The structural landscape and diversity of Pyricularia oryzae MAX effectors revisited.

Magnaporthe AVRs and ToxB-like (MAX) effectors constitute a family of secreted virulence proteins in the fungus Pyricularia oryzae (syn. Magnaporthe oryzae), which causes blast disease on numerous cereals and grasses. In spite of high sequence divergence, MAX effectors share a common fold characterized by a ß-sandwich core stabilized by a conserved disulfide bond.

Structure, dynamics and transferability of the metal-dependent polyhistidine tetramerization motif TetrHis for single-chain Fv antibodies.

The polyhistidine (6XHis) motif is one of the most ubiquitous protein purification tags. The 6XHis motif enables the binding of tagged proteins to various metals, which can be advantageously used for purification with immobilized metal affinity chromatography. Despite its popularity, protein structures encompassing metal-bound 6XHis are rare.

Structure-guided mutagenesis of the capsid protein indicates that a nanovirus requires assembled viral particles for systemic infection.

Nanoviruses are plant multipartite viruses with a genome composed of six to eight circular single-stranded DNA segments. The distinct genome segments are encapsidated individually in icosahedral particles that measure ≈18 nm in diameter. Recent studies on the model species Faba bean necrotic stunt virus (FBNSV) revealed that complete sets of genomic segments rarely occur in infected plant cells and that the function encoded by a given viral segment can complement the others across neighbouring cells, presumably by translocation of the gene products through unknown molecular processes.

The Syp1/FCHo2 protein induces septin filament bundling through its intrinsically disordered domain.

The septin collar of budding yeast is an ordered array of septin filaments that serves a scaffolding function for the cytokinetic machinery at the bud neck and compartmentalizes the membrane between mother and daughter cell. How septin architecture is aided by septin-binding proteins is largely unknown. Syp1 is an endocytic protein that was implicated in the timely recruitment of septins to the newly forming collar through an unknown mechanism.

Unravelling the Adaptation Mechanisms to High Pressure in Proteins.

Life is thought to have appeared in the depth of the sea under high hydrostatic pressure. Nowadays, it is known that the deep biosphere hosts a myriad of life forms thriving under high-pressure conditions. However, the evolutionary mechanisms leading to their adaptation are still not known.

A Flexible and Original Architecture of Two Unrelated Zinc Fingers Underlies the Role of the Multitask P1 in RYMV Spread.

Viruses of the sobemovirus genus are plant viruses, most of which generate very important agricultural and financial losses. Among them, the rice yellow mottle virus (RYMV) is one of the most damaging pathogens devastating rice fields in Africa. RYMV infectivity and propagation rely on its protein P1, identified as a key movement and potential long-distance RNA silencing suppressor.

Structural insights into recognition of chemokine receptors by leukotoxins.

(SA) leukocidin ED (LukED) belongs to a family of bicomponent pore forming toxins that play important roles in SA immune evasion and nutrient acquisition. LukED targets specific G protein-coupled chemokine receptors to lyse human erythrocytes (red blood cells) and leukocytes (white blood cells). The first recognition step of receptors is critical for specific cell targeting and lysis.

Agonists and allosteric modulators promote signaling from different metabotropic glutamate receptor 5 conformations.

Metabotropic glutamate receptors (mGluRs) are dimeric G-protein-coupled receptors activated by the main excitatory neurotransmitter, L-glutamate. mGluR activation by agonists binding in the venus flytrap domain is regulated by positive (PAM) or negative (NAM) allosteric modulators binding to the 7-transmembrane domain (7TM). We report the cryo-electron microscopy structures of fully inactive and intermediate-active conformations of mGlu receptor bound to an antagonist and a NAM or an agonist and a PAM, respectively, as well as the crystal structure of the 7TM bound to a photoswitchable NAM.

Structure of a human intramembrane ceramidase explains enzymatic dysfunction found in leukodystrophy.

Alkaline ceramidases (ACERs) are a class of poorly understood transmembrane enzymes controlling the homeostasis of ceramides. They are implicated in human pathophysiology, including progressive leukodystrophy, colon cancer as well as acute myeloid leukemia. We report here the crystal structure of the human ACER type 3 (ACER3).

Structural determinants of the catalytic mechanism of Plasmodium CCT, a key enzyme of malaria lipid biosynthesis.

