Structural and Functional Analysis of a β2-Adrenergic Receptor Complex with GRK5.

The phosphorylation of agonist-occupied G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) functions to turn off G-protein signaling and turn on arrestin-mediated signaling. While a structural understanding of GPCR/G-protein and GPCR/arrestin complexes has emerged in recent years, the molecular architecture of a GPCR/GRK complex remains poorly defined. We used a comprehensive integrated approach of cross-linking, hydrogen-deuterium exchange mass spectrometry (MS), electron microscopy, mutagenesis, molecular dynamics simulations, and computational docking to analyze GRK5 interaction with the β2-adrenergic receptor (β2AR). These studies revealed a dynamic mechanism of complex formation that involves large conformational changes in the GRK5 RH/catalytic domain interface upon receptor binding. These changes facilitate contacts between intracellular loops 2 and 3 and the C terminus of the β2AR with the GRK5 RH bundle subdomain, membrane-binding surface, and kinase catalytic cleft, respectively. These studies significantly contribute to our understanding of the mechanism by which GRKs regulate the function of activated GPCRs. PAPERCLIP

Cloning, expression, purification, crystallization and preliminary X-ray studies of the mannose-binding lectin domain of MSMEG_3662 from Mycobacterium smegmatis

The mannose-binding lectin domain of MSMEG_3662 from Mycobacterium smegmatis has been cloned, expressed, purified and crystallized and the crystals have been characterized using X-ray diffraction. The Matthews coefficient suggests the possibility of two lectin domains in the triclinic cell. The amino-acid sequence of the domain indicates structural similarity to well characterized beta-prism II fold lectins

Structure, interactions and evolutionary implications of a domain-swapped lectin dimer from Mycobacterium smegmatis

Crystal structure determination of the lectin domain of MSMEG_3662 from Mycobacterium smegmatis and its complexes with mannose and methyl-alpha-mannose, the first effort of its kind on a mycobacterial lectin, reveals a structure very similar to beta-prism II fold lectins from plant sources, but with extensive unprecedented domain swapping in dimer formation. The two subunits in a dimer often show small differences in structure, but the two domains, not always related by 2-fold symmetry, have the same structure. Each domain carries three sugar-binding sites, similar to those in plant lectins, one on each Greek key motif. The occurrence of beta-prism II fold lectins in bacteria, with characteristics similar to those from plants, indicates that this family of lectins is of ancient origin and had evolved into a mature system before bacteria and plants diverged. In plants, the number of binding sites per domain varies between one and three, whereas the number is two in the recently reported lectin domains from Pseudomonas putida and Pseudomonas aeruginosa. An analysis of the sequences of the lectins and the lectin domains shows that the level of sequence similarity among the three Greek keys in each domain has a correlation with the number of binding sites in it. Furthermore, sequence conservation among the lectins from different species is the highest for that Greek key which carries a binding site in all of them. Thus, it would appear that carbohydrate binding influences the course of the evolution of the lectin

Negative Cooperativity and High Affinity in Chitooligosaccharide Binding by a <i>Mycobacterium smegmatis</i> Protein Containing LysM and Lectin Domains

LysM domains have been recognized in bacteria and eukaryotes as carbohydrate-binding protein modules, but the mechanism of their binding to chitooligosaccharides has been underexplored. Binding of a <i>Mycobacterium smegmatis</i> protein containing a lectin (MSL) and one LysM domain to chitooligosaccharides has been studied using isothermal titration calorimetry and fluorescence titration that demonstrate the presence of two binding sites of nonidentical affinities per dimeric MSL-LysM molecule. The affinity of the molecule for chitooligosaccharides correlates with the length of the carbohydrate chain. Its binding to chitooligosaccharides is characterized by negative cooperativity in the interactions of the two domains. Apparently, the flexibility of the long linker that connects the LysM and MSL domains plays a facilitating role in this recognition. The LysM domain in the MSL-LysM molecule, like other bacterial domains but unlike plant LysM domains, recognizes equally well peptidoglycan fragments as well as chitin polymers. Interestingly, in the case presented here, two LysM domains are enough for binding to peptidoglycan in contrast to the three reportedly required by the LysM domains of <i>Bacillus subtilis</i> and <i>Lactococcus lactis</i>. Also, the affinity of the MSL-LysM molecule for chitooligosaccharides is higher than that of LysM–chitooligosaccharide interactions reported so far

