N-terminal Tetrapeptide Research Articles

Nonspecific N-terminal tetrapeptide insertions disrupt the translation arrest induced by ribosome-arresting peptide sequences

The nascent polypeptide chains passing through the ribosome tunnel not only serve as an intermediate of protein synthesis but also, in some cases, act as dynamic genetic information, controlling translation through interaction with the ribosome. One notable example is Escherichia coli SecM, in which translation of the ribosome arresting peptide (RAP) sequence in SecM leads to robust elongation arrest. Translation regulations, including the SecM-induced translation arrest, play regulatory roles such as gene expression control. Recent investigations have indicated that the insertion of a peptide sequence, SKIK (or MSKIK), into the adjacent N-terminus of the RAP sequence of SecM behaves as an "arrest canceler". As the study did not provide a direct assessment of the strength of translation arrest, we conducted detailed biochemical analyses. The results revealed that the effect of SKIK insertion on weakening SecM-induced translation arrest was not specific to the SKIK sequence, that is, other tetrapeptide sequences inserted just before the RAP sequence also attenuated the arrest. Our data suggest that SKIK or other tetrapeptide insertions disrupt the context of the RAP sequence rather than canceling or preventing the translation arrest.

Молекулярное моделирование конформационной подвижности молекулы гиламбатина

The features of the spatial organization of the hylambatin molecule were investigated by methods of molecular mechanics and molecular dynamics. Hylambatin consists of twelve amino acid residues in the sequence: Asp-Pro-Pro-Asp-Pro-Asn-Arg-Phe-Tyr-Gly-Met-Met-NH2. Unlike all other tachykinins, hylambatin has a Met residue replacing the usual Leu at penultimate position. The tachykinin peptide hylambatin has been isolated and chemically characterized from methanol extracts of the skin of Hylambates maculatus, an African rhacophorid frog. It has been shown that intravenously administered hylambatin significantly increases the level of glucose and insulin in blood plasma. In this paper, the conformational flexibility of the hylambatin molecule was studied by methods of molecular mechanics and molecular dynamics. The conformational calculation of the peptide took into account non-valent and electrostatic interactions, hydrogen bonds and torsion potentials. Based on fragmentary analysis, stable spatial structures of the hylambatin dodecapeptide were determined, which can be represented as a set of conformations characterized by a relatively labile N-terminal tetrapeptide and a conformationally rigid C-terminal octapeptide. In the calculated stable conformational states, the effective interactions of the side chains of residues and hydrogen bonds were refined and energetically evaluated. It has been shown that the hylambatin molecule preferably forms practically isoenergetic conformations with various structural types at the N-end of the peptide chain, passing into the alpha helix at the C-end. By the method of molecular dynamics, the pattern of intramolecular mobility of stable conformations of the hylambatin molecule was modeled both in vacuum and surrounded by water molecules. Based on the calculated values of the dihedral angles, molecular models of energetically preferred conformational states of the hylambatin dodecapeptide were constructed.

Unraveling the Dichotomy of Enigmatic Serine Protease HtrA2.

Mitochondrial high-temperature requirement protease A2 (HtrA2) is an integral member of the HtrA family of serine proteases that are evolutionarily conserved from prokaryotes to humans. Involvement in manifold intricate cellular networks and diverse pathophysiological functions make HtrA2 the most enigmatic moonlighting protease amongst the human HtrAs. Despite perpetuating the oligomeric architecture and overall structural fold of its homologs that comprises serine protease and regulatory PDZ domains, subtle conformational alterations and dynamic enzymatic regulation through the distinct allosteric mode of action lead to its functional diversity. This mitochondrial protease upon maturation, exposes its one-of-a-kind N-terminal tetrapeptide (AVPS) motif that binds and subsequently cleaves Inhibitor of Apoptosis Proteins (IAPs) thus promoting cell death, and posing as an important molecule for therapeutic intervention. Interestingly, unlike its other human counterparts, HtrA2 has also been implicated in maintaining the mitochondrial integrity through a bi-functional chaperone-protease activity, the on-off switch of which is yet to be identified. Furthermore, its ability to activate a wide repertoire of substrates through both its N- and C-terminal regions presumably has calibrated its association with several cellular pathways and hence diseases including neurodegenerative disorders and cancer. Therefore, the exclusive structural attributes of HtrA2 that involve multimodal activation, intermolecular PDZ-protease crosstalk, and an allosterically-modulated trimeric active-site ensemble have enabled the protease to evolve across species and partake functions that are fine-tuned for maintaining cellular homeostasis and mitochondrial proteome quality control in humans. These unique features along with its multitasking potential make HtrA2 a promising therapeutic target both in cancer and neurodegeneration.

