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In the indeterminate nodules of a model legume Medicago truncatula, ∼700 nodule-specific cysteine-rich (NCR) peptides with conserved cysteine signature are expressed. NCR peptides are highly diverse in sequence, and some of these cationic peptides exhibit antimicrobial activity in vitro and in vivo. However, there is a lack of knowledge regarding their structural architecture, antifungal activity, and modes of action against plant fungal pathogens. Here, the three-dimensional NMR structure of the 36-amino acid NCR044 peptide was solved. This unique structure was largely disordered and highly dynamic with one four-residue α-helix and one three-residue antiparallel β-sheet stabilized by two disulfide bonds. NCR044 peptide also exhibited potent fungicidal activity against multiple plant fungal pathogens, including Botrytis cinerea and three Fusarium spp. It inhibited germination in quiescent spores of B. cinerea. In germlings, it breached the fungal plasma membrane and induced reactive oxygen species. It bound to multiple bioactive phosphoinositides in vitro. Time-lapse confocal and superresolution microscopy revealed strong fungal cell wall binding, penetration of the cell membrane at discrete foci, followed by gradual loss of turgor, subsequent accumulation in the cytoplasm, and elevated levels in nucleoli of germlings. Spray-applied NCR044 significantly reduced gray mold disease symptoms caused by the fungal pathogen B. cinerea in tomato and tobacco plants, and postharvest products. Our work illustrates the antifungal activity of a structurally unique NCR peptide against plant fungal pathogens and paves the way for future development of this class of peptides as a spray-on fungistat/fungicide.

RecQ C-terminal (RQC) domain is known as the main DNA binding module of RecQ helicases such as Bloom syndrome protein (BLM) and Werner syndrome protein (WRN) that recognizes various DNA structures. Even though BLM is able to resolve various DNA structures similarly to WRN, BLM has different binding preferences for DNA substrates from WRN. In this study, we determined the solution structure of the RQC domain of human BLM. The structure shares the common winged-helix motif with other RQC domains. However, half of the N-terminal has unstructured regions (α1–α2 loop and α3 region), and the aromatic side chain on the top of the β-hairpin, which is important for DNA duplex strand separation in other RQC domains, is substituted with a negatively charged residue (D1165) followed by the polar residue (Q1166). The structurally distinctive features of the RQC domain of human BLM suggest that the DNA binding modes of the BLM RQC domain may be different from those of other RQC domains.

Uniformly [sup.13]C- and [sup.15]N-labeled samples ensure fast and reliable nuclear magnetic resonance (NMR) assignments of proteins and are commonly used for structure elucidation by NMR. However, the preparation of uniformly labeled samples is a labor-intensive and expensive step. Reducing the portion of [sup.13]C-labeled glucose by a factor of five using a fractional 20% [sup.13]C- and 100% [sup.15]N-labeling scheme could lower the total chemical costs, yet retaining sufficient structural information of uniformly [[sup.13]C, [sup.15]N]-labeled sample as a result of the improved sensitivity of NMR instruments. Moreover, fractional [sup.13]C-labeling can facilitate reliable resonance assignments of sidechains because of the biosynthetic pathways of each amino-acid. Preparation of only one [20% [sup.13]C, 100% [sup.15]N]-labeled sample for small proteins (<15 kDa) could also eliminate redundant sample preparations of 100% [sup.15]N-labeled and uniformly 100% [[sup.13]C, [sup.15]N]-labeled samples of proteins. We determined the NMR structures of a small alpha-helical protein, the C domain of IgG-binding protein A from Staphylococcus aureus (SpaC), and a small beta-sheet protein, CBM64 module using [20% [sup.13]C, 100% [sup.15]N]-labeled sample and compared with the crystal structures and the NMR structures derived from the 100% [[sup.13]C, [sup.15]N]-labeled sample. Our results suggest that one [20% [sup.13]C, 100% [sup.15]N]-labeled sample of small proteins could be routinely used as an alternative to conventional 100% [[sup.13]C, [sup.15]N]-labeling for backbone resonance assignments, NMR structure determination, [sup.15]N-relaxation analysis, and ligand–protein interaction.

The membrane-proximal external region (MPER) of the HIV-1 envelope glycoprotein (Env) bears epitopes of broadly neutralizing antibodies (bnAbs) from infected individuals; it is thus a potential vaccine target. We report an NMR structure of the MPER and its adjacent transmembrane domain in bicelles that mimic a lipid-bilayer membrane. The MPER lies largely outside the lipid bilayer. It folds into a threefold cluster, stabilized mainly by conserved hydrophobic residues and potentially by interaction with phospholipid headgroups. Antigenic analysis and comparison with published images from electron cryotomography of HIV-1 Env on the virion surface suggest that the structure may represent a prefusion conformation of the MPER, distinct from the fusionintermediate state targeted by several well-studied bnAbs. Very slow bnAb binding indicates that infrequent fluctuations of the MPER structure give these antibodies occasional access to alternative conformations of MPER epitopes. Mutations in the MPER not only impede membrane fusion but also influence presentation of bnAb epitopes in other regions. These results suggest strategies for developing MPER-based vaccine candidates.

The cAMP-mediated allosteric transition in the catabolite activator protein (CAP; also known as the cAMP receptor protein, CRP) is a textbook example of modulation of DNA-binding activity by small-molecule binding. Here we report the structure of CAP in the absence of cAMP, which, together with structures of CAP in the presence of cAMP, defines atomic details of the cAMP-mediated allosteric transition. The structural changes, and their relationship to cAMP binding and DNA binding, are remarkably clear and simple. Binding of cAMP results in a coil-to-helix transition that extends the coiled-coil dimerization interface of CAP by 3 turns of helix and concomitantly causes rotation, by [almost equal to]60°, and translation, by [almost equal to]7 Å, of the DNA-binding domains (DBDs) of CAP, positioning the recognition helices in the DBDs in the correct orientation to interact with DNA. The allosteric transition is stabilized further by expulsion of an aromatic residue from the cAMP-binding pocket upon cAMP binding. The results define the structural mechanisms that underlie allosteric control of this prototypic transcriptional regulatory factor and provide an illustrative example of how effector-mediated structural changes can control the activity of regulatory proteins.

