Structural and functional analysis of Cyanovirin-N homologs: Carbohydrate binding affinities and antiviral potential of cyanobacterial peptides.

Cyanobacteria, a group of photosynthetic prokaryotes, can sinthesize several substances due to their secondary metabolism, with notable properties, such as Cyanovirin-N(CVN), a carbohydrate-binding lectin, that exhibits antiviral activity against several pathogens, due to its ability to bind viral surface carbohydrates such as mannose, thus interfering with the viral entry on the cell. CVN has been described in several cyanobacterial strains and shows biotechnological potential for the development of drugs of pharmaceutical interest. This study focuses on the genomic exploration and characterization of Cyanovirin-N homologs to assess the conservation of carbohydrate-binding affinity within the group. The analysis of their antiviral properties was carried out using bioinformatics tools to study protein models through an in silico pipeline, following the steps of genomic prospection on public databases, homology modeling, docking, molecular dynamics and energetic analysis. Mannose served as the reference ligand, and the lectins' binding affinity with mannose was assessed across Cyanovirin-N homologs. Genomic mining identified 33 cyanobacterial lectin sequences, which underwent structural and functional characterization. The results obtained from this work indicate strong carbohydrate affinity on several homologs, pointing to the conservation of antiviral properties alongside the group. However, this affinity was not uniformly distributed among sequences, exhibiting significant heterogeneity in binding site residues, suggesting potential multi-ligand binding capabilities on the Cyanovirin-N homologs group. Studies focused on the properties involved in these molecules and the investigation of the genetic diversity of Cyanovirin-N homologs could provide valuable insights into the discovery of new drug candidates, harvesting the potential of bioinformatics for large-scale functional and structural analysis.

The transport activity of the multidrug ABC transporter BmrA does not require a wide separation of the nucleotide-binding domains.

ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins responsible for the translocation of a wide diversity of substrates across biological membranes. Some of them confer multidrug or antimicrobial resistance to cancer cells and pathogenic microorganisms, respectively. Despite a wealth of structural data gained in the last two decades, the molecular mechanism of these multidrug efflux pumps remains elusive, including the extent of separation between the two nucleotide-binding domains (NBDs) during the transport cycle. Based on recent outward-facing structures of BmrA, a homodimeric multidrug ABC transporter from Bacillus subtilis, we introduced a cysteine mutation near the C-terminal end of the NBDs to analyze the impact of disulfide-bond formation on BmrA function. Interestingly, the presence of the disulfide bond between the NBDs did not prevent the ATPase, nor did it affect the transport of Hoechst 33342 and doxorubicin. Yet, the 7-amino-actinomycin D was less efficiently transported, suggesting that a further opening of the transporter might improve its ability to translocate this larger compound. We solved by cryo-EM the apo structures of the cross-linked mutant and the WT protein. Both structures are highly similar, showing an intermediate opening between their NBDs while their C-terminal extremities remain in close proximity. Distance measurements obtained by electron paramagnetic resonance spectroscopy support the intermediate opening found in these 3D structures. Overall, our data suggest that the NBDs of BmrA function with a tweezers-like mechanism distinct from the related lipid A exporter MsbA.

Residues within the LptC transmembrane helix are critical for Escherichia coli LptB2 FG ATPase regulation.