Mitocryptide-2: Identification of Its Minimum Structure for Specific Activation of FPR2-Possible Receptor Switching from FPR2 to FPR1 by Its Physiological C-terminal Cleavages.

Mitocryptides are a novel family of endogenous neutrophil-activating peptides originating from various mitochondrial proteins. Mitocryptide-2 (MCT-2) is one of such neutrophil-activating peptides, and is produced as an N-formylated pentadecapeptide from mitochondrial cytochrome b. Although MCT-2 is a specific endogenous ligand for formyl peptide receptor 2 (FPR2), the chemical structure within MCT-2 that is responsible for FPR2 activation is still obscure. Here, we demonstrate that the N-terminal heptapeptide structure of MCT-2 with an N-formyl group is the minimum structure that specifically activates FPR2. Moreover, the receptor molecule for MCT-2 is suggested to be shifted from FPR2 to its homolog formyl peptide receptor 1 (FPR1) by the physiological cleavages of its C-terminus. Indeed, N-terminal derivatives of MCT-2 with seven amino acid residues or longer caused an increase of intracellular free Ca2+ concentration in HEK-293 cells expressing FPR2, but not in those expressing FPR1. Those MCT-2 derivatives also induced β-hexosaminidase secretion in neutrophilic/granulocytic differentiated HL-60 cells via FPR2 activation. In contrast, MCT-2(1–4), an N-terminal tetrapeptide of MCT-2, specifically activated FPR1 to promote those functions. Moreover, MCT-2 was degraded in serum to produce MCT-2(1–4) over time. These findings suggest that MCT-2 is a novel critical factor that not only initiates innate immunity via the specific activation of FPR2, but also promotes delayed responses by the activation of FPR1, which may include resolution and tissue regeneration. The present results also strongly support the necessity of considering the exact chemical structures of activating factors for the investigation of innate immune responses.

Evaluation of Dermorphin Metabolism Using Zebrafish Water Tank Model and Human Liver Microsomes.

Dermorphin is a heptapeptide with an analgesic potential higher than morphine that does not present the same risk for the development of tolerance. These pharmacological features make dermorphin a potential doping agent in competitive sports and it is already prohibited for racehorses. For athletes, the development of an efficient strategy to monitor for its abuse necessitates an investigation of the metabolism of dermorphin in humans. Here, human liver microsomes and zebrafish were utilized as model systems of human metabolism to evaluate the presence and kinetics of metabolites derived from dermorphin. Five hours after its administration, the presence of dermorphin metabolites could be detected in both models by liquid chromatography coupled to highresolution mass spectrometry. Although the two models showed common results, marked differences were also observed in relation to the formed metabolites. Six putative metabolites, based on their exact masses of m/z 479.1915, m/z 501.1733, m/z 495.1657, m/z 223.1073, m/z 180.1017 and m/z 457.2085, are proposed to represent the metabolic pattern of dermorphin. The major metabolite generated from the administration of dermorphin in both models was YAFG-OH (m/z 457.2085), which is the N-terminal tetrapeptide previously identified from studies on rats. Its extensive characterization and commercial availability suggest that it could serve as a primary target analyte for the detection of dermorphin misuse. The metabolomics approach also allowed the assignment of other confirmatory metabolites.

