Amphipathic Region Research Articles

An intracellular hydrophobic nexus critical for hERG1 channel slow deactivation

Slow deactivation is a critical property of voltage-gated K+ channels encoded by the human Ether-à-go-go-Related Gene 1 (hERG). hERG1 channel deactivation is modulated by interactions between intracellular N-terminal Per-Arnt-Sim (PAS) and C-terminal cyclic nucleotide-binding homology (CNBh) domains. The PAS domain is multipartite, comprising a globular domain (gPAS; residues 26–135) and an N-terminal PAS-cap that is further subdivided into an initial unstructured “tip” (residues 1–12) and an amphipathic α-helical region (residues 13–25). Although the PAS-cap tip has long been considered the effector of slow deactivation, how its position near the gating machinery is controlled has not been elucidated. Here, we show that a triad of hydrophobic interactions among the gPAS, PAS-cap α helix, and the CNBh domains is required to support slow deactivation in hERG1. The primary sequence of this “hydrophobic nexus” is highly conserved among mammalian ERG channels but shows key differences to fast-deactivating Ether-à-go-go 1 (EAG1) channels. Combining sequence analysis, structure-directed mutagenesis, electrophysiology, and molecular dynamics simulations, we demonstrate that polar serine substitutions uncover an intermediate deactivation mode that is also mimicked by deletion of the PAS-cap α helix. Molecular dynamics simulation analyses of the serine-substituted channels show an increase in distance among the residues of the hydrophobic nexus, a rotation of the intracellular gating ring, and a retraction of the PAS-cap tip from its receptor site near the voltage sensor domain and channel gate. These findings provide compelling evidence that the hydrophobic nexus coordinates the respective components of the intracellular gating ring and positions the PAS-cap tip to control hERG1 deactivation gating.

Structural basis of mammalian respiratory complex I inhibition by medicinal biguanides

The molecular mode of action of biguanides, including the drug metformin, which is widely used in the treatment of diabetes, is incompletely characterized. Here, we define the inhibitory drug-target interaction(s) of a model biguanide with mammalian respiratory complex I by combining cryo-electron microscopy and enzyme kinetics. We interpret these data to explain the selectivity of biguanide binding to different enzyme states. The primary inhibitory site is in an amphipathic region of the quinone-binding channel, and an additional binding site is in a pocket on the intermembrane-space side of the enzyme. An independent local chaotropic interaction, not previously described for any drug, displaces a portion of a key helix in the membrane domain. Our data provide a structural basis for biguanide action and enable the rational design of medicinal biguanides.

Soybean meal peptides regulated membrane phase of giant unilamellar vesicles: A key role for bilayer amphipathic region localization

Membrane phase separation forms liquid-ordered (Lo) and liquid-disordered (Ld) phases and is involved in cellular processes and functions. Our previous study has confirmed that peptides can regulate phase separation by increasing the Lo phase. However, the specific mechanisms underlying the phase separation regulation of peptides remain poorly understood. This study aimed to explore the effect of soybean meal peptides on phase separation and illustrate the correlation between phase regulation and membrane localization of the peptides. Phase separation was studied by giant unilamellar vesicles (GUVs), and membrane localization of the peptides was detected by steady-state fluorescence quenching. Our results revealed that peptides YYK, CLA, and SLW enhanced the Lo phase while WLQ decreased the Lo phase. The localization in the membrane amphiphilic region of the peptides played a crucial role in their regulation of phase separation. The more localization of the peptides (YYK, CLA, and SLW) in the membrane amphiphilic region, the stronger the capacity to increase the Lo phase.