Lipopolysaccharide (LPS) synthesis in Gram-negative bacteria is completed at the outer leaflet of the inner membrane (IM). Following synthesis, seven LPS transport (Lpt) proteins facilitate the movement of LPS to the outer membrane (OM), an essential process that if disrupted at any stage has lethal effects on bacterial viability. LptB2 FG, the IM component of the Lpt bridge system, is a type VI ABC transporter that provides the driving force for LPS extraction from the IM and subsequent transport across a stable protein bridge to the outer leaflet of the OM. LptC is a periplasmic protein anchored to the IM by a single transmembrane (TM) helix intercalating within the lateral gate formed by LptF TM5 and LptG TM1. LptC facilitates the hand-off of LPS from LptB2 FG to the periplasmic protein LptA and has been shown to regulate the ATPase activity of LptB2 FG. Here, using an engineered chromosomal knockout system in Escherichia coli to assess the effects of LptC mutations in vivo, we identified six partial loss of function LptC mutations in the first unbiased alanine screen of this essential protein. To investigate the functional effects of these mutations, nanoDSF (differential scanning fluorimetry) and site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy in combination with an in vitro ATPase assay show that specific residues in the TM helix of LptC destabilize the LptB2 FGC complex and regulate the ATPase activity of LptB.

Contributions from ClpS surface residues in modulating N-terminal peptide binding and their implications for NAAB development.

Numerous technologies are currently in development for use in next-generation protein sequencing platforms. A notable published approach employs fluorescently-tagged binding proteins to identity the N-terminus of immobilized peptides, in-between rounds of digestion. This approach makes use of N-terminal amino acid binder (NAAB) proteins, which would identify amino acids by chemical and shape complementarity. One source of NAABs is the ClpS protein family, which serve to recruit proteins to bacterial proteosomes based on the identity of the N-terminal amino acid. In this study, a Thermosynechococcus vestitus (also known as Thermosynechococcus elongatus) ClpS2 protein was used as the starting point for direct evolution of an NAAB with affinity and specificity for N-terminal leucine. Enriched variants were analyzed and shown to improve the interaction between the ClpS surface and the peptide chain, without increasing promiscuity. Interestingly, interactions were found that were unanticipated which favor different charged residues located at position 5 from the N-terminus of a target peptide.

Structure and function of a dual antagonist of the human growth hormone and prolactin receptors with site-specific PEG conjugates.

Human growth hormone (hGH) is a pituitary-derived endocrine protein that regulates several critical postnatal physiologic processes including growth, organ development, and metabolism. Following adulthood, GH is also a regulator of multiple pathologies like fibrosis, cancer, and diabetes. Therefore, there is a significant pharmaceutical interest in developing antagonists of hGH action. Currently, there is a single FDA-approved antagonist of the hGH receptor (hGHR) prescribed for treating patients with acromegaly and discovered in our laboratory almost 3 decades ago. Here, we present the first data on the structure and function of a new set of protein antagonists with the full range of hGH actions-dual antagonists of hGH binding to the GHR as well as that of hGH binding to the prolactin receptor. We describe the site-specific PEG conjugation, purification, and subsequent characterization using MALDI-TOF, size-exclusion chromatography, thermostability, and biochemical activity in terms of ELISA-based binding affinities with GHR and prolactin receptor. Moreover, these novel hGHR antagonists display distinct antagonism of GH-induced GHR intracellular signaling in vitro and marked reduction in hepatic insulin-like growth factor 1 output in vivo. Lastly, we observed potent anticancer biological efficacies of these novel hGHR antagonists against human cancer cell lines. In conclusion, we propose that these new GHR antagonists have potential for development towards multiple clinical applications related to GH-associated pathologies.

Combining Solid-State NMR with Structural and Biophysical Techniques to Design Challenging Protein-Drug Conjugates.

Several protein-drug conjugates are currently being used in cancer therapy. These conjugates rely on cytotoxic organic compounds that are covalently attached to the carrier proteins or that interact with them via non-covalent interactions. Human transthyretin (TTR), a physiological protein, has already been identified as a possible carrier protein for the delivery of cytotoxic drugs. Here we show the structure-guided development of a new stable cytotoxic molecule based on a known strong binder of TTR and a well-established anticancer drug. This example is used to demonstrate the importance of the integration of multiple biophysical and structural techniques, encompassing microscale thermophoresis, X-ray crystallography and NMR. In particular, we show that solid-state NMR has the ability to reveal effects caused by ligand binding which are more easily relatable to structural and dynamical alterations that impact the stability of macromolecular complexes.

