Gly Residues Research Articles

Multiple docking analysis and In silico absorption, distribution, metabolism, excretion, and toxicity screening of anti-leprosy phytochemicals and dapsone against dihydropteroate synthase of Mycobacterium leprae

Leprosy is a neglected tropical disease affecting millions of people. The current treatment against leprosy includes various antibacterial drugs of which dapsone is known to bind to dihydropteroate synthase of Mycobacterium leprae. Dapsone is an expensive antibacterial drug with many side effects. A natural alternative for dapsone having less to no side effects and cheaper in production is needed. The three-dimensional protein structure of dihydropteroate synthase of M. leprae is not available. Protein homology modeling of target protein was carried out, and protein structure validation and energy minimization were performed. Phytochemicals mentioned in literature having anti-leprosy properties were studied for absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties and that which passed ADMET filters were further carried for comparative in silico docking analysis along with dapsone. Preliminary docking analysis was carried using AutoDock Vina, and results obtained were validated using AutoDock 4.2.6 and SwissDock. Neobavaisoflavone was predicted to be ten times safer for administration than dapsone. On performing in silico docking, it was found that neobavaisoflavone has better binding affinity than dapsone and forms a stable protein-ligand complex. Residues GLY.50, THR.88, and VAL.107 play an important role as binding site residues. Further, in vitro and in vivo experimental studies are required to confirm anti-leprosy properties of neobavaisoflavone over drug dapsone.

Fish SUMO3 functions as a critical antiviral molecule against iridovirus and nodavirus

Protein SUMOylation (SUMO is small ubiquitin-related modifier) is a dynamic process that is strictly regulated under physiological and pathological conditions. We previously cloned and characterized two SUMO homologue genes (EcSUMO1 and EcSUMO2) from orange-spotted grouper (Epinephelus coioides). In the present study, the SUMO3 homologue from E. coioides (EcSUMO3) was cloned and its possible roles in fish immunity were analyzed. The open reading frame of EcSUMO3 contains 285 base pairs encoding a 94 amino acid protein with a predicted molecular mass of 10.73 kDa. The protein sequence of EcSUMO3 revealed similar domains with mammals, including the UBQ (ubiquitin-like proteins) domain, the hydrophobic surface, the Ulp1-Smt3 interaction sites, a VKTE motif and the C-terminal Gly residues. EcSUMO3 shares 46.83% and 89.58% identity with EcSUMO1 and EcSUMO2, respectively, and it shares 94%, 98%, and 98% identity with SUMO3 from Oreochromis niloticus, Danio rerio, and Homo sapiens, respectively. Quantitative real-time polymerase chain reaction analysis indicated that EcSUMO3 was constitutively expressed in all of the analyzed tissues in healthy grouper. EcSUMO3 expression levels were remarkably (p < 0.01) up-regulated in grouper spleen (GS) cells in response to stimulation with red-spotted grouper nervous necrosis virus (RGNNV) and Singapore grouper iridovirus (SGIV). EcSUMO3 was distributed in both the cytoplasm and nucleus in GS cells. EcSUMO3 enhanced SGIV and RGNNV replication during viral infection in vitro. These results are important for better understanding of the SUMO pathway in fish and provide insights into the regulatory mechanism of viral infection in E. coioides under farmed conditions.

Transmembrane Helix Integrity versus Fraying To Expose Hydrogen Bonds at a Membrane-Water Interface.