Spatial Structure of the Acth-(6-9)-PGP Molecule

The spatial structure of ACTH-(6-9)-PGP molecule has been investigated using theoretical conformational analysis method. Amino acid sequence of the N-terminal pentapeptide fragment of His-Phe-Arg-Trp-Pro of this molecule conforms to the fragment 6-9 of ACTH hormone. Calculations of conformational states of this molecule are carried out regarding nonvalent, electrostatic and torsional interactions and the energy of hydrogen bonds. The spatial structure of the His-Phe-Arg-Trp-Pro-Gly-Pro molecule was estimated on the low–energy conformations of the N-terminal tetrapeptide fragment His-Phe-Arg-Trp and C-terminal tripeptide fragment Pro-Gly-Pro of this molecule. It is shown that the spatial structure of heptapeptide molecule can be presented by 11 low-energy forms of the main chain. The low–energy conformations of this molecule, the values of dihedral angles of the backbone and side chains of the amino acid residues were founded and the energies of intra- and inter-residual interactions were determined.

Spatial Structure of the Acth-(6-9)-PGP Molecule

The spatial structure of ACTH-(6-9)-PGP molecule has been investigated using theoretical conformational analysis method. Amino acid sequence of the N-terminal pentapeptide fragment of His-Phe-Arg-Trp-Pro of this molecule conforms to the fragment 6-9 of ACTH hormone. Calculations of conformational states of this molecule are carried out regarding nonvalent, electrostatic and torsional interactions and the energy of hydrogen bonds. The spatial structure of the His-Phe-Arg-Trp-Pro-Gly-Pro molecule was estimated on the low–energy conformations of the N-terminal tetrapeptide fragment His-Phe-Arg-Trp and C-terminal tripeptide fragment Pro-Gly-Pro of this molecule. It is shown that the spatial structure of heptapeptide molecule can be presented by 11 low-energy forms of the main chain. The low–energy conformations of this molecule, the values of dihedral angles of the backbone and side chains of the amino acid residues were founded and the energies of intra- and inter-residual interactions were determined.

“Smac”ing the Life out of XIAP

X‐linked inhibitor of apoptosis protein (XIAP) is a key prosurvival protein that blocks apoptosis by inhibiting both the intrinsic and extrinsic pathways. Released by the mitochondria when under stress, XIAP is a homodimer, with 2 sets of repeating type I and type II baculoviral IAP domains: BIR1, BIR2 and BIR3, and RING. XIAP has a capacity to block apoptosis by directly inhibiting caspases 3,7 and 9. Second mitochondria‐derived activator of caspases (Smac) is a 184 amino acid chain. Smac antagonizes XIAP by attaching to the BIR3 domain inhibiting the binding of caspase‐9. Synthetic Smac mimetics (SMs) have been designed to duplicate the N‐terminal tetrapeptide (AVPI) or the IAP‐binding motif (IBM). Our model aims to show two BIR3 domain of a homodimer XIAP, bound to the IAP‐binding motif (IBM) of a Smac dimer. Selective antagonism of XIAP, specifically the BIR3 by Smac/SMs or monotherapy is not enough to kill cancer cells, however, it could be combined with immunotherapy, specifically virotherapy. This tricks the immune system to think that the cancer cells have a virus, which stimulates the immune antiviral response and creates an overproduction of immune cells known as a “cytokine storm”, to kill cancer cells.Support or Funding InformationThe Ashbury College MSOE Center for BioMolecular Modeling SMART Team used 3D modeling and printing technology to examine structure‐function relationships between Smac and BIR3 of XIAP.

Investigation of Hot Spot Region in XIAP Inhibitor Binding Site by Fragment Molecular Orbital Method