Long-range HNCO NMR spectra for proteins show crosspeaks due to 1JNC′, 2JNC′, 3JNCγ, and h3JNC′ couplings. The h3JNC′ couplings are transmitted through hydrogen bonds and their sizes are correlated to hydrogen bond lengths. We collected long-range HNCO data at a series of temperatures for four protein structures. P22i and CUS-3i are six-stranded beta-barrel I-domains from phages P22 and CUS-3 that share less than 40% sequence identity. The cis and trans states of the C-terminal domain from pore-forming toxin hemolysin ΙΙ (HlyIIC) arise from the isomerization of a single G404-P405 peptide bond. For P22i and CUS-3i, hydrogen bonds detected by NMR agree with those observed in the corresponding domains from cryoEM structures of the two phages. Hydrogen bond lengths derived from the h3JNC′ couplings, however, are poorly conserved between the distantly related CUS-3i and P22i domains and show differences even between the closely related cis and trans state structures of HlyIIC. This is consistent with hydrogen bond lengths being determined by local differences in structure rather than the overall folding topology. With increasing temperature, hydrogen bonds typically show an apparent increase in length that has been attributed to protein thermal expansion. Some hydrogen bonds are invariant with temperature, however, while others show apparent decreases in length, suggesting they become stabilized with increasing temperature. Considering the data for the three proteins in this study and previously published data for ubiquitin and GB3, lowered protein folding stability and cooperativity corresponds with a larger range of temperature responses for hydrogen bonds. This suggests a partial uncoupling of hydrogen bond energetics from global unfolding cooperativity as protein stability decreases.

NMR chemical shifts provide important local structural information for proteins. Consistent structure generation from NMR chemical shift data has recently become feasible for proteins with sizes of up to 130 residues, and such structures are of a quality comparable to those obtained with the standard NMR protocol. This study investigates the influence of the completeness of chemical shift assignments on structures generated from chemical shifts. The Chemical-Shift-Rosetta (CS-Rosetta) protocol was used for de novo protein structure generation with various degrees of completeness of the chemical shift assignment, simulated by omission of entries in the experimental chemical shift data previously used for the initial demonstration of the CS-Rosetta approach. In addition, a new CS-Rosetta protocol is described that improves robustness of the method for proteins with missing or erroneous NMR chemical shift input data. This strategy, which uses traditional Rosetta for pre-filtering of the fragment selection process, is demonstrated for two paramagnetic proteins and also for two proteins with solid-state NMR chemical shift assignments.

Immune checkpoints are crucial in the maintenance of antitumor immune responses. The activation or blockade of immune checkpoints is dependent on the interactions between receptors and ligands; such interactions can provide inhibitory or stimulatory signals, including the enhancement or suppression of T-cell proliferation, differentiation, and/or cytokine secretion. B-and T-lymphocyte attenuator (BTLA) is a lymphoid-specific cell surface receptor which is present on T-cells and interacts with herpes virus entry mediator (HVEM), which is present on tumor cells. The binding of HVEM to BTLA triggers an inhibitory signal which attenuates the immune response. This feature is interesting for studying the molecular interactions between HVEM and BTLA, as they may be targeted for novel immunotherapies. This work was based on the crystal structure of the BTLA/HVEM complex showing that BTLA binds the N-terminal cysteine-rich domain of HVEM. We investigated the amino acid sequence of HVEM and used molecular modeling methods to develop inhibitors of the BTLA/HVEM interaction. We synthesized novel compounds and determined their ability to interact with the BTLA protein and inhibit the formation of the BTLA/HVEM complex. Our results suggest that the HVEM (14–39) peptide is a potent inhibitor of the formation of the BTLA/HVEM protein complex.

Out of the 14 avian β-defensins identified in the Gallus gallus genome, only 3 are present in the chicken egg, including the egg-specific avian β-defensin 11 (Gga-AvBD11). Given its specific localization and its established antibacterial activity, Gga-AvBD11 appears to play a protective role in embryonic development. Gga-AvBD11 is an atypical double-sized defensin, predicted to possess 2 motifs related to β-defensins and 6 disulfide bridges. The 3-dimensional NMR structure of the purified Gga-AvBD11 is a compact fold composed of 2 packed β-defensin domains. This fold is the archetype of a structural family, dubbed herein as avian-double-β-defensins (Av-DBD). We speculate that AvBD11 emanated from a monodomain gene ancestor and that similar events might have occurred in arthropods, leading to another structural family of less compact DBDs. We show that Gga-AvBD11 displays antimicrobial activities against gram-positive and gram-negative bacterial pathogens, the avian protozoan Eimeria tenella, and avian influenza virus. Gga-AvBD11 also shows cytotoxic and antiinvasive activities, suggesting that it may not only be involved in innate protection of the chicken embryo, but also in the (re)modeling of embryonic tissues. Finally, the contribution of either of the 2 Gga-AvBD11 domains to these biological activities was assessed, using chemically synthesized peptides. Our results point to a critical importance of the cationic N-terminal domain in mediating antibacterial, antiparasitic, and antiinvasive activities, with the C-terminal domain potentiating the 2 latter activities. Strikingly, antiviral activity in infected chicken cells, accompanied by marked cytotoxicity, requires the full-length protein.