Structural insight into the interaction of proteins containing NPF,DPF, and GPF motifs with the C‐terminal EH‐domain of EHD1

Eps15 homology (EH)‐domain containing proteins are regulators of endocytic membrane trafficking. EH‐domain binding to proteins containing the tripeptide NPF has been well characterized, but recent studies have shown that EH‐domains are also able to interact with ligands containing DPF or GPF motifs. We demonstrate that the three motifs interact in a similar way with the EH‐domain of EHD1, with the NPF motif having the highest affinity due to the presence of an intermolecular hydrogen bond. The weaker affinity for the DPF and GPF motifs suggests that if complex formation occurs in vivo, they may require high ligand concentrations, the presence of successive motifs and/or specific flanking residues.     

Hysteresis in poly‐2′‐deoxycytidine i‐motif folding is impacted by the method of analysis as well as loop and stem lengths

In DNA, i‐motif (iM) folds occur under slightly acidic conditions when sequences rich in 2′‐deoxycytidine (dC) nucleotides adopt consecutive dC self base pairs. The pH stability of an iM is defined by the midpoint in the pH transition (pH_T) between the folded and unfolded states. Two different experiments to determine pH_T values via circular dichroism (CD) spectroscopy were performed on poly‐dC iMs of length 15, 19, or 23 nucleotides. These experiments demonstrate two points: (1) pH_T values were dependent on the titration experiment performed, and (2) pH‐induced denaturing or annealing processes produced isothermal hysteresis in the pH_T values. These results in tandem with model iMs with judicious mutations of dC to thymidine to favor particular folds found the hysteresis was maximal for the shorter poly‐dC iMs and those with an even number of base pairs, while the hysteresis was minimal for longer poly‐dC iMs and those with an odd number of base pairs. Experiments to follow the iM folding via thermal changes identified thermal hysteresis between the denaturing and annealing cycles. Similar trends were found to those observed in the CD experiments. The results demonstrate that the method of iM analysis can impact the pH_T parameter measured, and hysteresis was observed in the pH_T and T_m values.     

Examination of the active secondary structure of the peptide 101.10, an allosteric modulator of the interleukin‐1 receptor,by positional scanning using β‐amino γ‐lactams

The relationship between the conformation and biological activity of the peptide allosteric modulator of the interleukin‐1 receptor 101.10 (D ‐Arg‐D ‐Tyr‐D ‐Thr‐D ‐Val‐D ‐Glu‐D ‐Leu‐D ‐Ala‐NH₂) has been studied using (R)‐ and (S)‐Bgl residues. Twelve Bgl peptides were synthesized using (R)‐ and (S)‐cyclic sulfamidate reagents derived from L ‐ and D ‐aspartic acid in an optimized Fmoc‐compatible protocol for efficient lactam installment onto the supported peptide resin. Examination of these (R)‐ and (S)‐Bgl 101.10 analogs for their potential to inhibit IL‐1β‐induced thymocyte cell proliferation using a novel fluorescence assay revealed that certain analogs exhibited retained and improved potency relative to the parent peptide 101.10. In light of previous reports that Bgl residues may stabilize type II′β‐turn‐like conformations in peptides, CD spectroscopy was performed on selected compounds to identify secondary structure necessary for peptide biological activity. Results indicate that the presence of a fold about the central residues of the parent peptide may be important for activity. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Peptide selectivity between the PDZ domains of human pregnancy‐related serine proteases (HtrA1, HtrA2, HtrA3, and HtrA4) can be reshaped by different halogen probes

The human HtrA family of serine proteases (HtrA1, HtrA2, HtrA3, and HtrA4) are the key enzymes associated with pregnancy and closely related to the development and progression of many pathological events. Previously, it was found that halogen substitution at the indole moiety of peptide Trp‐1 residue can form a geometrically satisfactory halogen bond with the Drosophila discs large, zona occludens‐1 (PDZ) domain of HtrA proteases. Here, we attempt to systematically investigate the effect of substitution with 4 halogen types and 2 indole positions on the binding affinity and specificity of peptide ligands to the 4 HtrA PDZ domains. The complex structures, interaction energies, halogen‐bonding strength, and binding affinity of domain‐peptide systems were modeled, analyzed, and measured via computational modeling and fluorescence‐based assay. It is revealed that there is a compromise between the local rearrangement of halogen bond involving different halogen atoms and the global optimization of domain‐peptide interaction; the substitution position is fundamentally important for peptide‐binding affinity, while the halogen type can effectively shift peptide selectivity between the 4 domains. The HtrA1‐PDZ and HtrA4‐PDZ as well as HtrA2‐PDZ and HtrA3‐PDZ respond similarly to different halogen substitutions of peptide; –Br substitution at R2‐position and –I substitution at R4‐position are most effective in improving peptide selectivity for HtrA1‐PDZ/HtrA4‐PDZ and HtrA2‐PDZ/HtrA3‐PDZ, respectively; –F substitution would not address substantial effect on peptide selectivity for all the 4 domains. Consequently, the binding affinities of a native peptide ligand DSRIWWV^–COOH as well as its 4 R2‐halogenated counterparts were determined as 1.9, 1.4, 0.5, 0.27, and 0.92 μM, which are basically consistent with computational analysis. This study would help to rationally design selective peptide inhibitors of HtrA family members by using different halogen substitutions.     