Transmembrane helices dominate the landscape for many membrane proteins. Often flanked by interfacial aromatic residues, these transmembrane helices also contain loops and interhelix segments, which could help in stabilizing a transmembrane orientation. Using 2H nuclear magnetic resonance spectroscopy to monitor bilayer-incorporated model GWALP23 family peptides, we address systematically the issue of helix fraying in relation to the dynamics and orientation of highly similar individual transmembrane helices. We inserted aromatic (Phe, Trp, Tyr, and His) or non-aromatic residues (Ala and Gly) into positions 4 and 5 adjacent to a core transmembrane helix to examine the side-chain dependency of the transmembrane orientation, dynamics, and helix integrity (extent and location of unraveling). Incorporation of [2H]alanine labels enables one to assess the helicity of the core sequence and the peptide termini. For most of the helices, we observed substantial unwinding involving at least three residues at both ends. For the unique case of histidine at positions 4 and 5, an extended N-terminal unwinding was observed up to residue 7. For further investigation of the onset of fraying, we employed A4,5GWALP23 with 2H labels at residues 4 and 5 and found that the number of terminal residues involved in the unwinding depends on bilayer thicknesses and helps to govern the helix dynamics. The combined results enable us to compare and contrast the extent of fraying for each related helix, as reflected by the deviation of experimental 2H quadrupolar splitting magnitudes of juxta-terminal alanines A3 and A21 from those represented by an ideal helix geometry.

The Singular NMR Fingerprint of a Polyproline II Helical Bundle.

Polyproline II (PPII) helices play vital roles in biochemical recognition events and structures like collagen and form part of the conformational landscapes of intrinsically disordered proteins (IDPs). Nevertheless, this structure is generally hard to detect and quantify. Here, we report the first thorough NMR characterization of a PPII helical bundle protein, the Hypogastrura harveyi "snow flea" antifreeze protein (sfAFP). J-couplings and nuclear Overhauser enhancement spectroscopy confirm a natively folded structure consisting of six PPII helices. NMR spectral analyses reveal quite distinct Hα2 versus Hα3 chemical shifts for 28 Gly residues as well as 13Cα, 15N, and 1HN conformational chemical shifts (Δδ) unique to PPII helical bundles. The 15N Δδ and 1HN Δδ values and small negative 1HN temperature coefficients evince hydrogen-bond formation. 1H-15N relaxation measurements reveal that the backbone structure is generally highly rigid on ps-ns time scales. NMR relaxation parameters and biophysical characterization reveal that sfAFP is chiefly a dimer. For it, a structural model featuring the packing of long, flat hydrophobic faces at the dimer interface is advanced. The conformational stability, measured by amide H/D exchange to be 6.24 ± 0.2 kcal·mol-1, is elevated. These are extraordinary findings considering the great entropic cost of fixing Gly residues and, together with the remarkable upfield chemical shifts of 28 Gly 1Hα, evidence significant stabilizing contributions from CαHα ||| O═C hydrogen bonds. These stabilizing interactions are corroborated by density functional theory calculations and natural bonding orbital analysis. The singular conformational chemical shifts, J-couplings, high hNOE ratios, small negative temperature coefficients, and slowed H/D exchange constitute a unique set of fingerprints to identify PPII helical bundles, which may be formed by hundreds of Gly-rich motifs detected in sequence databases. These results should aid the quantification of PPII helices in IDPs, the development of improved antifreeze proteins, and the incorporation of PPII helices into novel designed proteins.

Isotopically Site-Selected Dynamics of a Three-Stranded β-Sheet Peptide Detected with Temperature-Jump Infrared-Spectroscopy.

Infrared detected temperature-jump (T-jump) spectroscopy and site-specific isotopic labeling were applied to study a model three-stranded β-sheet peptide with the goal of individually probing the dynamics of strand and turn structural elements. This peptide had two DPro-Gly (pG) turn sequences to stabilize the two component hairpins, which were labeled with 13C═O on each of the Gly residues to resolve them spectroscopically. Labeling the second turn on the amide preceding the DPro (Xxx-DPro amide) provided an alternate turn label as a control. Placing 13C═O labels on specific in-strand residues gave shifted modes that overlap the Xxx-DPro amide I' modes. Their impact could be separated from the turn dynamics by a novel difference transient analysis approach. Fourier-transform infrared spectra were modeled with density functional theory-computations which showed the local, isotope-selected vibrations were effectively uncoupled from the other amide I modes. Our T-jump dynamics results, combined with nuclear magnetic resonance structures and equilibrium spectral measurements, showed the first turn to be most stable and best formed with the slowest dynamics, whereas the second turn and first strand (N-terminus) had similar dynamics, and the third strand (C-terminus) had the fastest dynamics and was the least structured. The relative dynamics of the strands, Xxx-DPro amides, and 13C-labeled Gly residues on the turns also qualitatively corresponded to molecular dynamics (MD) simulations of turn and strand fluctuations. MD trajectories indicated the turns to be bistable, with the first turn being Type I' and the second turn flipping from I' to II'. The differences in relaxation times for each turn and the separate strands revealed that the folding process of this turn-stabilized β-sheet structure proceeds in a multistep process.

