pT118 -H1.0 contain two helical regions, a long amphipathic α helix spanning residues 104-115 and a short α/310 helix (residues 119-123), that are almost perpendicular in T118 -H1.0 but have a poorly defined orientation in pT118 -H1.0. Peptides T140 -H1.0 and pT140 -H1.0 form very similar α helices between residues 141-147. The TPKK and TPVK motifs show the same backbone conformation, but differ in their side-chain contacts; the Thr and pThr side chains interact with the i+2 Lys side chain in the TPKK motif, and with the i+3 Lys side chain in the TPVK motif. The pT phosphate group in pT118 -H1.0 and pT140 -H1.0 has pKa values below the intrinsic values, which can be explained by non-specific charge-charge interactions with nearby Lys. The non-polar Val in the TPVK motif accounts for the pT140 pKa being closer to the intrinsic pKa value than the pT118 pKa . Altogether, these results validate that minimalist strategies using model peptides can provide structural details difficult to obtain in short-lived intrinsically disordered proteins and domains.

A new allele in the BMP15 gene (FecXRA) that affects prolificacy co-segregates with FecXR and FecXGR in Rasa aragonesa sheep.

A FecX-mutated allele called FecXR in the BMP15 gene has been described in Rasa aragonesa sheep. FecXR causes increased prolificacy when heterozygous and sterility when homozygous in ewes. However, highly prolific ewes without the FecXR allele have been found in this breed. Therefore, a genome-wide association study (GWAS) was performed in 158 ewes (tail H: N = 73, mean prolificacy ± standard deviation = 2.14 ± 0.26; tail L: N = 85, mean prolificacy = 1.06 ± 0.08) with the Ovine HD SNP BeadChip. In this analysis, the FecXGR allele was found to have genome-wide significance associated with prolificacy, first described in the Grivette sheep breed. We also identified a novel polymorphism in exon 2 of BMP15 in 9 high prolific ewes by Sanger sequencing. This new mutation, called FecXRA, is a SNP (Oar3.1_X: g. 50970948C > T; NM_001114767.1: c.1172C > T) that produces an amino acid substitution (ENSOART00000010201: p.T400I) that is predicted to be deleterious and to alter the predicted secondary structure of the mature protein. To confirm if this SNP had any the effect on prolificacy, we genotyped sires with known EBVs (Estimated Breeding Values), finding one hemizygous sire for the FecXRA allele with the highest EBV in the breeding program (effect on litter size at + 0.39 lamb per lambing). A very low frequency, ranging from 0.13 to 2%, was found for the FecXGR and FecXRA alleles in 3428 animals belonging to four different flocks. Finally, an association study was performed to validate and quantify the effects of the FecXGR and FecXRA alleles. Significant increased prolificacy of 0.52 ± 0.05, 0.42 ± 0.05 and 0.32 ± 0.01 were found when comparing FecXGR, FecXRA and FecXR heterozygous ewes to wild type homozygous ones. These effects are of the same order of magnitude as the effect of most of other known major genes for prolificacy. Only significant differences between FecXGR and FecXR were found among the three alleles associated with increased prolificacy. However, we cannot confirm the effect of the FecXRA allele at homozygous state because we did not find any homozygous ewes. These results confirm that these three alleles in the BMP15 gene that affect prolificacy co-segregate in Rasa aragonesa sheep.

Turncoat Polypeptides: We Adapt to Our Environment.

A common interpretation of Anfinsen's hypothesis states that one amino acid sequence should fold into a single, native, ordered state, or a highly similar set thereof, coinciding with the global minimum in the folding-energy landscape, which, in turn, is responsible for the function of the protein. However, this classical view is challenged by many proteins and peptide sequences, which can adopt exchangeable, significantly dissimilar conformations that even fulfill different biological roles. The similarities and differences of concepts related to these proteins, mainly chameleon sequences, metamorphic proteins, and switch peptides, which are all denoted herein "turncoat" polypeptides, are reviewed. As well as adding a twist to the conventional view of protein folding, the lack of structural definition adds clear versatility to the activity of proteins and can be used as a tool for protein design and further application in biotechnology and biomedicine.