Tumor protein D54 binds intracellular nanovesicles via an extended amphipathic region

Tumor protein D54 (TPD54) is an abundant cytosolic protein that belongs to the TPD52 family, a family of four proteins (TPD52, 53, 54, and 55) that are overexpressed in several cancer cells. Even though the functions of these proteins remain elusive, recent investigations indicate that TPD54 binds to very small cytosolic vesicles with a diameter of ca. 30 nm, half the size of classical (e.g., COPI and COPII) transport vesicles. Here, we investigated the mechanism of intracellular nanovesicle capture by TPD54. Bioinformatical analysis suggests that TPD54 contains a small coiled-coil followed by four amphipathic helices (AH1-4), which could fold upon binding to lipid membranes. Limited proteolysis, CD spectroscopy, tryptophan fluorescence, and cysteine mutagenesis coupled to covalent binding of a membrane-sensitive probe showed that binding of TPD54 to small liposomes is accompanied by large structural changes in the amphipathic helix region. Furthermore, site-directed mutagenesis indicated that AH2 and AH3 have a predominant role in TPD54 binding to membranes both in cells and using model liposomes. We found that AH3 has the physicochemical features of an amphipathic lipid packing sensor (ALPS) motif, which, in other proteins, enables membrane binding in a curvature-dependent manner. Accordingly, we observed that binding of TPD54 to liposomes is very sensitive to membrane curvature and lipid unsaturation. We conclude that TPD54 recognizes nanovesicles through a combination of ALPS-dependent and ALPS-independent mechanisms.

A Novel Antimicrobial Peptide Derived from Bony Fish IFN1 Exerts Potent Antimicrobial and Anti-Inflammatory Activity in Mammals.

ABSTRACTType I interferons (IFN-Is) are critical antiviral cytokine in innate immunity but with limited direct defense ability against bacterial infections in mammals. In bony fish, despite all the IFN-Is (IFN1-4) act in antiviral immunity, studies demonstrate that IFN1 can remarkably contribute to host defense against bacterial infections. In this study, we found that IFN1 from grass carp (Ctenopharyngodon idella) contains an unusual cationic and amphipathic α-helical region (named as gcIFN-20, sequence: SYEKKINRHFKILKKNLKKK). The synthesized peptide gcIFN-20 could form α-helical structure in a membrane environment and exerts potent antimicrobial activity against multiple species of Gram-negative (G−) and Gram-positive (G+) bacteria with negligible toxicity. Mechanism studies showed gcIFN-20 kills G+ bacteria through membrane disruption and cytoplasm outflow while G− bacteria through membrane permeation and protein synthesis inhibition. In two mouse bacterial infection models, gcIFN-20 therapy could significantly reduce tissue bacterial loads and mortalities. In addition to the direct antibacterial activity, we also found that gcIFN-20 could significantly suppress the lipopolysaccharide (LPS)-induced pro-inflammatory cytokines in vitro and in vivo, obviously alleviated lung lesions in a mouse endotoxemia model. The mechanism is that gcIFN-20 interacts with LPS, causes LPS aggregation and neutralization. The antimicrobial and anti-inflammatory activities in vivo of gcIFN-20 in mammalian models suggested a promising agent for developing peptide-based antibacterial therapy.IMPORTANCE Type I interferons play crucial role in antiviral immunity in both vertebrates and invertebrates. The powerful antimicrobial activity is recently reported in nonmammalian vertebrates. The present study identified a novel antimicrobial peptide (gcIFN-20) derived from grass carp interferon 1, found gcIFN-20 exhibits forceful bactericidal and anti-inflammatory activity in mammals, and efficient therapeutic effect against two clinical severe extraintestinal pathogenic Escherichia coli and a mouse endotoxemia models. The antimicrobial mechanisms are membrane disruption and cytoplasm overflow for Gram-positive bacteria, while membrane permeation and protein synthesis inhibition for Gram-negative bacteria. The anti-inflammatory mechanisms can be aggregating and neutralizing lipopolysaccharide to attenuate the binding with receptors and facilitate phagocytosis. The results indicate that gcIFN-20 can be a promising novel therapeutic agent for bacterial diseases and inflammatory disorders, especially as a potential weapon for multidrug resistant strain infections.

PaP1, a Broad-Spectrum Lysin-Derived Cationic Peptide to Treat Polymicrobial Skin Infections.