Liquid and Hydrogel Phases of PrPC Linked to Conformation Shifts and Triggered by Alzheimer’s Amyloid-β Oligomers

Protein phase separation by low-complexity, intrinsically disordered domains generates membraneless organelles and links to neurodegeneration. Cellular prion protein (PrPC) contains such domains, causes spongiform degeneration, and is a receptor for Alzheimer's amyloid-β oligomers (Aβo). Here, we show that PrPC separates as a liquid phase, in which α-helical Thr become unfolded. At the cell surface, PrPC Lys residues interact with Aβo to create a hydrogel containing immobile Aβo and relatively mobile PrPC. The Aβo/PrP hydrogel has a well-defined stoichiometry and dissociates with excess Aβo. NMR studies of hydrogel PrPC reveal a distinct α-helical conformation for natively unfolded amino-terminal Gly and Ala residues. Aβo/PrP hydrogel traps signal-transducing mGluR5 on the plasma membrane. Recombinant PrPC extracts endogenous Aβo from human Alzheimer's soluble brain lysates into hydrogel, and a PrPC antagonist releases Aβo from endogenous brain hydrogel. Thus, coupled phase and conformational transitions of PrPC are driven by Aβ species from Alzheimer's disease.

The several facets of Trichogin GA IV: High affinity Tb(III) binding properties. A spectroscopic and molecular dynamics simulation study

AbstractTrichogin GA IV (TrGA) is an antimicrobial peptide isolated from Trichoderma longibrachiatum. The amino acid sequence of TrGA is rather peculiar, because it is characterized by three Aib and four Gly residues, which confer unique dynamic and structural properties. In a previous study, we found that TrGA shows excellent binding properties to Ca(II) and lanthanide Gd(III) ions in acetonitrile solutions. Within the lanthanide ions, Tb(III) ions possess fascinating optical characteristics, such as luminescence which greatly improves after coordination. Here, we present the results of our spectroscopic and molecular dynamics investigations on the Tb(III) ion‐binding properties of an Nα‐Fmoc functionalized analog of Trichogin GA IV (F0TrOMe). The high Tb(III) ion‐F0TrOMe affinity, together with the proteolytic resistance and membrane affinity of the natural compound, confers to this system potentially promising applications in several fields, such as bioimaging and bioanalytical assays.

Functional Analysis of Novicidin Peptide: Coordinated Delivery System for Zinc via Schiff Base Ligand.

Novicidin (NVC), is a membrane-penetrating peptide, which forms a stable complex with Zn-Schiff base with interesting antitumor selectivity. We studied NVC derivatives to determine functional roles of key amino acids in toxicity, helicity, and binding of the Zn-Schiff base complex. Trimmed derivatives highlighted the role of peptide length and helicity in toxicity and membrane penetration. The removal of Lys from position 1 and 2 strongly increases the ability to disrupt the membranes. The trimming of the N-terminal residues significantly increases the stability of peptide helicity enhancing penetrating properties. Gly residue derivatives undermined a role of peptide bending in membrane penetration and toxicity. After the substitution of the central Gly derivatives with Ile or Lys, the peptides retained toxicity. These results illustrate the minor role of central helix bending in NVC toxicity. Binding-site-peptide derivatives identified His residue as the sole Zn-Schiff base binding site and eliminated the role of other aromatic residues.

