A Potent Peptidomimetic Inhibitor of Botulinum Neurotoxin Serotype A Has a Very Different Conformation than SNAP-25 Substrate

SummaryBotulinum neurotoxin serotype A is the most lethal of all known toxins. Here, we report the crystal structure, along with SAR data, of the zinc metalloprotease domain of BoNT/A bound to a potent peptidomimetic inhibitor (Ki = 41 nM) that resembles the local sequence of the SNAP-25 substrate. Surprisingly, the inhibitor adopts a helical conformation around the cleavage site, in contrast to the extended conformation of the native substrate. The backbone of the inhibitor's P1 residue displaces the putative catalytic water molecule and concomitantly interacts with the “proton shuttle” E224. This mechanism of inhibition is aided by residue contacts in the conserved S1′ pocket of the substrate binding cleft and by the induction of new hydrophobic pockets, which are not present in the apo form, especially for the P2′ residue of the inhibitor. Our inhibitor is specific for BoNT/A as it does not inhibit other BoNT serotypes or thermolysin

The Mediterranean Island Wetlands (MedIsWet) inventory: strengths and shortfalls of the currently available floristic data

MedIsWet (Conservation of the island wetlands of the Mediterranean Basin) is a MAVA funded project which aims at investigating all seasonal or permanent island wetlands both natural and artificial, with a minimum extent of 0.1 hectares. More than 16,000 wetlands from almost all the Mediterranean, including islands from France, Italy, Malta, Croatia, Cyprus, Tunisia, Turkey, Greece and Spain were mapped. Over 2,500 of them were inventoried in the field and more than 500 scientific contributions catalogued. In total, more than 35,000 plant occurrences were uploaded, in a standardised and comparable way, on the national open-source web portals. These can be related to the recorded threats, uses and other spatially retrievable information. Here, we show strengths and shortfalls of the already available information about the floristic records. Although further improvements are needed, we discuss how these data can be used for research and policy actions and to develop conservation projects

A new MR-SAD algorithm for the automatic building of protein models from low-resolution X-ray data and a poor starting model

Determining macromolecular structures from X-ray data with resolution worse than 3 Å remains a challenge. Even if a related starting model is available, its incompleteness or its bias together with a low observation-to-parameter ratio can render the process unsuccessful or very time-consuming. Yet, many biologically important macromolecules, especially large macromolecular assemblies, membrane proteins and receptors, tend to provide crystals that diffract to low resolution. A new algorithm to tackle this problem is presented that uses a multivariate function to simultaneously exploit information from both an initial partial model and low-resolution single-wavelength anomalous diffraction data. The new approach has been used for six challenging structure determinations, including the crystal structures of membrane proteins and macromolecular complexes that have evaded experts using other methods, and large structures from a 3.0 Å resolution F_1-ATPase data set and a 4.5 Å resolution SecYEG–SecA complex data set. All of the models were automatically built by the method to R_(free) values of between 28.9 and 39.9% and were free from the initial model bias

The expanding functional roles and signaling mechanisms of adhesion G protein–coupled receptors

The adhesion class of G protein–coupled receptors (GPCRs) is the second largest family of GPCRs (33 members in humans). Adhesion GPCRs (aGPCRs) are defined by a large extracellular N‐terminal region that is linked to a C‐terminal seven transmembrane (7TM) domain via a GPCR‐autoproteolysis inducing (GAIN) domain containing a GPCR proteolytic site (GPS). Most aGPCRs undergo autoproteolysis at the GPS motif, but the cleaved fragments stay closely associated, with the N‐terminal fragment (NTF) bound to the 7TM of the C‐terminal fragment (CTF). The NTFs of most aGPCRs contain domains known to be involved in cell–cell adhesion, while the CTFs are involved in classical G protein signaling, as well as other intracellular signaling. In this workshop report, we review the most recent findings on the biology, signaling mechanisms, and physiological functions of aGPCRs

A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis

Crystallographic structures encompassing GPCR autoproteolytic sequences (GPS) delineate a novel conserved structural domain called GAIN, which is found in cell-adhesion GPCRs, polycystic kidney disease proteins conserved throughout evolution

A split horseradish peroxidase for detection of intercellular protein-protein interactions and sensitive visualization of synapses

Intercellular protein-protein interactions (PPIs) enable communication between cells in diverse biological processes, including cell proliferation, immune responses, infection and synaptic transmission, but they are challenging to visualize because existing techniques1,2,3 have insufficient sensitivity and/or specificity. Here we report split horseradish peroxidase (sHRP) as a sensitive and specific tool for detection of intercellular PPIs. The two sHRP fragments, engineered through screening of 17 cut sites in HRP followed by directed evolution, reconstitute into an active form when driven together by an intercellular PPI, producing bright fluorescence or contrast for electron microscopy. Fusing the sHRP fragments to the proteins neurexin (NRX) and neuroligin (NLG), which bind each other across the synaptic cleft4, enabled sensitive visualization of synapses between specific sets of neurons, including two classes of synapses in the mouse visual system. sHRP should be widely applicable for studying mechanisms of communication between a variety of cell types