Fused radical SAM and αKG-HExxH domain proteins contain a distinct structural fold and catalyse cyclophane formation and β-hydroxylation.

Two of nature's recurring binding motifs in metalloproteins are the CxxxCxxC motif in radical SAM enzymes and the 2-His-1-carboxylate motif found both in zincins and α-ketoglutarate and non-haem iron enzymes. Here we show the confluence of these two domains in a single post-translational modifying enzyme containing an N-terminal radical S-adenosylmethionine domain fused to a C-terminal 2-His-1-carboxylate (HExxH) domain. The radical SAM domain catalyses three-residue cyclophane formation and is the signature modification of triceptides, a class of ribosomally synthesized and post-translationally modified peptides.

Genome- and metabolome-guided discovery of marine BamA inhibitors revealed a dedicated darobactin halogenase.

Darobactins represent a class of ribosomally synthesized and post-translationally modified peptide (RiPP) antibiotics featuring a rare bicyclic structure. They target the Bam-complex of Gram-negative bacteria and exhibit in vivo activity against drug-resistant pathogens. First isolated from Photorhabdus species, the corresponding biosynthetic gene clusters (BGCs) are widespread among γ-proteobacteria, including the genera Vibrio, Yersinia, and Pseudoalteromonas (P.

Characterization of a Radical SAM Oxygenase for the Ether Crosslinking in Darobactin Biosynthesis.

Darobactin A is a ribosomally synthesized, post-translationally modified peptide (RiPP) with potent and broad-spectrum anti-Gram-negative antibiotic activity. The structure of darobactin A is characterized by an ether and C-C crosslinking. However, the specific mechanism of the crosslink formation, especially the ether crosslink, remains elusive.

Rational Control of Off-State Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast Enhancement.

Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, one of the key parameters that dictate the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain.

Nanoscale architecture of a VAP-A-OSBP tethering complex at membrane contact sites.

Membrane contact sites (MCS) are subcellular regions where two organelles appose their membranes to exchange small molecules, including lipids. Structural information on how proteins form MCS is scarce. We designed an in vitro MCS with two membranes and a pair of tethering proteins suitable for cryo-tomography analysis.

Author Correction: Human ESCRT-III polymers assemble on positively curved membranes and induce helical membrane tube formation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Human ESCRT-III polymers assemble on positively curved membranes and induce helical membrane tube formation.

Endosomal sorting complexes for transport-III (ESCRT-III) assemble in vivo onto membranes with negative Gaussian curvature. How membrane shape influences ESCRT-III polymerization and how ESCRT-III shapes membranes is yet unclear. Human core ESCRT-III proteins, CHMP4B, CHMP2A, CHMP2B and CHMP3 are used to address this issue in vitro by combining membrane nanotube pulling experiments, cryo-electron tomography and AFM.

Chiral Separation, X-ray Structure, and Biological Evaluation of a Potent and Reversible Dual Binding Site AChE Inhibitor.

Acetylcholinesterase (AChE) inhibitors (AChEIs) still remain the leading therapeutic options for the symptomatic treatment of cognitive deficits associated with mild-to-moderate Alzheimer's disease. The search for new AChEIs benefits from well-established knowledge of the molecular interactions of selective AChEIs, such as donepezil and related dual binding site inhibitors. Starting from a previously disclosed coumarin-based inhibitor (±)--, active as racemate in the nanomolar range toward AChE, we proceeded on a double track by (i) achieving chiral resolution of the enantiomers of by HPLC and (ii) preparing two close achiral analogues of , i.

Radiation damage and dose limits in serial synchrotron crystallography at cryo- and room temperatures.

Radiation damage limits the accuracy of macromolecular structures in X-ray crystallography. Cryogenic (cryo-) cooling reduces the global radiation damage rate and, therefore, became the method of choice over the past decades. The recent advent of serial crystallography, which spreads the absorbed energy over many crystals, thereby reducing damage, has rendered room temperature (RT) data collection more practical and also extendable to microcrystals, both enabling and requiring the study of specific and global radiation damage at RT.

Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy.

Reversibly switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Photoswitching from a non-fluorescent off-state to a fluorescent on-state involves trans-to-cis chromophore isomerization and proton transfer. Whereas excited-state events on the ps timescale have been structurally characterized, conformational changes on slower timescales remain elusive.

Structure-Based Optimization of Nonquaternary Reactivators of Acetylcholinesterase Inhibited by Organophosphorus Nerve Agents.