Molecular mechanisms governing the evolutionary conservation of Glycine in the 6th position of loops ΙΙΙ and ΙV in photoprotein mnemiopsin 2

Photoproteins in their functional form are complexed noncovalently with 2-hydroperoxycoelenterazine. A conformational change upon coordination of Ca+2 ions with their EF-hand loops leads to oxidation of substrate and emission of light. In all photoproteins, EF-hand loops Ι, ΙΙΙ and ΙV have standard sequence for binding to Ca+2 ion, however the second one is not able for Ca+2 coordination. Sequence analysis of Mnemiopsin 2 and other known photoproteins shows that Glutamate (Glu) is occurred in the 6th position of its first EF-hand loop, but this position in other loops of mnemiopsin 2 and all functional loops of other photoproteins is occupied by Glycine (Gly). Here we designed and made single and double mutants where Gly residue at the 6th positions of loops ΙΙΙ and ΙV of mnemiopsin 2 was replaced with Glu. According to the activity measurements, wild-type (WT) and G142E variants have more initial luminescence intensity than G176E and double mutants; while WT and G176E have higher values of half decay time when compared with G142E and double mutants. According to the isothermal denaturation experiments, all protein variants are structurally more stable than WT mnemiopsin 2 and that the stabilizing effects of single mutants are paired resulting in more stability of double mutant against urea denaturation. We concluded that simultaneous occurrence of Gly in the 6th position of loops ΙΙΙ and ΙV is essential for evolutionary adjustment of initial intensity and decay rate of luminescence emission via affecting the interaction of the core structure of photoprotein with coelenteramide and binding affinity of Ca+2 to the corresponding loops, respectively.

Modifying the Lantibiotic Mutacin 1140 for Increased Yield, Activity, and Stability.

Mutacin 1140 belongs to the epidermin family of type AI lantibiotics. This family has a broad spectrum of activity against Gram-positive bacteria. The binding of mutacin 1140 to lipid II leads to the inhibition of cell wall synthesis. Pharmacokinetic experiments with type AI lantibiotics are generally discouraging for clinical applications due to the short half-life of these compounds. The unprotected dehydrated and protease-susceptible residues outside the lanthionine rings may play a role in the short half-life in physiological settings. Previous mutagenesis work on mutacin 1140 has been limited to the lanthionine-forming residues, the C-terminally decarboxylated residue, and single amino acid substitutions at residues Phe1, Trp4, Dha5, and Arg13. To study the importance of the dehydrated (Dha5 and Dhb14) and protease-susceptible (Lys2 and Arg13) residues within mutacin 1140 for stability and bioactivity, each of these residues was evaluated for its impact on production and inhibitory activity. More than 15 analogs were purified, enabling direct comparison of the activities against a select panel of Gram-positive bacteria. The efficiency of the posttranslational modification (PTM) machinery of mutacin 1140 is highly restricted on its substrate. Analogs in the various intermediate stages of PTMs were observed as minor products following single point mutations at the 2nd, 5th, 13th, and 14th positions. The combination of alanine substitutions at the Dha5 and Dhb14 positions abolished mutacin 1140 production, while the production was restored by substitution of a Gly residue at one of these positions. Analogs with improved activity, productivity, and proteolytic stability were identified.IMPORTANCE Our findings show that the efficiency of mutacin 1140 PTMs is highly dependent on the core peptide sequence. Analogs in various intermediate stages of PTMs can be transported by the bacterium, which indicates that PTMs and transport are finely tuned for the native mutacin 1140 core peptide. Only certain combinations of amino acid substitutions at the Dha5 and Dhb14 dehydrated residue positions were tolerated. Observation of glutamylated core peptide analogs shows that dehydrations occur in a glutamate-dependent manner. Interestingly, mutations at positions outside rings A and B, the lipid II binding domain, would interfere with lipid II binding. Purified mutacin 1140 analogs have various activities and selectivities against different genera of bacteria, supporting the effort to generate analogs with higher specificity against pathogenic bacteria. The discovery of analogs with improved inhibitory activity against pathogenic bacteria, increased stability in the presence of protease, and higher product yields may promote the clinical development of this unique antimicrobial compound.