Most skin infections, including those complicating burns, are polymicrobial involving multiple causative bacteria. Add to this the fact that many of these organisms may be antibiotic-resistant, and a simple skin lesion or burn could soon become life-threatening. Membrane-acting cationic peptides from Gram-negative bacteriophage lysins can potentially aid in addressing the urgent need for alternative therapeutics. Such peptides natively constitute an amphipathic region within the structural composition of these lysins and function to permit outer membrane permeabilization in Gram-negative bacteria when added externally. This consequently allows the lysin to access and degrade the peptidoglycan substrate, resulting in rapid hypotonic lysis and bacterial death. When separated from the lysin, some of these cationic peptides kill sensitive bacteria more effectively than the native molecule via both outer and cytoplasmic membrane disruption. In this study, we evaluated the antibacterial properties of a modified cationic peptide from the broad-acting lysin PlyPa01. The peptide, termed PaP1, exhibited potent in vitro bactericidal activity toward numerous high priority Gram-positive and Gram-negative pathogens, including all the antibiotic-resistant ESKAPE pathogens. Both planktonic and biofilm-state bacteria were sensitive to the peptide, and results from time-kill assays revealed PaP1 kills bacteria on contact. The peptide was bactericidal over a wide temperature and pH range and could withstand autoclaving without loss of activity. However, high salt concentrations and complex matrices were found to be largely inhibitory, limiting its use to topical applications. Importantly, unlike other membrane-acting antimicrobials, PaP1 lacked cytotoxicity toward human cells. Results from a murine burn wound infection model using methicillin-resistant Staphylococcus aureus or multidrug-resistant Pseudomonas aeruginosa validated the in vivo antibacterial efficacy of PaP1. In these studies, the peptide enhanced the potency of topical antibiotics used clinically for treating chronic wound infections. Despite the necessity for additional preclinical drug development, the collective data from our study support PaP1 as a potential broad-spectrum monotherapy or adjunctive therapy for the topical treatment of polymicrobial infections and provide a foundation for engineering future lysin-derived peptides with improved antibacterial properties.

The protective role of the flanking domains in α-synuclein monomers

Large aggregates of the intrinsically disordered protein α-synuclein, known as Lewy bodies, are one of the key indicators of Parkinson’s disease (PD). They can occur in both solution and in the membrane, and form by the oligomerization and fibrilization of misfolded α-synuclein proteins. The α-synuclein protein consists of three distinct protein domains: an amphipathic N-terminal region, the hydrophobic core NAC region, and the proline rich C-terminal region. Within Lewy bodies, most α-synuclein proteins show a truncated C-terminal domain, and removal of the domain has been shown to increase the propensity for aggregation.

TAOK2 is an ER-localized kinase that catalyzes the dynamic tethering of ER to microtubules.

SUMMARYThe endoplasmic reticulum (ER) depends on extensive association with the microtubule (MT) cytoskeleton for its structure and mitotic inheritance. However, mechanisms that underlie coupling of ER membranes to MTs are poorly understood. We have identified thousand and one amino acid kinase 2 (TAOK2) as a pleiotropic protein kinase that mediates tethering of ER to MTs. In human cells, TAOK2 localizes in distinct ER subdomains via transmembrane helices and an adjacent amphipathic region. Through its C-terminal tail, TAOK2 directly binds MTs, coupling ER membranes to the MT cytoskeleton. In TAOK2 knockout cells, although ER-membrane dynamics are increased, movement of ER along growing MT plus ends is disrupted. ER-MT tethering is tightly regulated by catalytic activity of TAOK2, perturbation of which leads to defects in ER morphology, association with MTs, and cell division. Our study identifies TAOK2 as an ER-MT tether and reveals a kinase-regulated mechanism for control of ER dynamics.