The development of the malaria parasite, Plasmodium falciparum, in the human erythrocyte, relies on phospholipid metabolism to fulfil the massive need for membrane biogenesis. Phosphatidylcholine (PC) is the most abundant phospholipid in Plasmodium membranes. PC biosynthesis is mainly ensured by the de novo Kennedy pathway that is considered as an antimalarial drug target.

C Ribbon-Like Structures in Hybrid Peptides Alternating α- and Thiazole-Based γ-Amino Acids.

According to their restricted conformational freedom, heterocyclic γ-amino acids are usually considered to be related to Z-vinylogous γ-amino acids. In this context, oligomers alternating α-amino acids and thiazole-based γ-amino acids (ATCs) were expected to fold into a canonical 12-helical shape as described for α/γ-hybrid peptides composed of cis-α/β-unsaturated γ-amino acids. However, through a combination of X-ray crystallography, NMR spectroscopy, FTIR experiments, and DFT calculations, it was determined that the folding behavior of ATC-containing hybrid peptides is much more complex.

Structural insights into adiponectin receptors suggest ceramidase activity.

Adiponectin receptors (ADIPORs) are integral membrane proteins that control glucose and lipid metabolism by mediating, at least in part, a cellular ceramidase activity that catalyses the hydrolysis of ceramide to produce sphingosine and a free fatty acid (FFA). The crystal structures of the two receptor subtypes, ADIPOR1 and ADIPOR2, show a similar overall seven-transmembrane-domain architecture with large unoccupied cavities and a zinc binding site within the seven transmembrane domain. However, the molecular mechanisms by which ADIPORs function are not known.

Combining 'dry' co-crystallization and in situ diffraction to facilitate ligand screening by X-ray crystallography.

X-ray crystallography is an established technique for ligand screening in fragment-based drug-design projects, but the required manual handling steps - soaking crystals with ligand and the subsequent harvesting - are tedious and limit the throughput of the process. Here, an alternative approach is reported: crystallization plates are pre-coated with potential binders prior to protein crystallization and X-ray diffraction is performed directly 'in situ' (or in-plate). Its performance is demonstrated on distinct and relevant therapeutic targets currently being studied for ligand screening by X-ray crystallography using either a bending-magnet beamline or a rotating-anode generator.

Aedesin: structure and antimicrobial activity against multidrug resistant bacterial strains.

Multidrug resistance, which is acquired by both Gram-positive and Gram-negative bacteria, causes infections that are associated with significant morbidity and mortality in many clinical settings around the world. Because of the rapidly increasing incidence of pathogens that have become resistant to all or nearly all available antibiotics, there is a need for a new generation of antimicrobials with a broad therapeutic range for specific applications against infections. Aedesin is a cecropin-like anti-microbial peptide that was recently isolated from dengue virus-infected salivary glands of the Aedes aegypti mosquito.

Structural and biochemical characterization of the Cop9 signalosome CSN5/CSN6 heterodimer.

The Cop9 signalosome complex (CSN) regulates the functional cycle of the major E3 ubiquitin ligase family, the cullin RING E3 ubiquitin ligases (CRLs). Activated CRLs are covalently modified by the ubiquitin-like protein Nedd8 (neural precursor cell expressed developmentally down-regulated protein 8). CSN serves an essential role in myriad cellular processes by reversing this modification through the isopeptidase activity of its CSN5 subunit.

Fragment-based identification of a locus in the Sec7 domain of Arno for the design of protein-protein interaction inhibitors.

By virtual screening using a fragment-based drug design (FBDD) approach, 33 fragments were selected within small pockets around interaction hot spots on the Sec7 surface of the nucleotide exchange factor Arno, and then their ability to interfere with the Arno-catalyzed nucleotide exchange on the G-protein Arf1 was evaluated. By use of SPR, NMR, and fluorescence assays, the direct binding of three of the identified fragments to Arno Sec7 domain was demonstrated and the promiscuous aggregate behavior evaluated. Then the binding mode of one fragment and of a more active analogue was solved by X-ray crystallography.

Insights into the regulation of the human COP9 signalosome catalytic subunit, CSN5/Jab1.