Local structural motifs in proteins: Detection and characterization of fragments inserted in helices

The global/local fold of protein structures is stabilized by a variety of specific interactions. A primary role in this context is played by hydrogen bonds. In order to identify novel motifs in proteins, we searched Protein Data Bank structures looking for backbone H-bonds formed by NH groups of two (or more) consecutive residues with consecutive CO groups of distant residues in the sequence. The present analysis unravels the occurrence of recurrent structural motifs that, to the best of our knowledge, had not been characterized in literature. Indeed, these H-bonding patterns are found (i) in a specific parallel β-sheet capping, (ii) in linking of β-hairpins to α-helices, and (iii) in α-helix insertions. Interestingly, structural analyses of these motifs indicate that Gly residues frequently occupy prominent positions. The formation of these motifs is likely favored by the limited propensity of Gly to be embodied in helices/sheets. Of particular interest is the motif corresponding to insertions in helices that was detected in 1% of analyzed structures. Inserted fragments may assume different structures and aminoacid compositions and usually display diversified evolutionary conservation. Since inserted regions are physically separated from the rest of the protein structure, they represent hot spots for ad-hoc protein functionalization.

Contribution of an unusual CDR2 element of a single domain antibody in ricin toxin binding affinity and neutralizing activity.

Ricin toxin's enzymatic subunit (RTA) has been subjected to intensive B cell epitope mapping studies using a combination of competition ELISAs, hydrogen exchange-mass spectrometry and X-ray crystallography. Those studies identified four spatially distinct clusters (I-IV) of toxin-neutralizing epitopes on the surface of RTA. Here we describe A9, a new single domain camelid antibody (VHH) that was proposed to recognize a novel epitope on RTA that straddles clusters I and III. The X-ray crystal structure of A9 bound to RTA (2.6 Å resolution) revealed extensive antibody contact with RTA's β-strand h (732 Å2 buried surface area; BSA), along with limited engagement with α-helix D (90 Å2) and α-helix C (138 Å2). Collectively, these contacts explain the overlap between epitope clusters I and III, as identified by competition ELISA. However, considerable binding affinity, and, consequently, toxin-neutralizing activity of A9 is mediated by an unusual CDR2 containing five consecutive Gly residues that interact with α-helix B (82 Å2), a known neutralizing hotspot on RTA. Removal of a single Gly residue from the penta-glycine stretch in CDR2 reduced A9's binding affinity by 10-fold and eliminated toxin-neutralizing activity. Computational modeling indicates that removal of a Gly from CDR2 does not perturb contact with RTA per se, but results in the loss of an intramolecular hydrogen bond network involved in stabilizing CDR2 in the unbound state. These results reveal a novel configuration of a CDR2 element involved in neutralizing ricin toxin.

Helix Fraying and Lipid-Dependent Structure of a Short Amphipathic Membrane-Bound Peptide Revealed by Solid-State NMR.