Design and structural characterisation of monomeric water-soluble α-helix and ß-hairpin peptides: State-of-the-art.

Peptides are not only useful models for the structural understanding of protein folding and stability but also provide promising therapeutic avenues for the treatment of numerous diseases, and as biomaterials. The field has been very active over the last decades, but the complex conformational behaviour of peptides still poses challenges to the characterisation and rational design of defined structures. In this context, we aim to provide a comprehensive overview of linear water-soluble monomeric peptides able to form the two simplest structural motifs: α-helices and ß-hairpins. For both structures, we describe the geometry features, and the main contributions to stability: intrinsic propensities, position dependence of specific residues, particular capping motifs and side chain interactions. They should be considered to design α-helical or ß-hairpin peptides. Solvent influence on peptide stability and selected in silico design approaches are also discussed. Moreover, we provide guidelines for structural characterisation of α-helical and ß-hairpin-forming peptides by NMR and circular dichroism. We also highlight recently reported designed peptides and current strategies developed to improve their stability, bioactivity and bioavailability. The information gathered herein may aid peptide design and characterisation of stable α-helical and ß-hairpin motifs in the search of biological constructs or improved peptide therapeutics.

A CON-based NMR assignment strategy for pro-rich intrinsically disordered proteins with low signal dispersion: the C-terminal domain of histone H1.0 as a case study.

The C-terminal domain of histone H1.0 (C-H1.0) is involved in DNA binding and is a main determinant of the chromatin condensing properties of histone H1.0. Phosphorylation at the (S/T)-P-X-(K/R) motifs affects DNA binding and is crucial for regulation of C-H1.0 function. Since C-H1.0 is an intrinsically disordered domain, solution NMR is an excellent approach to characterize the effect of phosphorylation on the structural and dynamic properties of C-H1.0. However, its very repetitive, low-amino acid-diverse and Pro-rich sequence, together with the low signal dispersion observed at the 1H-15N HSQC spectra of both non- and tri-phosphorylated C-H1.0 preclude the use of standard 1H-detected assignment strategies. We have achieved an essentially complete assignment of the heavy backbone atoms (15N, 13C' and 13Cα), as well as 1HN and 13Cß nuclei, of non- and tri-phosphorylated C-H1.0 by applying a novel 13C-detected CON-based strategy. No C-H1.0 region with a clear secondary structure tendency was detected by chemical shift analyses, confirming at residue level that C-H1.0 is disordered in aqueous solution. Phosphorylation only affected the chemical shifts of phosphorylated Thr's, and their adjacent residues. Heteronuclear {1H}-15N NOEs were also essentially equal in the non- and tri-phosphorylated states. Hence, structural tendencies and dynamic properties of C-H1.0 free in aqueous solution are unmodified by phosphorylation. We propose that the assignment strategy used for C-H1.0, which is based on the acquisition of only a few 3D spectra, is an excellent choice for short-lived intrinsically disordered proteins with repetitive sequences.

Genetic heterogeneity of dolphin morbilliviruses detected in the Spanish Mediterranean in inter-epizootic period.

BACKGROUND: In the last 20 years, Cetacean Morbillivirus (CeMV) has been responsible for many die-offs in marine mammals worldwide, as clearly exemplified by the three dolphin morbillivirus (DMV) epizootics of 1990-1992, 2006-2008 and 2011 that affected Mediterranean striped dolphins (Stenella coeruleoalba). Systemic infection caused by DMV in the Mediterranean has been reported only during these outbreaks. RESULTS: We report the infection of five striped dolphins (Stenella coeruleoalba) stranded on the Spanish Mediterranean coast of Valencia after the last DMV outbreak that ended in 2011. Animal 1 stranded in late 2011 and Animal 2 in 2012. Systemic infection affecting all tissues was found based on histopathology and positive immunohistochemical and polymerase chain reaction positive results. Animal 3 stranded in 2014; molecular and immunohistochemical detection was positive only in the central nervous system. Animals 4 and 5 stranded in 2015, and DMV antigen was found in several tissues. Partial sequences of the DMV phosphoprotein (P), nucleoprotein (N), and hemagglutinin (H) genes were identical for Animals 2, 3, 4, and 5, and were remarkably different from those in Animal 1. The P sequence from Animal 1 was identical to that of the DMV strain that caused the epizootic of 2011 in the Spanish Mediterranean. The corresponding sequence from Animals 2-5 was identical to that from a striped dolphin stranded in 2011 on the Canary Islands and to six dolphins stranded in northeastern Atlantic of the Iberian Peninsula. CONCLUSIONS: These results suggest the existence of an endemic infection cycle among striped dolphins in the Mediterranean that may lead to occasional systemic disease presentations outside epizootic periods. This cycle involves multiple pathogenic viral strains, one of which may have originated in the Atlantic Ocean.