Bovine serum albumin interacts with silver nanoparticles with a ``side on'' or ``end on'' conformation

As the nanoparticles (NPs) enter into the biological interface, they have to encounter immediate and first exposure to many proteins of different concentrations. The physicochemical interaction of NPs and proteins is greatly influenced not only by the number and type of proteins; but also the surface chemistry of NPs. To analyze the effects of NPs on proteins, the interaction between bovine serum albumin (BSA) and silver nanoparticles (AgNPs) at different concentrations were investigated. The interaction, BSA conformations, kinetics and adsorption were analyzed by UV-Visible spectrophotometer, dynamic light scattering (DLS), FT-IR spectroscopy and fluorescence quenching. DLS, FTIR and UV-visible spectrophotometric analysis confirms the interaction with minor alterations in size of the protein. Fluorescence quenching analysis confirms the side-on or end-on interaction of 1.5 molecules of BSA to AgNP. Further, pseudo-second order kinetics was determined with equilibrium contact-time of 30 min. The data of the present study determines the detailed evaluation of BSA adsorption on AgNP along with mechanism, kinetics and isotherm of the adsorption. (C) 2016 Elsevier Ireland Ltd. All rights reserved

Structural Basis for Regulated Proteolysis by the α-Secretase ADAM10

International audienceCleavage of membrane-anchored proteins by ADAM (a disintegrin and metalloproteinase) endopeptidases plays a key role in a wide variety of biological signal transduction and protein turnover processes. Among ADAM family members, ADAM10 stands out as particularly important because it is both responsible for regulated proteolysis of Notch receptors and catalyzes the non-amyloidogenic α-secretase cleavage of the Alzheimer’s precursor protein (APP). We present here the X-ray crystal structure of the ADAM10 ectodomain, which, together with biochemical and cellular studies, reveals how access to the enzyme active site is regulated. The enzyme adopts an unanticipated architecture in which the C-terminal cysteine-rich domain partially occludes the enzyme active site, preventing unfettered substrate access. Binding of a modulatory antibody to the cysteine-rich domain liberates the catalytic domain from autoinhibition, enhancing enzymatic activity toward a peptide substrate. Together, these studies reveal a mechanism for regulation of ADAM activity and offer a roadmap for its modulation

Toward a Framework for Evaluating Software Success: A Proposed First Step

<p>Software is a particularly critical technology in many computational science and engineering (CSE) sectors. Consequently, software is increasingly becoming an important component in the evaluation of competitive grants and the execution of research projects. As a result, software can be viewed as a scholarly contribution and has been proposed as a new factor to consider in tenure and promotion processes. However, existing metrics for evaluating the capability, use, reusability, or success of software are sorely lacking. This lack of software metrics permits the development of software based on poor development practices, which in turn allows poorly written software to “fly under the radar” in the scientific community and persist undetected. The absence of evaluation by knowledgeable peers often leads to the establishment and adoption of tools based on aggressive promotion by developers, ease-of-use, and other peripheral factors, hindering the sustainability, usefulness, and uptake of software and even leading to unreliable scientific findings. All of these factors mean that addressing the current lack of software evaluation metrics and methods is not just a question of increasing scientific productivity, but also a matter of preventing poor science.</p> <p>As a first step toward creating a methodology and framework for developing and evolving software success metrics for the CSE community, we propose the creation of a software “peer-review group.” This group, comprised of grant recipients funded to develop sustainable software, would meet periodically to evaluate their own and each others’ software, developing and refining success metrics along the way. We envision the group as a pilot test for a potential larger-scale effort to establish a more formal framework for software success metrics and evaluation.</p