The amphipathic α-helical peptide KIA14 [(KIAGKIA)2-NH2] was studied in membranes using circular dichroism and solid-state NMR spectroscopy to obtain global as well as local structural information. By analyzing 2H NMR data from 10 analogues of KIA14 that were selectively labeled with Ala- d3, those positions that are properly folded into a helix could be determined within the membrane-bound peptide. The N-terminus was found to be unraveled, whereas positions 4-14 formed an ideal helix all the way to the C-terminus. The helicity did not change when Gly residues were replaced by Ala- d3 but was reduced when Ile was replaced, indicating that large hydrophobic residues are required for membrane binding and helix formation. The reduced helicity was strongly correlated with a decrease in peptide-induced leakage from lipid vesicles. The orientation of the short KIA14 peptide was assessed in several lipid systems and compared with that of the longer KIA21 sequence [(KIAGKIA)3-NH2]. In 1,2-dioleoyl- sn-glycero-3-phosphatidylcholine, both peptides are aligned flat on the membrane surface, whereas in 1,2-dimyristoyl- sn-glycero-3-phosphatidylcholine (DMPC)/1-myristoyl-2-hydroxy- sn-glycero-3-phosphatidylcholine (lyso-MPC) both are inserted into the membrane in an upright orientation. These two types of lipid systems had been selected for their strongly negative and positive spontaneous curvature, respectively. We propose that in these cases, the peptide orientation is largely determined by the lipid properties. On the other hand, in plain DMPC and 1,2-dilauroyl- sn-glycero-3-phosphatidylcholine, which have only a slight positive curvature, a marked difference in orientation is evident: the short KIA14 lies almost flat on the membrane surface, whereas the longer KIA21 is more tilted. We thus propose that out of the lipid systems tested here, DMPC (with hardly any curvature) is the least biased lipid system in which peptide orientation and realignment can be studied, allowing to compare and discriminate the intrinsic effects of the properties of the peptides as such.

Structural insights on the efficient catalysis of hydroperoxide reduction by Ohr: Crystallographic and molecular dynamics approaches.

Organic hydroperoxide resistance (Ohr) enzymes are highly efficient Cys-based peroxidases that play central roles in bacterial response to fatty acid hydroperoxides and peroxynitrite, two oxidants that are generated during host-pathogen interactions. In the active site of Ohr proteins, the conserved Arg (Arg19 in Ohr from Xylella fastidiosa) and Glu (Glu51 in Ohr from Xylella fastidiosa) residues, among other factors, are involved in the extremely high reactivity of the peroxidatic Cys (Cp) toward hydroperoxides. In the closed state, the thiolate of Cp is in close proximity to the guanidinium group of Arg19. Ohr enzymes can also assume an open state, where the loop containing the catalytic Arg is far away from Cp and Glu51. Here, we aimed to gain insights into the putative structural switches of the Ohr catalytic cycle. First, we describe the crystal structure of Ohr from Xylella fastidiosa (XfOhr) in the open state that, together with the previously described XfOhr structure in the closed state, may represent two snapshots along the coordinate of the enzyme-catalyzed reaction. These two structures were used for the experimental validation of molecular dynamics (MD) simulations. MD simulations employing distinct protonation states and in silico mutagenesis indicated that the polar interactions of Arg19 with Glu51 and Cp contributed to the stabilization of XfOhr in the closed state. Indeed, Cp oxidation to the disulfide state facilitated the switching of the Arg19 loop from the closed to the open state. In addition to the Arg19 loop, other portions of XfOhr displayed high mobility, such as a loop rich in Gly residues. In summary, we obtained a high correlation between crystallographic data, MD simulations and biochemical/enzymatic assays. The dynamics of the Ohr enzymes are unique among the Cys-based peroxidases, in which the active site Arg undergoes structural switches throughout the catalytic cycle, while Cp remains relatively static.

Collagen Gly missense mutations: Effect of residue identity on collagen structure and integrin binding.

Gly missense mutations in type I collagen, which replace a conserved Gly in the repeating (Gly-Xaa-Yaa)n sequence with a larger residue, are known to cause Osteogenesis Imperfecta (OI). The clinical consequences of such mutations range from mild to lethal, with more serious clinical severity associated with larger Gly replacement residues. Here, we investigate the influence of the identity of the residue replacing Gly within and adjacent to the integrin binding 502GFPGER507 sequence on triple-helix structure, stability and integrin binding using a recombinant bacterial collagen system. Recombinant collagens were constructed with Gly substituted by Ala, Ser or Val at four positions within the integrin binding region. All constructs formed a stable triple-helix structure with a small decrease in melting temperature. Trypsin was used to probe local disruption of the triple helix, and Gly to Val replacements made the triple helix trypsin sensitive at three of the four sites. Any mutation at Gly505, eliminated integrin binding, while decreased integrin binding affinity was observed in the replacement of Gly residues at Gly502 following the order Val > Ser > Ala. Molecular dynamics simulations indicated that all Gly replacements led to transient disruption of triple-helix interchain hydrogen bonds in the region of the Gly replacement. These computational and experimental results lend insight into the complex molecular basis of the varying clinical severity of OI.