Acetylcholinesterase (AChE), a key enzyme in the central and peripheral nervous systems, is the principal target of organophosphorus nerve agents. Quaternary oximes can regenerate AChE activity by displacing the phosphyl group of the nerve agent from the active site, but they are poorly distributed in the central nervous system. A promising reactivator based on tetrahydroacridine linked to a nonquaternary oxime is also an undesired submicromolar reversible inhibitor of AChE.

Potent 3-Hydroxy-2-Pyridine Aldoxime Reactivators of Organophosphate-Inhibited Cholinesterases with Predicted Blood-Brain Barrier Penetration.

A new series of 3-hydroxy-2-pyridine aldoxime compounds have been designed, synthesised and tested in vitro, in silico, and ex vivo as reactivators of human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE) inhibited by organophosphates (OPs), for example, VX, sarin, cyclosarin, tabun, and paraoxon. The reactivation rates of three oximes (16-18) were determined to be greater than that of 2-PAM and comparable to that of HI-6, two pyridinium aldoximes currently used by the armies of several countries. The interactions important for a productive orientation of the oxime group within the OP-inhibited enzyme have been clarified by molecular-modelling studies, and by the resolution of the crystal structure of the complex of oxime 17 with Torpedo californica AChE.

Photochemical Mechanism of an Atypical Algal Phytochrome.

Phytochromes are bilin-containing photoreceptors that are typically sensitive to the red/far-red region of the visible spectrum. Recently, phytochromes from certain eukaryotic algae have become attractive targets for optogenetic applications because of their unique ability to respond to multiple wavelengths of light. Herein, a combination of time-resolved spectroscopy and structural approaches across picosecond to second timescales have been used to map photochemical mechanisms and structural changes in this atypical group of phytochromes.

Chromophore twisting in the excited state of a photoswitchable fluorescent protein captured by time-resolved serial femtosecond crystallography.

Chromophores absorb light in photosensitive proteins and thereby initiate fundamental biological processes such as photosynthesis, vision and biofluorescence. An important goal in their understanding is the provision of detailed structural descriptions of the ultrafast photochemical events that they undergo, in particular of the excited states that connect chemistry to biological function. Here we report on the structures of two excited states in the reversibly photoswitchable fluorescent protein rsEGFP2.

Discovery and Structure-Activity Relationships of a Highly Selective Butyrylcholinesterase Inhibitor by Structure-Based Virtual Screening.

Structure-based virtual screening of two libraries containing 567 981 molecules was used to discover novel, selective BuChE inhibitors, which are potentially superior symptomatic treatments in late-stage Alzheimer's disease. Compound 16 was identified as a highly selective submicromolar inhibitor of BuChE (huBuChE IC50 = 0.443 μM) with high permeability in the PAMPA-BBB model.

Defective Guanine Nucleotide Exchange in the Elongation Factor-like 1 (EFL1) GTPase by Mutations in the Shwachman-Diamond Syndrome Protein.

Ribosome biogenesis is orchestrated by the action of several accessory factors that provide time and directionality to the process. One such accessory factor is the GTPase EFL1 involved in the cytoplasmic maturation of the ribosomal 60S subunit. EFL1 and SBDS, the protein mutated in the Shwachman-Diamond syndrome (SBDS), release the anti-association factor eIF6 from the surface of the ribosomal subunit 60S.

Crystallization and X-ray diffraction studies of crustacean proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA), a member of the sliding clamp family of proteins, interacts specifically with DNA replication and repair proteins through a small peptide motif called the PCNA-interacting protein or PIP box. PCNA is recognized as one of the key proteins involved in DNA metabolism. In the present study, the recombinant PCNA from Litopenaeus vannamei (LvPCNA) was heterologously overexpressed and purified using metal ion-affinity chromatography.

Structural changes caused by radiation-induced reduction and radiolysis: the effect of X-ray absorbed dose in a fungal multicopper oxidase.

X-ray radiation induces two main effects at metal centres contained in protein crystals: radiation-induced reduction and radiolysis and a resulting decrease in metal occupancy. In blue multicopper oxidases (BMCOs), the geometry of the active centres and the metal-to-ligand distances change depending on the oxidation states of the Cu atoms, suggesting that these alterations are catalytically relevant to the binding, activation and reduction of O(2). In this work, the X-ray-determined three-dimensional structure of laccase from the basidiomycete Coriolopsis gallica (Cg L), a high catalytic potential BMCO, is described.

Effective scavenging at cryotemperatures: further increasing the dose tolerance of protein crystals.

The rate of radiation damage to macromolecular crystals at both room temperature and 100 K has previously been shown to be reduced by the use of certain radical scavengers. Here the effects of sodium nitrate, an electron scavenger, are investigated at 100 K. For sodium nitrate at a concentration of 0.