X-linked inhibitor of apoptosis protein (XIAP) is an important regulator of cancer cell survival whose BIR3 domain (XIAP-BIR3) recognizes the Smac N-terminal tetrapeptide sequence (AVPI), making it an attractive protein-protein interaction (PPI) target for cancer therapies. We used the fragment molecular orbital (FMO) method to study the binding modes and affinities between XIAP-BIR3 and a series of its inhibitors (1–8) that mimic the AVPI binding motif; the inhibitors had common interactions with key residues in a hot spot region of XIAP-BIR3 (P1–P4 subpockets) with increased binding affinity mainly attributed to specific interactions with the P1 and P4 subpockets. Based on the structural information from FMO results, we proposed a novel XIAP natural product inhibitor, neoeriocitrin 10, which was derived from our preciously reported XIAP-BIR3 inhibitor 9, can be used as a highly potent candidate for XIAP-BIR3 inhibition. We also performed pair interaction energy decomposition analysis to investigate the binding energies between specific binding residues and individual ligands, showing that the novel natural product neoeriocitrin 10 had a higher binding affinity than epicatechin gallate 9. Molecular docking and dynamics simulations were performed to explore the mode of binding between 10 and XIAP-BIR3, demonstrating that 10 binds more strongly to the P1 and P4 pockets than 9. Overall, we present a novel natural product, neoeriocitrin 10, and demonstrate that the FMO method can be used to identify hot spots in PPIs and design new compounds for XIAP inhibition.

χ-Space Screening of Dermorphin-Based Tetrapeptides through Use of Constrained Arylazepinone and Quinolinone Scaffolds.

Herein, the synthesis of novel conformationally constrained amino acids, 4-amino-8-bromo-2-benzazepin-3-one (8-Br-Aba), 3-amino-3,4-dihydroquinolin-2-one, and regioisomeric 4-amino-naphthoazepinones (1- and 2-Ana), is described. Introduction of these constricted scaffolds into the N-terminal tetrapeptide of dermorphin (i.e., H-Tyr-d-Ala-Phe-Gly-NH2) induced significant shifts in binding affinity, selectivity, and in vitro activity at the μ- and δ-opioid receptors (MOP and DOP, respectively). A reported constrained μ-/δ-opioid lead tetrapeptide H-Dmt-d-Arg-Aba-Gly-NH2 was modified through application of various constrained building blocks to identify optimal spatial orientations in view of activity at the opioid receptors. Interestingly, when the aromatic moieties were turned toward the C-terminus of the peptide sequences, (partial) (ant)agonism at MOP and weak (ant)agonism at DOP were noticed, whereas the incorporation of the 1-Ana residue led toward balanced low nanomolar MOP/DOP binding and in vitro agonism.

N-terminal tetrapeptide T/SPLH motifs contribute to multimodal activation of human TRPA1 channel.

Human transient receptor potential ankyrin channel 1 (TRPA1) is a polymodal sensor implicated in pain, inflammation and itching. An important locus for TRPA1 regulation is the cytoplasmic N-terminal domain, through which various exogenous electrophilic compounds such as allyl-isothiocyanate from mustard oil or cinnamaldehyde from cinnamon activate primary afferent nociceptors. This major region is comprised of a tandem set of 17 ankyrin repeats (AR1-AR17), five of them contain a strictly conserved T/SPLH tetrapeptide motif, a hallmark of an important and evolutionarily conserved contribution to conformational stability. Here, we characterize the functional consequences of putatively stabilizing and destabilizing mutations in these important structural units and identify AR2, AR6, and AR11-13 to be distinctly involved in the allosteric activation of TRPA1 by chemical irritants, cytoplasmic calcium, and membrane voltage. Considering the potential involvement of the T/SP motifs as putative phosphorylation sites, we also show that proline-directed Ser/Thr kinase CDK5 modulates the activity of TRPA1, and that T673 outside the AR-domain is its only possible target. Our data suggest that the most strictly conserved N-terminal ARs define the energetics of the TRPA1 channel gate and contribute to chemical-, calcium- and voltage-dependence.

Electrophilic Oxidation and [1,2]-Rearrangement of the Biindole Core of Birinapant.