Abstract 83: Structural and biochemical properties of DIRAS3 N-terminal extension that permit its function as a tumor suppressor of oncogenic K-RAS

Abstract DIRAS3 is an imprinted tumor suppressor gene that encodes a small GTPase of the RAS superfamily. In contrast to oncogenic RAS proteins, DIRAS3 inhibits tumor growth and migration, while inducing autophagy and tumor dormancy. DIRAS3 inhibits PI3K/AKT/mTOR and RAS/MAP signaling. DIRAS3 inhibits oncogenic, mutant K-RAS signaling by binding directly to mutant K-RAS and disrupting K-RAS dimers and clusters on the inner leaflet of the plasma membrane. DIRAS3 bears 60% amino acid homology to K-RAS, differing by the addition of a distinctive 34 amino acid N-terminal extension (NTE). Both the NTE and an intact C-terminal CAAX box are required for the suppressor function of DIRAS3 in breast and ovarian cancers. While prenylation of the CAAX box is thought to facilitate binding of DIRAS3 to the inner leaflet of the cell surface membrane through a lipid tail, how the NTE influences membrane association is not well understood. In this study, we apply biochemical and biophysical methods to investigate NTE structure and ligand binding activities using synthetic wild type and mutated NTE peptides. Using a lipid overlay assay and surface plasmon resonance (SPR) analysis, we found that the DIRAS3 NTE has affinity for phosphatidylinositol phosphate lipids, especially PI(3,4,5)P3 and PI(4,5)P2. SPR demonstrated a strong interaction between NTE and PIP3 (dissociation constant of ~36 nM) with a cooperative self-association of NTE on the PIP3 sensor surface. Isothermal titration calorimetry revealed that NTE-PIP3 binding is complex, involving multiple sites and cooperativity, that appeared dominated by entropic interaction. Structural analysis of NTE mutants with circular dichroism spectroscopy indicated that the NTE is mainly unstructured in solution, however PIP3-binding induces a conformational rearrangement of the NTE that involves a hydrophobic surface of an amphipathic helix region. Furthermore, mass spectrometry identified N-myrisoylation of glycine in the NTE region of DIRAS3, which is unique among RAS family proteins. In addition, SPR screening of a K-RAS peptide library and kinetic analysis of the interaction between DIRAS3 NTE and K-RAS revealed engagement of the NTE with the effector lobe of K-RAS and a conformational change in the NTE•K-RAS complex. Together, our study suggests that DIRAS3 NTE may play an important role in inhibiting K-RAS and AKT signaling by facilitating specific protein-lipid interactions at the plasma membrane. The affinity of the NTE for PIP3 could contribute to the known regulation of PI3K by DIRAS3. Moreover, functional DIRAS3 may be tethered to the inner leaflet of the cell membrane at two points, by prenylation of the CAAX box at the C-terminal and by myristoylation of the NTE. Citation Format: Xiaowen Liang, Kwangkook Lee, Sung Y. Jung, Gamze Bildik, Weiqun Mao, Joshua P. Gray, Steven W. Millward, Zhen Lu, Robert C. Bast. Structural and biochemical properties of DIRAS3 N-terminal extension that permit its function as a tumor suppressor of oncogenic K-RAS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 83.

Living in a Foster Home: The Single Subpolar Flagellum Fla1 of Rhodobacter sphaeroides.

Rhodobacter sphaeroides is an α-proteobacterium that has the particularity of having two functional flagellar systems used for swimming. Under the growth conditions commonly used in the laboratory, a single subpolar flagellum that traverses the cell membrane, is assembled on the surface. This flagellum has been named Fla1. Phylogenetic analyses have suggested that this flagellar genetic system was acquired from an ancient γ-proteobacterium. It has been shown that this flagellum has components homologous to those present in other γ-proteobacteria such as the H-ring characteristic of the Vibrio species. Other features of this flagellum such as a straight hook, and a prominent HAP region have been studied and the molecular basis underlying these features has been revealed. It has also been shown that FliL, and the protein MotF, mainly found in several species of the family Rhodobacteraceae, contribute to remodel the amphipathic region of MotB, known as the plug, in order to allow flagellar rotation. In the absence of the plug region of MotB, FliL and MotF are dispensable. In this review we have covered the most relevant aspects of the Fla1 flagellum of this remarkable photosynthetic bacterium.

Critical Role for the Extended N Terminus of Chlamydial MreB in Directing Its Membrane Association and Potential Interaction with Divisome Proteins.