Trifluoroethanol‐containing RP‐HPLC mobile phases for the separation of transmembrane peptides human glycophorin‐A,integrin alpha‐1, and p24: analysis and prevention of potential side reactions due to formic acid

Reversed‐phase high‐pressure liquid chromatography analysis and purification of three hydrophobic, aggregation‐prone peptides, composed mainly of the transmembrane (TM) sequence, were performed using elution systems containing 2,2,2‐trifluoroethanol (TFE). The addition of 10–16% TFE to a common mobile phase, such as a water/acetonitrile/propanol (PrOH) or a water/PrOH/formic acid system, markedly improved the chromatographic separation of these peptides. The superior performance of TFE‐containing systems in separating peptides over water/PrOH/formic acid systems [Bollhagen R. et al., J. Chromatogr. A, 1995; 711 : 181–186.] clearly demonstrated that adding TFE to the mobile phase is one of best methods for TM‐peptide purification. Characterization of the potential side reactions using MALDI and ESI‐LIT/Orbitrap mass spectrometry indicated that prolonged incubation of peptides in a mixture of TFE–formic acid possibly induces O‐formylation of the Ser residue and N‐formylation of the N‐terminus of peptides. The conditions for selective removal of the formyl groups from TM peptides were also screened. We believe that these results will expand our ability to analyze and prepare hydrophobic, aggregation‐prone TM peptides and proteins. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

OsASR2 regulates the expression of a defence‐related gene,Os2H16, by targeting the GT‐1 cis‐element

The GT‐1 cis‐element widely exists in many plant gene promoters. However, the molecular mechanism that underlies the response of the GT‐1 cis‐element to abiotic and biotic stresses remains elusive in rice. We previously isolated a rice short‐chain peptide‐encoding gene, Os2H16, and demonstrated that it plays important roles in both disease resistance and drought tolerance. Here, we conducted a promoter assay of Os2H16 and identified GT‐1 as an important cis‐element that mediates Os2H16 expression in response to pathogen attack and osmotic stress. Using the repeated GT‐1 as bait, we characterized an abscisic acid, stress and ripening 2 (ASR2) protein from yeast‐one hybridization screening. Sequence alignments showed that the carboxy‐terminal domain of OsASR2 containing residues 80–138 was the DNA‐binding domain. Furthermore, we identified that OsASR2 was specifically bound to GT‐1 and activated the expression of the target gene Os2H16, as well as GFP driven by the chimeric promoter of 2 × GT‐1‐35S mini construct. Additionally, the expression of OsASR2 was elevated by pathogens and osmotic stress challenges. Overexpression of OsASR2 enhanced the resistance against Xanthomonas oryzae pv. oryzae and Rhizoctonia solani, and tolerance to drought in rice. These results suggest that the interaction between OsASR2 and GT‐1 plays an important role in the crosstalk of the response of rice to biotic and abiotic stresses.     

Structural and functional analysis of the phosphoryl transfer reaction mediated by the human small C‐terminal domain phosphatase,Scp1

Human small C‐terminal domain phosphatase 1 (Scp1) modulates the phosphorylation state of the C‐terminal domain (CTD) of eukaryotic RNA polymerase II (RNAP II), with preference for phosphorylated Ser5 in the tandem heptad repeats of the CTD. Additionally, Scp1 was identified as a conserved regulator of neuronal stem cell development. Scp1 is a member of haloacid dehalogenase (HAD) superfamily, whose catalysis depends on a Mg²⁺ ion and a DXDX(T/V) motif. The first Asp of the motif is identified as the nucleophile that is subject to phosphorylation leading to a phosphoryl‐aspartate intermediate. This high‐energy mixed anhydride intermediate is subsequently hydrolyzed to regenerate the enzyme. In the present study, we successfully captured the phosphoryl‐aspartate intermediate in the crystal structure of a Scp1D206A mutant soaked with para‐nitrophenyl phosphate (pNPP), providing strong evidence for the proposed mechanism. Furthermore, steady‐state kinetic analysis of a variety of Scp1 mutants revealed the importance of Asp206 in Mg²⁺ coordination mediated by a water molecule. Overall, we captured the snapshots of the phosphoryl transfer reaction at each stage of Scp1‐mediated catalysis. Through structural‐based sequence alignment, we show that the spatial position of the D206 side chain is strictly conserved throughout HAD family. Our results strongly suggest that Asp206 and its equivalent residues in other HAD family members play important structural and possible mechanistic roles.     