The COP9 (Constitutive photomorphogenesis 9) signalosome (CSN), a large multiprotein complex that resembles the 19S lid of the 26S proteasome, plays a central role in the regulation of the E3-cullin RING ubiquitin ligases (CRLs). The catalytic activity of the CSN complex, carried by subunit 5 (CSN5/Jab1), resides in the deneddylation of the CRLs that is the hydrolysis of the cullin-neural precursor cell expressed developmentally downregulated gene 8 (Nedd8)isopeptide bond. Whereas CSN-dependent CSN5 displays isopeptidase activity, it is intrinsically inactive in other physiologically relevant forms.

An integrated chemical biology approach provides insight into Cdk2 functional redundancy and inhibitor sensitivity.

Cdk2 promotes DNA replication and is a promising cancer therapeutic target, but its functions appear redundant with Cdk1, an essential Cdk affected by most Cdk2 inhibitors. Here, we present an integrated multidisciplinary approach to address Cdk redundancy. Mathematical modeling of enzymology data predicted conditions allowing selective chemical Cdk2 inhibition.

In-plate protein crystallization, in situ ligand soaking and X-ray diffraction.

X-ray crystallography is now a recognized technique for ligand screening, especially for fragment-based drug design. However, protein crystal handling is still tedious and limits further automation. An alternative method for the solution of crystal structures of proteins in complex with small ligands is proposed.

Structural insights into the molecular mechanisms of cauliflower mosaic virus transmission by its insect vector.

Cauliflower mosaic virus (CaMV) is transmitted from plant to plant through a seemingly simple interaction with insect vectors. This process involves an aphid receptor and two viral proteins, P2 and P3. P2 binds to both the aphid receptor and P3, itself tightly associated with the virus particle, with the ensemble forming a transmissible viral complex.

Removing the invariant salt bridge of parvalbumin increases flexibility in the AB-loop structure.

Parvalbumins (PVs) are calcium-buffer proteins that belong to the EF-hand family. Their N-terminal domain consists of two antiparallel helices A and B that make up a flat hydrophobic surface that is associated with the opposite side of the CD and EF binding sites. A single conserved Arg75-Glu81 salt bridge is buried in this hydrophobic interface.

Structure of PlcR: Insights into virulence regulation and evolution of quorum sensing in Gram-positive bacteria.

Gram-positive bacteria use a wealth of extracellular signaling peptides, so-called autoinducers, to regulate gene expression according to population densities. These "quorum sensing" systems control vital processes such as virulence, sporulation, and gene transfer. Using x-ray analysis, we determined the structure of PlcR, the major virulence regulator of the Bacillus cereus group, and obtained mechanistic insights into the effects of autoinducer binding.

Role of Asn(2) and Glu(7) residues in the oxidative folding and on the conformation of the N-terminal loop of apamin.

The X-ray structure of [N-acetyl]-apamin has been solved at 0.95 A resolution. It consists of an 1-7 N-terminal loop stabilized by an Asn-beta-turn motif (2-5 residues) and a helical structure spanning the 9-18 residues tightly linked together by two disulfide bonds.

Structure of a liganded type 2 non-specific lipid-transfer protein from wheat and the molecular basis of lipid binding.

In plants, a family of ubiquitous proteins named non-specific lipid-transfer proteins (ns-LTPs) facilitates the transfer of fatty acids, phospholipids and steroids between membranes. Recent data suggest that these secreted proteins play a key role in the formation of cuticular wax layers and in defence mechanisms against pathogens. In this study, X-ray crystallography has been used to examine the structural details of the interaction between a wheat type 2 ns-LTP and a lipid, L-alpha-palmitoyl-phosphatidyl glycerol.

High-resolution X-ray structure of the unexpectedly stable dimer of the [Lys(-2)-Arg(-1)-des(17-21)]endothelin-1 peptide.

Previous structural studies on the [Lys((-2))-Arg((-1))]endothelin-1 peptide (KR-ET-1), 540-fold less potent than ET-1, strongly suggested the presence of an intramolecular Arg(-1)-Asp(8) (R(-1)-D(8)) salt bridge that was also observed in the shorter [Lys((-2))-Arg((-1))-des(17-21)]endothelin-1 derivative (KR-CSH-ET). In addition, for these two analogues, we have shown that the Lys-Arg dipeptide, which belongs to the prosequence, significantly improves the formation of the native disulfide bonds (>or=96% instead of approximately 70% for ET-1). In contrast to what was inferred from NMR data, molecular dynamics simulations suggested that such an intramolecular salt bridge would be unstable.