Determination of Local Structure of 13C Selectively Labeled 47-mer Peptides as a Model for Gly-Rich Region of Nephila clavipes Dragline Silk Using a Combination of 13C Solid-State NMR and MD Simulation

For the first time, we elucidate the complex structure of the Gly-rich regions in Nephila clavipes dragline silk through synergistic experimental and theoretical studies. First, the 13C selectively labeled 47-mer peptides selected from the glycine (Gly)-rich region of N. clavipes dragline silk were synthesized. The 13C CP/MAS NMR spectra were analyzed to determine the fractions of the conformations of individual Gly and Ala residues through 13C conformation-dependent chemical shifts and peak deconvolution. By comparing the 13C solid-state NMR spectra of several simple model peptides, the presence of 31 helix in the 47-mer peptides was disproved, and the (Ala)6 regions were shown to form β-sheet structure in the staggered arrangement. Although the fraction of β-sheet components tended to increase and the fraction of random coil component decrease toward both chain ends, significant change in the fractions was observed depending on the amino acid position. These results were successfully rationalized throug...

Structural basis for tankyrase-RNF146 interaction reveals noncanonical tankyrase-binding motifs.

Poly(ADP-ribosyl)ation (PARylation) catalyzed by the tankyrase enzymes (Tankyrase-1 and -2; a.k.a. PARP-5a and -5b) is involved in mitosis, telomere length regulation, GLUT-4 vesicle transport, and cell growth and differentiation. Together with the E3 ubiquitin ligase RNF146 (a.k.a. Iduna), tankyrases regulate the cellular levels of several important proteins including Axin, 3BP2, and angiomotins, which are key regulators of Wnt, Src and Hippo signaling, respectively. These tankyrase substrates are first PARylated and then ubiquitylated by RNF146, which is allosterically activated by binding to PAR polymer. Each tankyrase substrate is recognized by a tankyrase-binding motif (TBM). Here we show that RNF146 binds directly to tankyrases via motifs in its C-terminal region. Four of these RNF146 motifs represent novel, extended TBMs, that have one or two additional amino acids between the most conserved Arg and Gly residues. The individual RNF146 motifs display weak binding, but together mediate a strong multivalent interaction with the substrate-binding region of TNKS, forming a robust one-to-one complex. A crystal structure of the first RNF146 noncanonical TBM in complex with the second ankyrin repeat domain of TNKS shows how an extended motif can be accommodated in a peptide-binding groove on tankyrases. Overall, our work demonstrates the existence of a new class of extended TBMs that exist in previously uncharacterized tankyrase-binding proteins including those of IF4A1 and NELFE.

Genome mining for peptidases in heat-tolerant and mesophilic fungi and putative adaptations for thermostability

BackgroundPeptidases (EC 3.4) consist of a large group of hydrolytic enzymes that catalyze the hydrolysis of proteins accounting for approximately 65% of the total worldwide enzyme production. Peptidases from thermophilic fungi have adaptations to high temperature that makes them adequate for biotechnological application. In the present study, we profiled the genomes of heat-tolerant fungi and phylogenetically related mesophilic species for genes encoding for peptidases and their putative adaptations for thermostability.ResultsWe generated an extensive catalogue of these enzymes ranging from 241 to 820 peptidase genes in the genomes of 23 fungi. Thermophilic species presented the smallest number of peptidases encoding genes in relation to mesophilic species, and the peptidases families with a greater number of genes were the most affected. We observed differences in peptidases in thermophilic species in comparison to mesophilic counterparts, at (i) the genome level: a great reduction in the number of peptidases encoding genes that harbored a higher number of copies; (ii) in the primary protein structure: shifts in proportion of single or groups of amino acids; and (iii) in the three-dimensional structure: reduction in the number of internal cavities. Similar results were reported for extremely thermophilic proteins, but here we show for the first time that several changes also occurred on the moderate thermophilic enzymes of fungi. In regards to the amino acids composition, peptidases from thermophilic species in relation to the mesophilic ones, contained a larger proportion of Ala, Glu, Gly, Pro, Arg and Val residues and a lower number of Cys, His, Ile, Lys, Met, Asn, Gln, Ser, Thr and Trp residues (P < 0.05). Moreover, we observed an increase in the proportion of hydrophobic and charged amino acids and a decrease in polar amino acids.ConclusionsAlthough thermophilic fungi present less genes encoding for peptidases, these have adaptations that could play a role in thermal resistance from genome to protein structure level.