Cross-Linking of the Nonribosomal Peptide Synthetase Adenylation Domain with a Carrier Protein Using a Pantetheine-Type Probe.

Adenylation domains (A-domains) are responsible for the selective incorporation of carboxylic acid substrates in the biosynthesis of nonribosomal peptides and related natural products. The A-domain transfers an acyl substrate onto its cognate carrier protein (CP). The proper interactions between an A-domain and the cognate CP are important for functional substrate transfer. To stabilize the transient interactions sufficiently for structural analysis of A-domain-CP complex, vinylsulfonamide adenosine inhibitors have been traditionally used as molecular probes. Recently, we have developed an alternative strategy using a synthetic pantetheine-type probe that enables site-specific cross-linking between an A-domain and a CP. In this chapter, we describe the laboratory protocols for this cross-linking reaction.

Discovery of Nanomolar DCAF1 Small Molecule Ligands.

DCAF1 is a substrate receptor of two distinct E3 ligases (CRL4DCAF1 and EDVP), plays a critical physiological role in protein degradation, and is considered a drug target for various cancers. Antagonists of DCAF1 could be used toward the development of therapeutics for cancers and viral treatments. We used the WDR domain of DCAF1 to screen a 114-billion-compound DNA encoded library (DEL) and identified candidate compounds using similarity search and machine learning. This led to the discovery of a compound (Z1391232269) with an SPR KD of 11 µM. Structure-guided hit optimization led to the discovery of OICR-8268 (26e) with an SPR KD of 38 nM and cellular target engagement with EC50 of 10 µM as measured by cellular thermal shift assay (CETSA). OICR-8268 is an excellent tool compound to enable the development of next-generation DCAF1 ligands toward cancer therapeutics, further investigation of DCAF1 functions in cells, and the development of DCAF1-based PROTACs.

Two broadly conserved families of polyprenyl-phosphate transporters.

Peptidoglycan and almost all surface glycopolymers in bacteria are built in the cytoplasm on the lipid carrier undecaprenyl phosphate (UndP)1-4. These UndP-linked precursors are transported across the membrane and polymerized or directly transferred to surface polymers, lipids or proteins. UndP is then flipped to regenerate the pool of cytoplasmic-facing UndP. The identity of the flippase that catalyses transport has remained unknown. Here, using the antibiotic amphomycin that targets UndP5-7, we identified two broadly conserved protein families that affect UndP recycling. One (UptA) is a member of the DedA superfamily8; the other (PopT) contains the domain DUF368. Genetic, cytological and syntenic analyses indicate that these proteins are UndP transporters. Notably, homologues from Gram-positive and Gram-negative bacteria promote UndP transport in Bacillus subtilis, indicating that recycling activity is broadly conserved among family members. Inhibitors of these flippases could potentiate the activity of antibiotics targeting the cell envelope.

Free energy simulations to study mutational effect of a conserved residue, Trp24, on stability of human serum retinol-binding protein.

Human serum retinol-binding protein (RBP) is a plasma transport protein for vitamin A. RBP is a prime subclass of lipocalins, which bind nonpolar ligands within a ß-barrel. To understand the role of Trp 24, one of the highly conserved residues in RBP, free energy simulations have been carried out to understand the effects of the mutations from Trp at position 24 to Leu, Phe, and Tyr in the apo-RBP on its thermal stability. We examine various unfolded systems to study the dependence of the free energy differences on the denatured structure. Our calculated free energy difference values for the three mutations are in excellent agreement with the experimental values when the initial coordinates of the seven-residue peptide segments truncated from the crystal structure are used for the denatured systems. Our free energy change differences for the Trp→Leu, Trp→Phe, and Trp→Tyr mutations are 2.50 ± 0.69, 2.58 ± 0.50, and 2.49 ± 0.48 kcal/mol, respectively, when the native-like seven-residue peptides are used as models for the denatured systems. The main contributions to the free energy change differences for the Trp24→Leu and Trp24→Phe mutations are mainly from van der Waals and covalent interactions, respectively. Electrostatic, van der Waals and covalent terms equally contribute to the free energy change difference for the Trp24→Tyr mutation. The free energy simulation helps understand the detailed microscopic mechanism of the stability of the RBP mutants relative to the wild type and the role of the highly conserved residue, Trp24, of the human RBP.Communicated by Ramaswamy H. Sarma.