Birinapant/TL32711 (1) is a bivalent antagonist of the inhibitor of apoptosis (IAP) family of proteins and was designed to mimic AVPI, the N-terminal tetrapeptide of the second mitochondria-derived activator of caspases (Smac/DIABLO). Birinapant bound to the BIR3 domains of cIAP1, cIAP2, and XIAP with K i values of 1, 36, and 45 nM, respectively. Birinapant-mediated activation of cIAP1 resulted in cIAP1 autoubiquitylation and degradation and correlated with inhibition of TNF-mediated NF-κB activation, induction of tumor cell death in vitro, and tumor regression in vivo. Birinapant is being evaluated in Phase 1/2 trials for the treatment of cancer and hepatitis B virus (HBV) infection. After one year at accelerated storage conditions, a formulation of 1 afforded four degradants in >0.1% abundance by HPLC analysis. The primary degradants (2 and 3) were formed via oxidation of the biindole core, while the secondary degradants (5 and 6) arose via [1,2]-rearrangement of 3 and 2, respectively. Forced degradation conditions were developed, which allowed the isolation of 2 and 3 in multigram quantities. Novel deuterated analogues of 1 were prepared to determine the site of oxidation, and NMR experiments confirmed the chemical structures of 5 and 6. The de novo synthesis of 2, 3, 5, and 6 confirmed these experimental findings.

Bioactivity and structural properties of chimeric analogs of the starfish SALMFamide neuropeptides S1 and S2

The starfish SALMFamide neuropeptides S1 (GFNSALMFamide) and S2 (SGPYSFNSGLTFamide) are the prototypical members of a family of neuropeptides that act as muscle relaxants in echinoderms. Comparison of the bioactivity of S1 and S2 as muscle relaxants has revealed that S2 is ten times more potent than S1. Here we investigated a structural basis for this difference in potency by comparing the bioactivity and solution conformations (using NMR and CD spectroscopy) of S1 and S2 with three chimeric analogs of these peptides. A peptide comprising S1 with the addition of S2's N-terminal tetrapeptide (Long S1 or LS1; SGPYGFNSALMFamide) was not significantly different to S1 in its bioactivity and did not exhibit concentration-dependent structuring seen with S2. An analog of S1 with its penultimate residue substituted from S2 (S1(T); GFNSALTFamide) exhibited S1-like bioactivity and structure. However, an analog of S2 with its penultimate residue substituted from S1 (S2(M); SGPYSFNSGLMFamide) exhibited loss of S2-type bioactivity and structural properties. Collectively, our data indicate that the C-terminal regions of S1 and S2 are the key determinants of their differing bioactivity. However, the N-terminal region of S2 may influence its bioactivity by conferring structural stability in solution. Thus, analysis of chimeric SALMFamides has revealed how neuropeptide bioactivity is determined by a complex interplay of sequence and conformation.

Birinapant (TL32711), a Bivalent SMAC Mimetic, Targets TRAF2-Associated cIAPs, Abrogates TNF-Induced NF-κB Activation, and Is Active in Patient-Derived Xenograft Models

The acquisition of apoptosis resistance is a fundamental event in cancer development. Among the mechanisms used by cancer cells to evade apoptosis is the dysregulation of inhibitor of apoptosis (IAP) proteins. The activity of the IAPs is regulated by endogenous IAP antagonists such as SMAC (also termed DIABLO). Antagonism of IAP proteins by SMAC occurs via binding of the N-terminal tetrapeptide (AVPI) of SMAC to selected BIR domains of the IAPs. Small molecule compounds that mimic the AVPI motif of SMAC have been designed to overcome IAP-mediated apoptosis resistance of cancer cells. Here, we report the preclinical characterization of birinapant (TL32711), a bivalent SMAC-mimetic compound currently in clinical trials for the treatment of cancer. Birinapant bound to the BIR3 domains of cIAP1, cIAP2, XIAP, and the BIR domain of ML-IAP in vitro and induced the autoubiquitylation and proteasomal degradation of cIAP1 and cIAP2 in intact cells, which resulted in formation of a RIPK1:caspase-8 complex, caspase-8 activation, and induction of tumor cell death. Birinapant preferentially targeted the TRAF2-associated cIAP1 and cIAP2 with subsequent inhibition of TNF-induced NF-κB activation. The activity of a variety of chemotherapeutic cancer drugs was potentiated by birinapant both in a TNF-dependent or TNF-independent manner. Tumor growth in multiple primary patient-derived xenotransplant models was inhibited by birinapant at well-tolerated doses. These results support the therapeutic combination of birinapant with multiple chemotherapies, in particular, those therapies that can induce TNF secretion.