Antigenicity of Leishmania-Activated C-Kinase Antigen (LACK) in Human Peripheral Blood Mononuclear Cells, and Protective Effect of Prime-Boost Vaccination With pCI-neo-LACK Plus Attenuated LACK-Expressing Vaccinia Viruses in Hamsters.

Leishmania-activated C-kinase antigen (LACK) is a highly conserved protein among Leishmania species and is considered a viable vaccine candidate for human leishmaniasis. In animal models, prime-boost vaccination with LACK-expressing plasmids plus attenuated vaccinia viruses (modified vaccinia Ankara [MVA] and mutant M65) expressing LACK, has been shown to protect against cutaneous leishmaniasis (CL). Further, LACK demonstrated to induce the production of protective cytokines in patients with active CL or cured visceral leishmaniasis, as well as in asymptomatic individuals from endemic areas. However, whether LACK is capable to trigger cytokine release by peripheral blood mononuclear cells from patients cured of CL due to Leishmania infantum (L. infantum) or induce protection in L. infantum-infected hamsters [visceral leishmaniasis (VL) model], has not yet been analyzed. The present work examines the ex vivo immunogenicity of LACK in cured VL and CL patients, and asymptomatic subjects from an L. infantum area. It also evaluates the vaccine potential of LACK against L. infantum infection in hamsters, in a protocol of priming with plasmid pCI-neo-LACK (DNA-LACK) followed by a booster with the poxvirus vectors MVA-LACK or M65-LACK. LACK-stimulated PBMC from both asymptomatic and cured subjects responded by producing IFN-γ, TNF-α, and granzyme B (Th1-type response). Further, 78% of PBMC samples that responded to soluble Leishmania antigen showed IFN-γ secretion following stimulation with LACK. In hamsters, the protocol of DNA-LACK prime/MVA-LACK or M65-LACK virus boost vaccination significantly reduced the amount of Leishmania DNA in the liver and bone marrow, with no differences recorded between the use of MVA or M65 virus vector options. In summary, the Th1-type and cytotoxic responses elicited by LACK in PBMC from human subjects infected with L. infantum, and the parasite protective effect of prime/boost vaccination in hamsters with DNA-LACK/MVA-LACK and DNA-LACK/M65-LACK, revealed the significance of LACK in activating human and hamster immune responses and support LACK to be a valuable candidate for inclusion in a vaccine against human VL.

Roles of Amphipathicity and Hydrophobicity in the Micelle-Driven Structural Switch of a 14-mer Peptide Core from a Choline-Binding Repeat.

Choline-binding repeats (CBRs) are ubiquitous sequences with a ß-hairpin core that are found in the surface proteins of several microorganisms such as S. pneumoniae (pneumococcus). Previous studies on a 14-mer CBR sequence derived from the pneumoccal LytA autolysin (LytA239-252 peptide) have demonstrated a switch behaviour for this peptide, so that it acquires a stable, native-like ß-hairpin conformation in aqueous solution but is reversibly transformed into an amphipathic α-helix in the presence of detergent micelles. With the aim of understanding the factors responsible for this unusual ß-hairpin to α-helix transition, and to specifically assess the role of peptide hydrophobicity and helical amphipathicity in the process, we designed a series of LytA239-252 variants affecting these two parameters and studied their interaction with dodecylphosphocholine (DPC) micelles by solution NMR, circular dichroism and fluorescence spectroscopies. Our results indicate that stabilising cross-strand interactions become essential for ß-hairpin stability in the absence of optimal turn sequences. Moreover, both amphipathicity and hydrophobicity display comparable importance for helix stabilisation of CBR-derived peptides in micelles, indicating that these sequences represent a novel class of micelle/membrane-interacting peptides.