The Role of PHF6 in Hematopoiesis and Hematologic Malignancies.

Epigenetic regulation of gene expression represents an important mechanism in the maintenance of stem cell function. Alterations in epigenetic regulation contribute to the pathogenesis of hematological malignancies. Plant homeodomain finger protein 6 (PHF6) is a member of the plant homeodomain (PHD)-like zinc finger family of proteins that is involved in transcriptional regulation through the modification of the chromatin state. Germline mutation of PHF6 is the causative genetic alteration of the X-linked mental retardation Borjeson-Forssman-Lehmann syndrome (BFLS). Somatic mutations in PHF6 are identified in human leukemia, such as adult T-cell acute lymphoblastic leukemia (T-ALL, ~ 38%), pediatric T-ALL (~ 16%), acute myeloid leukemia (AML, ~ 3%), chronic myeloid leukemia (CML, ~ 2.5%), mixed phenotype acute leukemia (MPAL, ~ 20%), and high-grade B-cell lymphoma (HGBCL, ~ 3%). More recent studies imply an oncogenic effect of PHF6 in B-cell acute lymphoblastic leukemia (B-ALL) and solid tumors. These data demonstrate that PHF6 could act as a double-edged sword, either a tumor suppressor or an oncogene, in a lineage-dependent manner. However, the underlying mechanisms of PHF6 in normal hematopoiesis and leukemogenesis remain largely unknown. In this review, we summarize current knowledge of PHF6, emphasizing the role of PHF6 in hematological malignancies. Epigenetic regulation of PHF6 in B-ALL. PHF6 maintains a chromatin structure that is permissive to B-cell identity genes, but not T-cell-specific genes (left). Loss of PHF6 leads to aberrant expression of B-cell- and T-cell-specific genes resulting from lineage promiscuity and binding of T-cell transcription factors (right).

AAA+ protease-adaptor structures reveal altered conformations and ring specialization.

ClpAP, a two-ring AAA+ protease, degrades N-end-rule proteins bound by the ClpS adaptor. Here we present high-resolution cryo-EM structures of Escherichia coli ClpAPS complexes, showing how ClpA pore loops interact with the ClpS N-terminal extension (NTE), which is normally intrinsically disordered. In two classes, the NTE is bound by a spiral of pore-1 and pore-2 loops in a manner similar to substrate-polypeptide binding by many AAA+ unfoldases. Kinetic studies reveal that pore-2 loops of the ClpA D1 ring catalyze the protein remodeling required for substrate delivery by ClpS. In a third class, D2 pore-1 loops are rotated, tucked away from the channel and do not bind the NTE, demonstrating asymmetry in engagement by the D1 and D2 rings. These studies show additional structures and functions for key AAA+ elements. Pore-loop tucking may be used broadly by AAA+ unfoldases, for example, during enzyme pausing/unloading.

Knowledge-driven design and optimization of potent symmetric anticancer molecules: A case study on PKM2 activators.