Chlamydiae lack the conserved central coordinator protein of cell division FtsZ, a tubulin-like homolog. Current evidence indicates that Chlamydia uses the actin-like homolog, MreB, to substitute for the role of FtsZ in a polarized division mechanism. Interestingly, we observed MreB as a ring at the septum in dividing cells of Chlamydia We hypothesize that MreB, to substitute for FtsZ in Chlamydia, must possess unique properties compared to canonical MreB orthologs. Sequence differences between chlamydial MreB and orthologs in other bacteria revealed that chlamydial MreB possesses an extended N-terminal region, harboring predicted amphipathicity, as well as the conserved amphipathic helix found in other bacterial MreBs. The conserved amphipathic helix-directed green fluorescent protein (GFP) to label the membrane uniformly in Escherichia coli but the extended N-terminal region did not. However, the extended N-terminal region together with the conserved amphipathic region directed GFP to restrict the membrane label to the cell poles. In Chlamydia, the extended N-terminal region was sufficient to direct GFP to the membrane, and this localization was independent of an association with endogenous MreB. Importantly, mutating the extended N-terminal region to reduce its amphipathicity resulted in the accumulation of GFP in the cytosol of the chlamydiae and not in the membrane. The N-terminal domain of MreB was not required for homotypic interactions but was necessary for interactions with cell division components RodZ and FtsK. Our data provide mechanistic support for chlamydial MreB to serve as a substitute for FtsZ by forming a ringlike structure at the site of polarized division.IMPORTANCEChlamydia trachomatis is an obligate intracellular pathogen, causing sexually transmitted diseases and trachoma. The study of chlamydial physiology is important for developing novel therapeutic strategies for these diseases. Chlamydiae divide by a unique MreB-dependent polarized cell division process. In this study, we investigated unique properties of chlamydial MreB and observed that chlamydial species harbor an extended N-terminal region possessing amphipathicity. MreB formed a ring at the septum, like FtsZ in Escherichia coli, and its localization was dependent upon the amphipathic nature of its extended N terminus. Furthermore, this region is crucial for the interaction of MreB with cell division proteins. Given these results, chlamydial MreB likely functions at the septum as a scaffold for divisome proteins to regulate cell division in this organism.

Molecular design and optimization of hepatic cancer SLP76-derived PLCγ1 SH3-binding peptide with the systematic N-substitution of peptide PXXP motif.

The phospholipase Cγ1 (PLCγ1) is essential for T-cell signaling and activation in hepatic cancer immune response, which has a regulatory Src homology 3 (SH3) domain that can specifically recognize and interact with the PXXP-containing decapeptide segment (185 QPPVPPQRPM194 , termed as SLP76185-194 peptide) of adaptor protein SLP76 following T-cell receptor ligation. The isolated peptide can only bind to the PLCγ1 SH3 domain with a moderate affinity due to lack of protein context support. Instead of the traditional natural residue mutagenesis that is limited by low structural diversity and shifted target specificity, we herein attempt to improve the peptide affinity by replacing the two key proline residues Pro187 and Pro190 of SLP76185-194 PXXP motif with nonnatural N-substituted amino acids, as the proline is the only endogenous N-substituted amino acid. The replacement would increase peptide flexibility but can restore peptide activity by establishing additional interactions with the domain. Structural analysis reveals that the domain pocket can be divided into a large amphipathic region and a small negatively charged region; they accommodate hydrophobic, aromatic, polar, and moderate-sized N-substituted amino acid types. A systematic replacement combination profile between the peptide residues Pro187 and Pro190 is created by structural modeling, dynamics simulation, and energetics analysis, from which six improved and two reduced N-substituted peptides as well as native SLP76185-194 peptide are identified and tested for their binding affinity to the recombinant protein of the human PLCγ1 SH3 domain using fluorescence-based assays. Two N-substituted peptides, SLP76185-194 (N-Leu187/N-Gln190) and SLP76185-194 (N-Thr187/N-Gln190), are designed to have high potency (Kd =0.67±0.18 and 1.7±0.3μM, respectively), with affinity improvement by, respectively, 8.5-fold and 3.4-fold relative to native peptide (Kd =5.7±1.2μM).

Dual binding motifs underpin the hierarchical association of perilipins1-3 with lipid droplets.