Cellulose production,activated by cyclic di‐GMP through BcsA and BcsZ,is a virulence factor and an essential determinant of the three‐dimensional architectures of biofilms formed by Erwinia amylovora Ea1189

Bacterial biofilms are multicellular aggregates encased in an extracellular matrix mainly composed of exopolysaccharides (EPSs), protein and nucleic acids, which determines the architecture of the biofilm. Erwinia amylovora Ea1189 forms a biofilm inside the xylem of its host, which results in vessel plugging and water transport impairment. The production of the EPSs amylovoran and levan is critical for the formation of a mature biofilm. In addition, cyclic dimeric GMP (c‐di‐GMP) has been reported to positively regulate amylovoran biosynthesis and biofilm formation in E. amylovora Ea1189. In this study, we demonstrate that cellulose is synthesized by E. amylovora Ea1189 and is a major modulator of the three‐dimensional characteristics of biofilms formed by this bacterium, and also contributes to virulence during systemic host invasion. In addition, we demonstrate that the activation of cellulose biosynthesis in E. amylovora is a c‐di‐GMP‐dependent process, through allosteric binding to the cellulose catalytic subunit BcsA. We also report that the endoglucanase BcsZ is a key player in c‐di‐GMP activation of cellulose biosynthesis. Our results provide evidence of the complex composition of the extracellular matrix produced by E. amylovora and the implications of cellulose biosynthesis in shaping the architecture of the biofilm and in the expression of one of the main virulence phenotypes of this pathogen.     

Comparison of a Molecular and an Immobilized Gadolinium(III)‐tris[(1R,4S)‐3‐heptafluorobutanoyl‐camphor] as Catalyst in the Asymmetric Danishefsky‐Hetero‐Diels‐Alder‐Reaction

On‐column reaction gas chromatography combines the power of separation and rapid analysis of reactants and reaction products with screening of reactions in a single step. Not only conversions but the reaction rates at various temperatures can be obtained from single measurements, making this approach superior to the time‐consuming measurements typically performed in reaction progress analysis. However, this approach has only been used in the investigation of interconversion processes, rearrangement reactions, and only a few examples of higher‐order reactions are known. Here we present the screening of immobilized gadolinium(III)‐tris[(1R,4S)‐3‐heptafluorobutanoyl‐camphor] in the Danishefsky‐hetero‐Diels‐Alder‐reaction by enantioselective on‐column reaction gas chromatography utilizing cryogenic focusing to achieve catalytic conversions in this higher‐order reaction and subsequent separation of the enantiomeric product mixture to determine the enantiomeric ratio. The results obtained by this approach could be transferred to the conventional batch reaction at a larger scale, demonstrating that on‐column reaction chromatography provides reliable results in the screening of enantioselective reactions. Chirality 26:243–248, 2014. © 2014 Wiley Periodicals, Inc.     

BhSGAMP‐1, a gene that encodes an antimicrobial peptide,is developmentally regulated by the direct action of 20‐OH ecdysone in the salivary gland of Bradysia hygida (Diptera,Sciaridae)

Recently we have shown that BhSGAMP‐1 is a developmentally regulated reiterated gene that encodes an antimicrobial peptide (AMP) and is expressed exclusively in the salivary glands, at the end of the larval stage. We show, for the first time, that a gene for an AMP is directly activated by 20‐OH ecdysone. This control probably involves the participation of short‐lived repressor(s). We also found that the promoter of BhSGAMP‐1 is not equipped with elements that respond to infection, provoked by the injection of microorganisms, in the salivary glands or in the fat body. We produced polyclonal antibodies against the synthetic peptide and found that the BhSGAMP‐1 peptide is secreted in the saliva. The BhSGAMP‐1 gene was also activated during the third larval molt. These facts confirm our hypothesis that this preventive system of defense was selected to produce an environment free of harmful microorganisms in the insect's immediate vicinity, during molts. genesis 47:847–857, 2009. © 2009 Wiley‐Liss, Inc.