BACKGROUND: Pyruvate kinase M2 (PKM2) is preferentially expressed as a low-activity dimer over the active tetramer in proliferating tumor cells, resulting in metabolic reprogramming to achieve high energy requirements and nutrient uptake. This leads to a shift from the normal glycolytic pathway causing tumor cells to proliferate uncontrollably. This study utilizes knowledge-based drug discovery to determine the critical features from experimentally known PKM2 activators and design compounds that would significantly confer a stable structural and functional edge over the known compounds which are still at the preclinical stage. METHODS: Conscientious molecular modeling studies were carried out and critical structural features were identified and validated from the knowledge of experimentally known PKM2 activators to confer high-binding affinities. A virtual library of 200 palindromic and non-palindromic activators was designed based on these identified critical features to target a distinct activator binding-site. This binding would favor specific dimer-dimer association and subsequent protein tetramerization. The resultant compounds strongly correlated with identified structural features and binding affinities which further strengthened our findings. The designed activators were then subjected to pharmacokinetic profiling and toxicity prediction, followed by free-binding energy calculations and MD simulations. RESULTS: All the virtually designed activators comprising the identified critical features were observed to confer high-binding affinities ranging from -9.1 to -15.0 kcal/mol to the receptor protein. The designed activators also demonstrated optimum pharmacokinetic and toxicity profiles. CONCLUSION: The best activators selected for MD simulations studies were conclusively observed to stabilize the required tetrameric conformation suggesting that these activators could potentially target PKM2 tetramerization that might restore the normal glycolytic pathway and suppress tumor progression.

Engineering recombinantly expressed lectin-based antiviral agents.

Cyanovirin-N (CV-N), a lectin from Nostoc ellipsosporum was found an infusion inhibitory protein for human immunodeficiency virus (HIV)-1. A tandem-repeat of the engineered domain-swapped dimer bound specific sites at hemagglutinin (HA), Ebola and HIV spike glycoproteins as well as dimannosylated HA peptide, N-acetyl-D-glucosamine and high-mannose containing oligosaccharides. Among these, CV-N bound the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein at a dissociation constant (KD) of 18.6 µM (and KD=260 µM to RBD), which was low-affinity carbohydrate-binding as compared with the recognition of the other viral spikes. Binding of dimannosylated peptide to homo-dimeric CVN2 and variants of CVN2 that were pairing Glu-Arg residues sterically located close to its high-affinity carbohydrate binding sites, was measured using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Binding affinity increased with polar interactions, when the mutated residues were used to substitute a single, or two disulfide bonds, in CVN2. Site-specific N-linked glycans on spikes were mediating the infection with influenza virus by broadly neutralizing antibodies to HA and lectin binding to HA was further investigated via modes of saturation transfer difference (STD)-NMR. Our findings showed that stoichiometry and the lectin's binding affinity were revealed by an interaction of CVN2 with dimannose units and either the high- or low-affinity binding site. To understand how these binding mechanisms add to viral membrane fusion we compare our tested HA-derived peptides in affinity with SARS-CoV-2 glycoprotein and review lectins and their mechanisms of binding to enveloped viruses for a potential use to simulate neutralization ability.

Electrostatic Contributions to the Binding Free Energy of Nicotine to the Acetylcholine Binding Protein.

Biomolecular binding relies on specific attractive interactions between two partner molecules, including electrostatics, dispersion, hydrophobicity, and solvation. Assessing the contributions of electrostatic interactions to binding is key to the understanding of ligand binding mechanisms and the design of improved biomolecular binders. For example, nicotine is a well-known agonist of nicotinic acetylcholine receptors (nAChRs), but the molecular mechanisms for the differential action of nicotine on brain and muscle nAChRs remain elusive. In this work, we have chosen the acetylcholine binding protein (AChBP) in complex with nicotine as a model system to interrogate the electrostatic contributions to nicotine binding. Our absolute binding free energy simulations confirm that nicotine binds AChBP predominantly in its protonated (charged) form. By comparing energetic contributions from decomposed interactions for either neutral or charged nicotine, our calculations shed light on the nature of the binding of nicotine to the AChBP. The preferred binding of charged nicotine over neutral nicotine originates from its stronger electrostatic interactions with AChBP, a cation-π interaction to a tryptophan residue and a hydrogen bond between nicotine and the backbone carbonyl of the tryptophan, whereas the major force driving the binding process appears to be van der Waals interactions. The various nonelectrostatic terms can also indirectly modulate the electrostatic interactions through fine-tuning the binding pose of the ligand in the binding site, providing an explanation of why the binding specificity of nicotine to the brain versus muscle nAChRs is driven by electrostatic interaction, given that the immediate binding site residues, including the key tryptophan residue, are identical in the two receptors.