Lipid droplets (LDs) in all eukaryotic cells are coated with at least one of the perilipin (Plin) family of proteins. They all regulate key intracellular lipases but do so to significantly different extents. Where more than one Plin is expressed in a cell, they associate with LDs in a hierarchical manner. In vivo, this means that lipid flux control in a particular cell or tissue type is heavily influenced by the specific Plins present on its LDs. Despite their early discovery, exactly how Plins target LDs and why they displace each other in a “hierarchical” manner remains unclear. They all share an amino-terminal 11-mer repeat (11mr) amphipathic region suggested to be involved in LD targeting. Here, we show that, in vivo, this domain functions as a primary highly reversible LD targeting motif in Plin1–3, and, in vitro, we document reversible and competitive binding between a wild-type purified Plin1 11mr peptide and a mutant with reduced binding affinity to both “naked” and phospholipid-coated oil–water interfaces. We also present data suggesting that a second carboxy-terminal 4-helix bundle domain stabilizes LD binding in Plin1 more effectively than in Plin2, whereas it weakens binding in Plin3. These findings suggest that dual amphipathic helical regions mediate LD targeting and underpin the hierarchical binding of Plin1–3 to LDs.

Thylakoid-integrated recombinant Hcf106 participates in the chloroplast twin arginine transport system.

The chloroplast twin arginine transport (cpTat) system distinguishes itself as a protein transport pathway by translocating fully folded proteins, using the proton‐motive force (PMF) as the sole source of energy. The cpTat pathway is evolutionarily conserved with the Tat pathway found in the plasma membrane of many prokaryotes. The cpTat (Escherichia coli) system uses three proteins, Tha4 (TatA), Hcf106 (TatB), and cpTatC (TatC), to form a transient translocase allowing the passage of precursor proteins. Briefly, cpTatC and Hcf106, with Tha4, form the initial receptor complex responsible for precursor protein recognition and binding in an energy‐independent manner, while a separate pool of Tha4 assembles with the precursor‐bound receptor complex in the presence the PMF. Analysis by blue‐native polyacrylamide gel electrophoresis (BN‐PAGE) shows that the receptor complex, in the absence of precursor, migrates near 700 kDa and contains cpTatC and Hcf106 with little Tha4 remaining after detergent solubilization. To investigate the role that Hcf106 may play in receptor complex oligomerization and/or stability, systematic cysteine substitutions were made in positions from the N‐terminal transmembrane domain to the end of the predicted amphipathic helix of the protein. BN‐PAGE analysis allowed us to identify the locations of amino acids in Hcf106 that were critical for interacting with cpTatC. Oxidative cross‐linking allowed us to map interactions of the transmembrane domain and amphipathic helix region of Hcf106. In addition, we showed that in vitro expressed, integrated Hcf106 can interact with the precursor signal peptide domain and imported cpTatC, strongly suggesting that a subpopulation of the integrated Hcf106 is participating in competent cpTat complexes.

Antimicrobial peptides from C-terminal amphipathic region of E. coli FtsA

Antimicrobial peptides constitute an indispensable component of innate immune system in organisms ranging from bacteria to man. Despite this, peptides lag far behind the conventional antibiotics in treating infections. The menace of multidrug-resistant bacteria, however, has revived the antimicrobial peptide research. We reasoned that the membrane-binding regions of bacterial proteins could be purposed to combat them. Here, we identify potent antimicrobial peptides from the C-terminal amphipathic helix of E. coli FtsA protein. The 11 and 13-residue peptides exhibited activity against E. coli, gentamicin-resistant MRSA, and C. albicans. The activity is little affected by the presence of salt and divalent cations. The peptides preferentially bind to the negatively-charged membranes as indicated by tryptophan fluorescence studies. The peptides permeabilize the E. coli outer and inner membranes at very promising concentrations suggesting membrane-disruption as one of the mechanisms of killing.