Fragment based discovery of arginine isosteres through REPLACE: Towards non-ATP competitive CDK inhibitors

In order to develop non-ATP competitive CDK2/cyclin A inhibitors, the REPLACE strategy has been applied to generate fragment alternatives for the N-terminal tetrapeptide of the cyclin binding motif (HAKRRLIF) involved in substrate recruitment prior to phosphotransfer. The docking approach used for the prediction of small molecule mimics for peptide determinants was validated through reproduction of experimental binding modes of known inhibitors and provides useful information for evaluating binding to protein–protein interaction sites. Further to this, potential arginine isosteres predicted using the validated LigandFit docking method were ligated to the truncated C-terminal peptide, RLIF using solid phase synthesis and evaluated in a competitive binding assay. After testing, identified fragments were shown to represent not only appropriate mimics for a critical arginine residue but also to interact effectively with a minor hydrophobic pocket present in the binding groove. Further evaluation of binding modes was undertaken to optimize the potency of these compounds. Through further application of the REPLACE strategy in this study, peptide-small molecule hybrid CDK2 inhibitors were identified that are more drug-like and suitable for further optimization as anti-tumor therapeutics.

Involvement of spinal release of α-neo-endorphin on the antinociceptive effect of TAPA

The antinociceptive effect of i.t.-administered Tyr-d-Arg-Phe-β-Ala (TAPA), an N-terminal tetrapeptide analog of dermorphin, was characterized in ddY mice. In the mouse tail-flick test, TAPA administered i.t. produced a potent antinociception. The antinociception induced by TAPA was significantly attenuated by i.t. pretreatment with the κ-opioid receptor antagonist nor-binaltorphimine, as well as by the μ-opioid receptor antagonist β-funaltrexamine and the μ1-opioid receptor antagonist naloxonazine. TAPA-induced antinociception was also significantly suppressed by co-administration of the μ1-opioid receptor antagonist Tyr-d-Pro-Phe-Phe-NH2 (d-Pro2-endomorphin-2) but not by co-administration of the μ2-opioid receptor antagonists Tyr-d-Pro-Trp-Phe-NH2 (d-Pro2-endomorphin-1) and Tyr-d-Pro-Trp-Gly-NH2 (d-Pro2-Tyr-W-MIF-1). In CXBK mice whose μ1-opioid receptors were naturally reduced, the antinociceptive effect of TAPA was markedly suppressed compared to the parental strain C57BL/6ByJ mice. Moreover, the antinociception induced by TAPA was significantly attenuated by i.t. pretreatment with antiserum against the endogenous κ-opioid peptide α-neo-endorphin but not antisera against other endogenous opioid peptides. In prodynorphin-deficient mice, the antinociceptive effect of TAPA was significantly reduced compared to wild-type mice. These results suggest that the spinal antinociception induced by TAPA is mediated in part through the release of α-neo-endorphin in the spinal cord via activation of spinal μ1-opioid receptors.

Identification of Dmt-D-Lys-Phe-Phe-OH as a highly antinociceptive tetrapeptide metabolite of the opioid-neurotensin hybrid peptide PK20