Structure of Gas Phase Monohydrated Nicotine: Implications for Nicotine's Native Structure in the Acetylcholine Binding Protein.

We report a joint experimental-theoretical study of the never reported before structure and infrared spectra of gas phase monohydrated nicotine (NIC) and nornicotine (NOR) and use them to assign their protonation sites. NIC's biological activity is strongly affected by its protonation site, namely, the pyrrolidine (Pyrro-NICH+, anticipated active form) and pyridine (Pyri-NICH+) forms; however, these have yet to be directly experimentally determined in either the nicotinic acetylcholine receptor (nAChR, no water present) or the acetylcholine-binding protein (AChBP, a single water molecule is present) but can only be inferred to be Pyrro-NICH+ from the intermolecular distance to the neighboring residues (i.e., tryptophan). Our temperature-controlled double ion trap infrared spectroscopic experiments assisted by the collisional stripping method and high-level theoretical calculations yield the protonation ratio of Pyri:Pyrro = 8:2 at 240 K for the gas phase NICH+···(H2O) complex, which resembles the molecular cluster present in the AChBP. Therefore, a single water molecule in the gas phase enhances this ratio in NICH+ relative to the 3:2 for the nonhydrated gas phase NICH+ in a trend that contrasts with the almost exclusive presence of Pyrro-NICH+ in aqueous solution. In contrast, the Pyri-NORH+ protomer is exclusively observed, a fact that may correlate with its weaker biological activity.

The point mutation of the cholesterol trafficking membrane protein NPC1 may affect its proper function in more than a single step: Molecular dynamics simulation study.

The Niemann-Pick type C1 (NPC1) protein is one of the key players of cholesterol trafficking from the lysosome and its function is closely coupled with the Niemann-Pick type C2 (NPC2) protein. The dysfunction of one of these proteins can cause problems in the overall cholesterol homeostasis and leads to a disease, which is called the Niemann-Pick type C (NPC) disease. The parts of the cholesterol transport mechanism by NPC1 have begun to recently emerge, especially after the full-length NPC1 structure was determined from a cryo-EM study. However, many details about the overall cholesterol trafficking process by NPC1 still remain to be elucidated. Notably, the NPC1 could act as one of the target proteins for the control of infectious diseases due to its role as the virus entry point into the cells as well as for cancer treatment due to the inhibitory effect of tumor growth. A mutation of NPC1 can leads to dysfunctions and understanding this process can provide valuable insights into the mechanisms of the corresponding protein and the therapeutic strategies against the disease that are caused by the mutation. It has been found that patients with the point mutation R518W (or R518Q) on the NPC1 show the accumulation of lipids within the lysosomal lumen. In this paper, we report how the corresponding mutation can affect the cholesterol transport process by NPC1 in the different stages by the molecular dynamics simulations. The simulation results show that the point mutation intervenes at least at two different steps during the cholesterol transport by NPC1 and NPC2 in combination, which includes the association step of NPC2 with the NPC1, the cholesterol transfer step from NPC2 to NPC1-NTD while the cholesterol passage within the NPC1 via a channel is relatively unaffected by R518W mutation. The detailed analysis of the resulting simulation trajectories reveals the important structural features that are essential for the proper functioning of the NPC1 for the cholesterol transport, and it shows how the overall structure, which thereby includes the function, can be affected by a single mutation.

The UVSSA protein is part of a genome integrity homeostasis network with links to transcription-coupled DNA repair and ATM signaling.

