Semisynthetic LC3 Probes for Autophagy Pathways Reveal a Noncanonical LC3 Interacting Region Motif Crucial for the Enzymatic Activity of Human ATG3.

Macroautophagy is one of two major degradation systems in eukaryotic cells. Regulation and control of autophagy are often achieved through the presence of short peptide sequences called LC3 interacting regions (LIR) in autophagy-involved proteins. Using a combination of new protein-derived activity-based probes prepared from recombinant LC3 proteins, along with protein modeling and X-ray crystallography of the ATG3-LIR peptide complex, we identified a noncanonical LIR motif in the human E2 enzyme responsible for LC3 lipidation, ATG3.

Chimeric single α-helical domains as rigid fusion protein connections for protein nanotechnology and structural biology.

Chimeric fusion proteins are essential tools for protein nanotechnology. Non-optimized protein-protein connections are usually flexible and therefore unsuitable as structural building blocks. Here we show that the ER/K motif, a single α-helical domain (SAH), can be seamlessly fused to terminal helices of proteins, forming an extended, partially free-standing rigid helix.

Design, Expression, Purification, and Characterization of a YFP-Tagged 2019-nCoV Spike Receptor-Binding Domain Construct.

2019-nCoV is the causative agent of the serious, still ongoing, worldwide coronavirus disease (COVID-19) pandemic. High quality recombinant virus proteins are required for research related to the development of vaccines and improved assays, and to the general understanding of virus action. The receptor-binding domain (RBD) of the 2019-nCoV spike (S) protein contains disulfide bonds and N-linked glycosylations, therefore, it is typically produced by secretion.

Femtosecond-to-millisecond structural changes in a light-driven sodium pump.

Light-driven sodium pumps actively transport small cations across cellular membranes. These pumps are used by microorganisms to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. Although the resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved, it is unclear how structural alterations over time allow sodium to be translocated against a concentration gradient.

Seamless insert-plasmid assembly at sub-terminal homologous sequences.

The engineering of fusion proteins for structural biology and protein nanotechnology often requires seamless DNA assembly with slight variations in the domain boundaries. To improve the molecular biology workflow for such projects, we evaluated the use of sub-terminal homologous sequences (HS) for co-transformation cloning and for T5 exonuclease / Phusion DNA polymerase mediated in vitro assembly. To quantify the effects of different HS-to-ends distances on cloning efficiency, we designed a blue-white-pink screening system that allowed us to easily identify positive clones (blue colonies), negative clones resulting from circular template plasmid (pink colonies) and negative colonies originating from linearized plasmids that have recircularized without an insert (white colonies).

Botulinum Neurotoxin Diversity from a Gene-Centered View.

Botulinum neurotoxins (BoNTs) rank amongst the most potent toxins known. The factors responsible for the emergence of the many known and yet unknown BoNT variants remain elusive. It also remains unclear why anaerobic bacteria that are widely distributed in our environment and normally do not pose a threat to humans, produce such deadly toxins.

Crystal structure of the BoNT/A2 receptor-binding domain in complex with the luminal domain of its neuronal receptor SV2C.

A detailed molecular understanding of botulinum neurotoxin (BoNT)/host-cell-receptor interactions is fundamental both for developing strategies against botulism and for generating improved BoNT variants for medical applications. The X-ray crystal structure of the receptor-binding domain (H) of BoNT/A1 in complex with the luminal domain (LD) of its neuronal receptor SV2C revealed only few specific side-chain - side-chain interactions that are important for binding. Notably, two BoNT/A1 residues, Arg 1156 and Arg 1294, that are crucial for the interaction with SV2, are not conserved among subtypes.

Structural basis for misregulation of kinesin KIF21A autoinhibition by CFEOM1 disease mutations.

Tight regulation of kinesin activity is crucial and malfunction is linked to neurological diseases. Point mutations in the KIF21A gene cause congenital fibrosis of the extraocular muscles type 1 (CFEOM1) by disrupting the autoinhibitory interaction between the motor domain and a regulatory region in the stalk. However, the molecular mechanism underlying the misregulation of KIF21A activity in CFEOM1 is not understood.

Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost.

Seamless cloning methods, such as co-transformation cloning, sequence- and ligation-independent cloning (SLIC) or the Gibson assembly, are essential tools for the precise construction of plasmids. The efficiency of co-transformation cloning is however low and the Gibson assembly reagents are expensive. With the aim to improve the robustness of seamless cloning experiments while keeping costs low, we examined the importance of complementary single-stranded DNA ends for co-transformation cloning and the influence of single-stranded gaps in circular plasmids on SLIC cloning efficiency.

Structure of the BoNT/A1--receptor complex.

Botulinum neurotoxin A causes botulism but is also used for medical and cosmetic applications. A detailed molecular understanding of BoNT/A--host receptor interactions is therefore fundamental for improving current clinical applications and for developing new medical strategies targeting human disorders. Towards this end, we recently solved an X-ray crystal structure of BoNT/A1 in complex with its neuronal protein receptor SV2C.

Botulinum neurotoxins: new questions arising from structural biology.

Botulinum neurotoxins (BoNTs) are the most toxic substances known and cause botulism in vertebrates. They have also emerged as effective and powerful reagents for cosmetic and medical applications. One important prerequisite for understanding BoNT function in disease, and the further development of the toxins for cosmetic and medical applications, is a detailed knowledge of BoNT interactions with non-toxic neurotoxin-associated proteins and cell surface receptors.

Structural basis for recognition of synaptic vesicle protein 2C by botulinum neurotoxin A.

Botulinum neurotoxin A (BoNT/A) belongs to the most dangerous class of bioweapons. Despite this, BoNT/A is used to treat a wide range of common medical conditions such as migraines and a variety of ocular motility and movement disorders. BoNT/A is probably best known for its use as an antiwrinkle agent in cosmetic applications (including Botox and Dysport).

The x-ray crystal structure of the first RNA recognition motif and site-directed mutagenesis suggest a possible HuR redox sensing mechanism.

Hu-antigen R (HuR) is a ubiquitous RNA-binding protein that comprises three RNA recognition motifs (RRMs). The first two tandem RRMs are known to bind to AU-rich elements (AREs) in the 3'-untranslated region of many mRNAs. The third RRM is connected to the second RRM through a basic hinge region that contains a localization signal termed HuR nucleocytoplasmic shuttling.

Terminal adenosyl transferase activity of posttranscriptional regulator HuR revealed by confocal on-bead screening.

Posttranscriptional regulation and RNA metabolism have become central topics in the understanding of mammalian gene expression and cell signalling, with the 3' untranslated region emerging as the coordinating unit. The 3' untranslated region trans-acting factor Hu protein R (HuR) forms a central posttranscriptional pathway node bridging between AU-rich element-mediated processes and microRNA regulation. While (m)RNA control by HuR has been extensively characterized, the molecular mode of action still remains elusive.

An improved method for fast, robust, and seamless integration of DNA fragments into multiple plasmids.

We describe an improved, universal method for the seamless integration of DNA fragments into plasmids at any desired position. The protocol allows in vitro joining of insert and linearized plasmid at terminal homology regions using the BD In-Fusion cloning system. According to the standard BD In-Fusion protocol, vectors are linearized by restriction enzyme digestion.