BackgroundRecently, we presented a novel compound (PK20, Dmt-D-Lys-Phe-Phe-Lys-Lys-Pro-Phe-Tle-Leu-OH) that targets single entity opioid and neurotensin pharmacophores. This endomorphin-2-like opioid peptide was introduced as a highly active analgesic because it elicited a strong dose- and time-dependent antinociceptive response when administered centrally and peripherally. Its pain-relieving activity was observed as rapidly as 5min after drug injection. Such promising results led us to perform further studies, such as determining the resistance to enzymatic degradation, which resulted in obtaining a very stable opioid pharmacore PK20 metabolite. MethodsThe synthesis of PK20 and its N-terminal tetrapeptide fragment has been accomplished using solid phase peptide chemistry. The biological stability of peptides has been measured in human serum and analyzed by HPLC/MS. Peptides were pharmacologically characterized in in vitro MOP and DOP receptor binding as well as [35S]GTPγS receptor binding assays. Antinociceptive properties of compounds were measured by in vivo assays in C57Bl6 mice after intravenous or intrathecal applications. ResultsDmt-D-Lys-Phe-Phe-OH (PK20M), an N-terminal tetrapeptide metabolite of the opioid-neurotensin hybrid peptide PK20, is characterized by a long duration of action, as demonstrated by a preserved, long-lasting analgesic effect even 2h post-injection (average % MPE=69.33). In rat brain membranes, PK20M efficiently displaced both the MOP and DOP receptor selective radioprobes [3H]DAMGO and [3H]DIDI (pKi of 9.52 and 7.86, respectively) and potently stimulated [35S]GTPγS binding, proving full agonism at both receptor types. In the [35S]GTPγS assay, which measured the agonist-mediated G protein activation, PK20M together with PK20 and Met-enkephalin were potent stimulators of the regulatory G proteins. The relative affinities of PK20M for the μ and δ receptor subtypes revealed μ-receptor selectivity. ConclusionThe novel MOP receptor selective metabolite has been shown to possess opioid subtype receptor selectivity, high potency, and effective analgesic activities as measured in various bioassays.

The N-terminal tetra-peptide (IPDE) short extension of the U-box motif in rice SPL11 E3 is essential for the interaction with E2 and ubiquitin-ligase activity

Rice, a monocot model plant, contains at least 77 U-box E3 ubiquitin (Ub)-ligases and 48 E2 Ub-conjugating enzymes. Here, we investigated the minimal binding domain of rice SPL11 U-box E3 to its E2 partners. Serial deletions and site-directed mutagenesis analyses indicated that, in addition to an intact U-box motif, the N-terminal tetra-peptide (IPDE) short extension of the U-box was essential for the interaction of SPL11 with E2s and Ub-ligase activity. The Ile and Pro residues at the –4 and –3 positions of the U-box, respectively, were crucial for this interaction. These results suggest that the N-terminal tetra-peptide extension of the U-box participates in the specific interaction of SPL11 E3 with E2s in a sequence-specific manner in rice.

Structure and function of a cyanophage-encoded peptide deformylase.

Bacteriophages encode auxiliary metabolic genes that support more efficient phage replication. For example, cyanophages carry several genes to maintain host photosynthesis throughout infection, shuttling the energy and reducing power generated away from carbon fixation and into anabolic pathways. Photodamage to the D1/D2 proteins at the core of photosystem II necessitates their continual replacement. Synthesis of functional proteins in bacteria requires co-translational removal of the N-terminal formyl group by a peptide deformylase (PDF). Analysis of marine metagenomes to identify phage-encoded homologs of known metabolic genes found that marine phages carry PDF genes, suggesting that their expression during infection might benefit phage replication. We identified a PDF homolog in the genome of Synechococcus cyanophage S-SSM7. Sequence analysis confirmed that it possesses the three absolutely conserved motifs that form the active site in PDF metalloproteases. Phylogenetic analysis placed it within the Type 1B subclass, most closely related to the Arabidopsis chloroplast PDF, but lacking the C-terminal α-helix characteristic of that group. PDF proteins from this phage and from Synechococcus elongatus were expressed and characterized. The phage PDF is the more active enzyme and deformylates the N-terminal tetrapeptides from D1 proteins more efficiently than those from ribosomal proteins. Solution of the X-ray/crystal structures of those two PDFs to 1.95 Å resolution revealed active sites identical to that of the Type 1B Arabidopsis chloroplast PDF. Taken together, these findings show that many cyanophages encode a PDF with a D1 substrate preference that adds to the repertoire of genes used by phages to maintain photosynthetic activities.

Sugar furanoid trans-vicinal diacid as a γ-turn inducer: synthesis and conformational study

A simple method for the synthesis of a sugar furanoid trans vicinal diacid and its incorporation into the N-terminal tetrapeptide sequence (H-Phe-Trp-Lys-Thr-OH) to get glycopeptide has been described. 2D NMR and MD simulation studies of clearly show that the sugar diacid adopts a γ-turn conformation towards the N-terminus.