Antimicrobial and anti-inflammatory activities of chemokine CXCL14-derived antimicrobial peptide and its analogs

CXCL14 is a CXC chemokine family that exhibits antimicrobial activity and contains an amphipathic cationic α-helical region in the C-terminus, a characteristic structure of antimicrobial peptides (AMPs). In this study, we designed three analogs of CXCL1459–75 (named CXCL14-C17) corresponding to the C-terminal α-helix of CXCL14, which displayed potential antimicrobial activity against a wide variety of gram-negative and gram-positive bacteria with minimum inhibitory concentrations of 4−16 μM without mammalian cell toxicity. Furthermore, two CXCL14-C17 analogs (CXCL14-C17-a1 and CXCL14-C17-a3) with improved cell selectivity were engineered by introducing Lys, Arg, or Trp in CXCL14-C17. Additionally, CXCL14-C17 analogs showed much greater synergistic effect (FICI: 0.3125–0.375) with chloramphenicol and ciprofloxacin against multidrug-resistant Pseudomonas aeruginosa (MDRPA) than LL-37 did (FICI: 0.75–1.125). CXCL14-C17 analogs were more active against antibiotic-resistant bacteria including methicillin-resistant Staphylococcus aureus (MRSA), MDRPA, and vancomycin-resistant Enterococcus faecium (VREF) than LL-37 and melittin. In particular, CXCL14-C17-a2 and CXCL14-C17-a3 completely inhibited the biofilm formation at sub-MIC and all of the peptides were able to eliminate pre-formed biofilm as well. Membrane depolarization, flow cytometry, sytox green uptake, ONPG hydrolysis and confocal microscopy revealed the possible target of the native peptide (CXCL14-C17) to likely be intracellular, and the amphipathic designed analogs targeted the bacterial membrane. CXCL14-C17 also showed DNA binding characteristic activity similar to buforin-2. Interestingly, CXCL14-C17-a2 and CXCL14-C17-a3 effectively inhibited the production and expression of nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1 from lipopolysaccharide (LPS)-stimulated RAW264.7 cells, suggesting that these peptides could be promising anti-inflammatory and antimicrobial agents.

Light-Regulated Synthesis of Cyclic-di-GMP by a Bidomain Construct of the Cyanobacteriochrome Tlr0924 (SesA) without Stable Dimerization.

Phytochromes and cyanobacteriochromes (CBCRs) use double-bond photoisomerization of their linear tetrapyrrole (bilin) chromophores within cGMP-specific phosphodiesterases/adenylyl cyclases/FhlA (GAF) domain-containing photosensory modules to regulate activity of C-terminal output domains. CBCRs exhibit photocycles that are much more diverse than those of phytochromes and are often found in large modular proteins such as Tlr0924 (SesA), one of three blue light regulators of cell aggregation in the cyanobacterium Thermosynechococcus elongatus. Tlr0924 contains a single bilin-binding GAF domain adjacent to a C-terminal diguanylate cyclase (GGDEF) domain whose catalytic activity requires formation of a dimeric transition state presumably supported by a multidomain extension at its N-terminus. To probe the structural basis of light-mediated signal propagation from the photosensory input domain to a signaling output domain for a representative CBCR, these studies explore the properties of a bidomain GAF-GGDEF construct of Tlr0924 (Tlr0924Δ) that retains light-regulated diguanylate cyclase activity. Surprisingly, circular dichroism spectroscopy and size exclusion chromatography data do not support formation of stable dimers in either the blue-absorbing 15ZPb dark state or the green-absorbing 15EPg photoproduct state of Tlr0924Δ. Analysis of variants containing site-specific mutations reveals that proper signal transmission requires both chromophorylation of the GAF domain and individual residues within the amphipathic linker region between GAF and GGDEF domains. On the basis of these data, we propose a model in which bilin binding and light signals are propagated from the GAF domain via the linker to alter the equilibrium and interconversion dynamics between active and inactive conformations of the GGDEF domain to favor or disfavor formation of catalytically competent dimers.