SignificanceTranscription-coupled repair (TCR) involves four core proteins: CSA, CSB, USP7, and UVSSA. CSA and CSB are mutated in the severe human neurocutaneous disease Cockayne syndrome. In contrast UVSSA is a mild photosensitive disease in which a mutated protein sequence prevents recruitment of USP7 protease to deubiquitinate and stabilize CSB. We deleted the UVSSA protein using CRISPR-Cas9 in an aneuploid cell line, HEK293, and determined the functional consequences. The knockout cell line was sensitive to transcription-blocking lesions but not sensitive to oxidative agents or PARP inhibitors, unlike CSB. Knockout of UVSSA also activated ATM, like CSB, in transcription-arrested cells. The phenotype of UVSSA, especially its rarity, suggests that many TCR-deficient patients and tumors fail to be recognized clinically.

Characterization of silicon transporter gene family in Saccharum and functional analysis of the ShLsi6 gene in biotic stress.

Silicon, one of the most prevalent elements in the soil, is beneficial for plant growth and defense against different stresses. The silicon transporter gene (Lsi) plays an important role in the uptake and transport of silicon in higher plants. In this study, a total of 32 Lsi genes, including 20 SsLsi in sugarcane wild species Saccharum spontaneum, 5 ShLsi in Saccharum hybrid cultivar R570 and 7 SbLsi in sugarcane related species Sorghum bicolor, were identified and classified into three groups. Bioinformatics analysis showed that instability, hydrophobicity, localization of cell membranes and vacuoles were the main features of the Lsi proteins. Whole genome and segmental duplication contributed to the main expansion of Lsi gene family. Collinearity analysis of the Lsi genes showed that S. spontanum and R570 had a collinear relationship with monocotyledonous plants S. bicolor and Oryza sativa, but not with dicotyledonous plants Arabidopsis thaliana and Vitis vinifera. The replicated Lsi genes were mainly subjected to strong selection pressure for purification. The diverse cis-regulatory elements in the promoter of SsLsi, ShLsi and SbLsi genes suggested that they were widely involved in the response of plants to various stresses and the regulation of the growth and development. Transcriptome data and real time quantitative PCR analysis showed that the Lsi genes exhibited different expression profiles in sugarcane tissues and under Sporisorium scitamineum, drought and cold stresses. In addition, the cDNA and genomic DNA sequences of ShLsi6 that was homologous to SsLsi1b gene was cloned from Saccharum hybrid cultivar ROC22. Transient expression analysis showed that, compared with the control, Nicotiana benthamiana leaves which overexpressed the ShLsi6 gene showed a high sensitivity after inoculation with tobacco pathogens Ralstonia solanacearum and Fusarium solani var. coeruleum. This study provides important information for further functional analysis of Lsi genes and resistant breeding in sugarcane.

Structural basis for peptide recognition by archaeal oligopeptide permease A.

Oligopeptide permease A (OppA) plays an important role in the nutrition of cells and various signaling processes. In archaea, OppA is a major protein present in membrane vesicles of Thermococcales. Because there being no crystal structures of archaeal OppAs determined to date, we report the crystal structure of archaeal OppA from Thermococcus kodakaraensis (TkOppA) at 2.3 Å resolution by the single-wavelength anomalous dispersion method. TkOppA consists of three domains similarly to bacterial OppAs, and the inserted regions not present in bacterial OppAs are at the periphery of the core region. An endogenous pentapeptide was bound in the pocket of domains I and III of TkOppA by hydrogen bonds of main-chain atoms of the peptide and hydrophobic interactions. No hydrogen bonds of side-chain atoms of the peptide were observed; thus, TkOppA may have low peptide selectivity but some preference for residues 2 and 3. TkOppA has a relatively large pocket and can bind a nonapeptide; therefore, it is suitable for the binding of large peptides similarly to OppAs of Gram-positive bacteria.