InstructFeaturing expert speakers from Instruct Centres across Europe, Instruct-ERIC Webinar Series: Structure Meets Function highlights some of the latest developments in structural biology, demonstrating how integrative methods are enabling scientists to decipher the mechanisms that underpin health and disease.
Watch the previous webinars in the series here.
The 12th webinar in the series will be hosted by Instruct Latvia on 14 Sept 2021, 11:30 - 13:00 CEST
Agenda
Webinar moderator: Prof. Kaspars Tars, Latvian Biomedical Research and Study Centre and University of Latvia
Talk 1: ssRNA phages – structural studies and applications of their virus-like particles
Speaker: Kaspars Tars
Affiliation: Latvian Biomedical Research and Study Centre and University of Latvia
Abstract:ssRNA phages belonging to family Leviviridae are among the simplest known viruses. Historically, ssRNA phages have served as simple models to study various problems in molecular biology. Additionally, ssRNA phages have aided in construction of impressive toolbox for many practical applications such as MS2 tagging, armoured RNA technology, drug delivery, nanoreactor construction and vaccine development. Most of the applications depend on use of phage virus-like particles (VLPs). However, the available range of known ssRNA phages has remained very narrow, which has somewhat limited their applications. Due to advances in next generation sequencing, during past few years incomplete sequences of more than 1000 ssRNA phages from metagenome data have been described. Although the available metagenome sequences do not allow reconstruction of phages themselves, it is possible to produce the corresponding VLPs using data from coat protein (CP) sequences. We have attempted to produce more than 120 coat protein sequences, which have yielded about 80 novel VLPs of ssRNA phages. During our first characterization effort we identified VLPs with increased production level, solubility and stability. Our further goal was to select the most suitable VLPs for exposure of foreign antigens by genetic fusion. For this, we fused M2e protein fragment from influenza virus to C- and N- terminal parts of 40 selected coat proteins. In this way we were able to identify 10 VLPs, suitable for construction of vaccine candidates by genetic fusion (Lieknina et al., 2020). Finally, we solved crystal structures of 22 newly produced VLPs, which had several previously unseen and surprising features. For example, one of the identified VLPs had elongated shape, possibly allowing accommodation of larger genome in the corresponding phage. In another case we found clear electron density for dsRNA fragment bound to coat protein, illustrating novel ways of protein-RNA interactions in ssRNA phages. Another of the examined VLPs Beihai14 had a very unusual CP fold with structures of N-terminal region in different CP subunits resembling that of totally unrelated plant viruses and providing an example of convergent evolution in building of icosahedral particles.
Talk 2: Structural and functional insights into GRM2 type choline degrading type bacterial microcompartments
Speaker: Gints Kalnins
Affiliation:Latvian Biomedical Research and Study Centre
Abstract: Bacterial microcompartments (BMCs) are prokaryotic organelles consisting of a protein shell and an encapsulated enzymatic core. A subtype of bacterial microcompartments, metabolosomes, are involved in such biochemical processes as choline, glycerol, ethanolamine, fucose and rhamnose degradation. Encapsulation of the enzymatic pathway has the benefit of increasing the local substrate concentrations and containing toxic and/or volatile metabolites. Since nonnative enzymes can also be encapsulated in BMCs, an improved understanding of BMC shell assembly and encapsulation processes could be useful for synthetic biology applications.
We isolated and expressed BMC genes of a choline degrading metabolosome from Klebsiella pneumoniae GRM2 locus and discovered that the native enzymatic core is encapsulated in a hierarchical manner and that one of the key core enzymes CutC choline lyase could play a secondary role as an adaptor protein. We discovered that the shell particle size is dependable on the particular composition of BMC-H shell proteins. We have also solved a cryo-EM structure of a pT=4 quasi-symmetric icosahedral BMC shell particle at 3.3 Å resolution. In a further study we have obtained lower resolution maps of several minor BMC shell forms and demonstrated the particular modes of the larger BMC shell particle formation.
Talk 3: Spider silk: from NMR structural studies to mechanism of formation and artificial fibres
Speaker: Kristaps Jaudzems
Affiliation: Latvian Institute of Organic Synthesis and University of Latvia
Abstract: Spider silk, one of the toughest biomaterials known, is produced through the assembly of large proteins (spidroins) that consist of three structural units: a central repetitive region which accounts for spider silk's exceptional mechanical properties and two terminal domains (NT and CT) implicated in the silk formation process. The spidroins are soluble up to concentrations of 30-50% (w/v), when stored in the sac of the spider silk gland but form solid fibres upon passage through an elongated and narrowing duct. During this transition spidroins experience changes in pH, ion composition and shear forces, that have been shown to be of importance for the silk fibre formation. Despite many efforts, the mechanical properties of current artificial spider silks lag behind their natural counterparts. The main reason for this is the inability to reproduce the complex molecular mechanisms of native silk spinning. We used NMR and other biophysical techniques to study how the structure of the different spidroin domains is affected by the changing environment conditions in the duct.
Our studies revealed the structural changes experienced by each of the spidroin domains upon fibre spinning. Solution NMR spectroscopy allowed probing of inter-domain interactions and structural changes that occur before fibre formation in response to low pH and altered ion composition. The extremely high solubility of spidroins is achieved by forming micellar structures, with hydrophobic poly-alanine segments of the repetitive domain in the micelle core and the terminal domains outlining the micelle shell. This protein stabilization mechanism can be used for efficient production of other aggregation-prone proteins by fusion with an engineered spider silk N-terminal domain. Solid-state NMR spectroscopy of artificial fibres revealed the degree of conversion to beta-sheet structure and the conformation of the terminal domains that relates to the mimicry of the mechanism of native silk spinning.