Highly potent antimicrobial modified peptides derived from the Acinetobacter baumannii phage endolysin LysAB2

The increase in the prevalence of multidrug-resistant Acinetobacter baumannii (MDRAB) strains is a serious public health concern. Antimicrobial peptides (AMPs) are a possible solution to this problem. In this study, we examined whether AMPs could be derived from phage endolysins. We synthesized four AMPs based on an amphipathic helical region in the C-terminus of endolysin LysAB2 encoded by the A. baumannii phage ΦAB2. These peptides showed potent antibacterial activity against A. baumannii (minimum inhibitory concentration, 4–64 μM), including some MDR and colistin-resistant A. baumannii. Of the four peptides, LysAB2 P3, with modifications that increased its net positive charge and decreased its hydrophobicity, showed high antibacterial activity against A. baumannii but little haemolytic and no cytotoxic activity against normal eukaryotic cells. The results of electron microscopy experiments and a fluorescein isothiocyanate staining assay indicated that this peptide killed A. baumannii through membrane permeabilization. Moreover, in a mouse intraperitoneal infection model, at 4 h after the bacterial injection, LysAB2 P3 decreased the bacterial load by 13-fold in ascites and 27-fold in blood. Additionally, LysAB2 P3 rescued sixty percent of mice heavily infected with A. baumannii from lethal bacteremia. Our results confirmed that bacteriophage endolysins are a promising resource for developing effective AMPs.

Evolutionary Divergence of the C-terminal Domain of Complexin Accounts for Functional Disparities between Vertebrate and Invertebrate Complexins.

Complexin is a critical presynaptic protein that regulates both spontaneous and calcium-triggered neurotransmitter release in all synapses. Although the SNARE-binding central helix of complexin is highly conserved and required for all known complexin functions, the remainder of the protein has profoundly diverged across the animal kingdom. Striking disparities in complexin inhibitory activity are observed between vertebrate and invertebrate complexins but little is known about the source of these differences or their relevance to the underlying mechanism of complexin regulation. We found that mouse complexin 1 (mCpx1) failed to inhibit neurotransmitter secretion in Caenorhabditis elegans neuromuscular junctions lacking the worm complexin 1 (CPX-1). This lack of inhibition stemmed from differences in the C-terminal domain (CTD) of mCpx1. Previous studies revealed that the CTD selectively binds to highly curved membranes and directs complexin to synaptic vesicles. Although mouse and worm complexin have similar lipid binding affinity, their last few amino acids differ in both hydrophobicity and in lipid binding conformation, and these differences strongly impacted CPX-1 inhibitory function. Moreover, function was not maintained if a critical amphipathic helix in the worm CPX-1 CTD was replaced with the corresponding mCpx1 amphipathic helix. Invertebrate complexins generally shared more C-terminal similarity with vertebrate complexin 3 and 4 isoforms, and the amphipathic region of mouse complexin 3 significantly restored inhibitory function to worm CPX-1. We hypothesize that the CTD of complexin is essential in conferring an inhibitory function to complexin, and that this inhibitory activity has been attenuated in the vertebrate complexin 1 and 2 isoforms. Thus, evolutionary changes in the complexin CTD differentially shape its synaptic role across phylogeny.

Molecular Characterization of Caveolin-induced Membrane Curvature

The generation of caveolae involves insertion of the cholesterol-binding integral membrane protein caveolin-1 (Cav1) into the membrane, however, the precise molecular mechanisms are as yet unknown. We have speculated that insertion of the caveolin scaffolding domain (CSD), a conserved amphipathic region implicated in interactions with signaling proteins, is crucial for caveola formation. We now define the core membrane-juxtaposed region of Cav1 and show that the oligomerization domain and CSD are protected by tight association with the membrane in both mature mammalian caveolae and a model prokaryotic system for caveola biogenesis. Cryoelectron tomography reveals the core membrane-juxtaposed domain to be sufficient to maintain oligomerization as defined by polyhedral distortion of the caveolar membrane. Through mutagenesis we demonstrate the importance of the membrane association of the oligomerization domain/CSD for defined caveola biogenesis and furthermore, highlight the functional significance of the intramembrane domain and the CSD for defined caveolin-induced membrane deformation. Finally, we define the core structural domain of Cav1, constituting only 66 amino acids and of great potential to nanoengineering applications, which is required for caveolin-induced vesicle formation in a bacterial system. These results have significant implications for understanding the role of Cav1 in caveola formation and in regulating cellular signaling events.