NMR Insights into the Structure-Function Relationships in the Binding of Melanocortin Analogues to the MC1R Receptor.

Linear and cyclic analogues of the α-melanocyte stimulating hormone (α-MSH) targeting the human melanocortin receptor 1 (MC1R) are of pharmacological interest for detecting and treating melanoma. The central sequence of α-MSH (His-Phe-Arg-Trp) has been identified as being essential for receptor binding. To deepen current knowledge on the molecular basis for α-MSH bioactivity, we aimed to understand the effect of cycle size on receptor binding. To that end, we synthesised two macrocyclic isomeric α-MSH analogues, c[NH-NO2-C6H3-CO-His-DPhe-Arg-Trp-Lys]-Lys-NH2 (CycN-K6) and c[NH-NO2-C6H3-CO-His-DPhe-Arg-Trp-Lys-Lys]-NH2 (CycN-K7). Their affinities to MC1R receptor were determined by competitive binding assays, and their structures were analysed by ¹H and 13C NMR. These results were compared to those of the previously reported analogue c[S-NO2-C6H3-CO-His-DPhe-Arg-Trp-Cys]-Lys-NH2 (CycS-C6). The MC1R binding affinity of the 22-membered macrocyclic peptide CycN-K6 (IC50 = 155 ± 16 nM) is higher than that found for the 25-membered macrocyclic analogue CycN-K7 (IC50 = 495 ± 101 nM), which, in turn, is higher than that observed for the 19-membered cyclic analogue CycS-C6 (IC50 = 1770 ± 480 nM). NMR structural study indicated that macrocycle size leads to changes in the relative dispositions of the side chains, particularly in the packing of the Arg side chain relative to the aromatic rings. In contrast to the other analogues, the 22-membered cycle's side chains are favorably positioned for receptor interaction.

First-in-class inhibitor of the T cell receptor for the treatment of autoimmune diseases.

Modulating T cell activation is critical for treating autoimmune diseases but requires avoiding concomitant opportunistic infections. Antigen binding to the T cell receptor (TCR) triggers the recruitment of the cytosolic adaptor protein Nck to a proline-rich sequence in the cytoplasmic tail of the TCR's CD3ε subunit. Through virtual screening and using combinatorial chemistry, we have generated an orally available, low-molecular weight inhibitor of the TCR-Nck interaction that selectively inhibits TCR-triggered T cell activation with an IC50 (median inhibitory concentration) ~1 nM. By modulating TCR signaling, the inhibitor prevented the development of psoriasis and asthma and, furthermore, exerted a long-lasting therapeutic effect in a model of autoimmune encephalomyelitis. However, it did not prevent the generation of a protective memory response against a mouse pathogen, suggesting that the compound might not exert its effects through immunosuppression. These results suggest that inhibiting an immediate TCR signal has promise for treating a broad spectrum of human T cell-mediated autoimmune and inflammatory diseases.

Structural basis for broad neutralization of HIV-1 through the molecular recognition of 10E8 helical epitope at the membrane interface.

The mechanism by which the HIV-1 MPER epitope is recognized by the potent neutralizing antibody 10E8 at membrane interfaces remains poorly understood. To solve this problem, we have optimized a 10E8 peptide epitope and analyzed the structure and binding activities of the antibody in membrane and membrane-like environments. The X-ray crystal structure of the Fab-peptide complex in detergents revealed for the first time that the epitope of 10E8 comprises a continuous helix spanning the gp41 MPER/transmembrane domain junction (MPER-N-TMD; Env residues 671-687). The MPER-N-TMD helix projects beyond the tip of the heavy-chain complementarity determining region 3 loop, indicating that the antibody sits parallel to the plane of the membrane in binding the native epitope. Biophysical, biochemical and mutational analyses demonstrated that strengthening the affinity of 10E8 for the TMD helix in a membrane environment, correlated with its neutralizing potency. Our research clarifies the molecular mechanisms underlying broad neutralization of HIV-1 by 10E8, and the structure of its natural epitope. The conclusions of our research will guide future vaccine-design strategies targeting MPER.