Molecular characterization of DXCF cyanobacteriochromes from the cyanobacterium Acaryochloris marina identifies a blue-light power sensor

Cyanobacteriochromes (CBCRs) are linear tetrapyrrole-binding photoreceptors that sense a wide range of wavelengths from ultraviolet to far-red. The primary photoreaction in these reactions is a Z/E isomerization of the double bond between rings C and D. After this isomerization, various color-tuning events establish distinct spectral properties of the CBCRs. Among the various CBCRs, the DXCF CBCR lineage is widely distributed among cyanobacteria. Because the DXCF CBCRs from the cyanobacterium Acaryochloris marina vary widely in sequence, we focused on these CBCRs in this study. We identified seven DXCF CBCRs in A. marina and analyzed them after isolation from Escherichia coli that produces phycocyanobilin, a main chromophore for the CBCRs. We found that six of these CBCRs covalently bound a chromophore and exhibited variable properties, including blue/green, blue/teal, green/teal, and blue/orange reversible photoconversions. Notably, one CBCR, AM1_1870g4, displayed unidirectional photoconversion in response to blue-light illumination, with a rapid dark reversion that was temperature-dependent. Furthermore, the photoconversion took place without Z/E isomerization. This observation indicated that AM1_1870g4 likely functions as a blue-light power sensor, whereas typical CBCRs reversibly sense two light qualities. We also found that AM1_1870g4 possesses a GDCF motif in which the Asp residue is swapped with the next Gly residue within the DXCF motif. Site-directed mutagenesis revealed that this swap is essential for the light power-sensing function of AM1_1870g4. This is the first report of a blue-light power sensor from the CBCR superfamily and of photoperception without Z/E isomerization among the bilin-based photoreceptors.

Molecular Mechanism of Synergy between the Antimicrobial Peptides PGLa and Magainin 2 in Membranes

The two antimicrobial peptides PGLa and magainin 2 (MAG2), both found in the skin of the African frog Xenopus laevis, are known to exhibit strong synergistic effects in bacterial killing, lipid vesicle leakage, and other membrane-related activities [1]. Here, we studied the synergy of the two peptides in an attempt to find the molecular mechanism behind their synergistic effect [2]. A large number of mutants of the two peptides were investigated, and the synergy tested was using three complementary methods: (i) a checkerboard assay against two bacteria; (ii) a vesicle leakage assay; and (iii) 15N-NMR, showing the insertion of PGLa into a transmembrane orientation (suggesting the formation of a pore) in the presence of MAG2. Several mutations were identified that reduced or inhibited the synergy of the peptides, and a single mutation on PGLa from Gly to Ala was enough to completely abolish synergy. At the same time, the designer-made and related but completely non-synergistic peptide MSI-103 could be modified by two point mutations to become strongly synergistic with MAG2. Also in this case Gly residues were involved, indicating that a GxxxG motif on PGLa is critical and necessary for synergy. Based on these results, a 3D model could be developed of the PGLa-MAG2 complex in the membrane that is synergistically active. An oligomeric pore is likely formed from antiparallel dimers of transmembrane PGLa peptides, wherein each PGLa molecule makes contact to a MAG2 peptide that is aligned on the membrane surface.