Random mutagenesis of the prokaryotic peptide transporter YdgR identifies potential periplasmic gating residues

The peptide transporter (PTR) family represents a group of proton-coupled secondary transporters responsible for bulk uptake of amino acids in the form of di- and tripeptides, an essential process employed across species ranging from bacteria to humans. To identify amino acids critical for peptide transport in a prokaryotic PTR member, we have screened a library of mutants of the Escherichia coli peptide transporter YdgR using a high-throughput substrate uptake assay.Wehave identified 35 single point mutations that result in a full or partial loss of transport activity. Additional analysis, including homology modeling based on the crystal structure of the Shewanella oneidensis peptide transporter PepT so, identifies Glu 56 and Arg 305 as potential periplasmic gating residues. In addition to providing new insights into transport by members of the PTR family, these mutants provide valuable tools for further study of the mechanism of peptide transport

HUWE1 employs a giant substrate-binding ring to feed and regulate its HECT E3 domain

HUWE1 is a universal quality-control E3 ligase that marks diverse client proteins for proteasomal degradation. Although the giant HECT enzyme is an essential component of the ubiquitin–proteasome system closely linked with severe human diseases, its molecular mechanism is little understood. Here, we present the crystal structure of Nematocida HUWE1, revealing how a single E3 enzyme has specificity for a multitude of unrelated substrates. The protein adopts a remarkable snake-like structure, where the C-terminal HECT domain heads an extended alpha-solenoid body that coils in on itself and houses various protein–protein interaction modules. Our integrative structural analysis shows that this ring structure is highly dynamic, enabling the flexible HECT domain to reach protein targets presented by the various acceptor sites. Together, our data demonstrate how HUWE1 is regulated by its unique structure, adapting a promiscuous E3 ligase to selectively target unassembled orphan proteins

An extracellular network of Arabidopsis leucine-rich repeat receptor kinases

The cells of multicellular organisms receive extracellular signals using surface receptors. The extracellular domains (ECDs) of cell surface receptors function as interaction platforms, and as regulatory modules of receptor activation1,2. Understanding how interactions between ECDs produce signal-competent receptor complexes is challenging because of their low biochemical tractability3,4. In plants, the discovery of ECD interactions is complicated by the massive expansion of receptor families, which creates tremendous potential for changeover in receptor interactions5. The largest of these families in Arabidopsis thaliana consists of 225 evolutionarily related leucine-rich repeat receptor kinases (LRR-RKs)5, which function in the sensing of microorganisms, cell expansion, stomata development and stem-cell maintenance6,7,8,9. Although the principles that govern LRR-RK signalling activation are emerging1,10, the systems-level organization of this family of proteins is unknown. Here, to address this, we investigated 40,000 potential ECD interactions using a sensitized high-throughput interaction assay3, and produced an LRR-based cell surface interaction network (CSILRR) that consists of 567 interactions. To demonstrate the power of CSILRR for detecting biologically relevant interactions, we predicted and validated the functions of uncharacterized LRR-RKs in plant growth and immunity. In addition, we show that CSILRR operates as a unified regulatory network in which the LRR-RKs most crucial for its overall structure are required to prevent the aberrant signalling of receptors that are several network-steps away. Thus, plants have evolved LRR-RK networks to process extracellular signals into carefully balanced responses

The HEV Ventilator: at the interface between particle physics and biomedical engineering.

A high-quality, low-cost ventilator, dubbed HEV, has been developed by the particle physics community working together with biomedical engineers and physicians around the world. The HEV design is suitable for use both in and out of hospital intensive care units, provides a variety of modes and is capable of supporting spontaneous breathing and supplying oxygen-enriched air. An external air supply can be combined with the unit for use in situations where compressed air is not readily available. HEV supports remote training and post market surveillance via a Web interface and data logging to complement standard touch screen operation, making it suitable for a wide range of geographical deployment. The HEV design places emphasis on the ventilation performance, especially the quality and accuracy of the pressure curves, reactivity of the trigger, measurement of delivered volume and control of oxygen mixing, delivering a global performance which will be applicable to ventilator needs beyond the COVID-19 pandemic. This article describes the conceptual design and presents the prototype units together with a performance evaluation