Synthetic antibodies against BRIL as universal fiducial marks for single−particle cryoEM structure determination of membrane proteins

We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins

Synthetic antibodies against BRIL as universal fiducial marks for single-particle cryoEM structure determination of membrane proteins

We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins

Cryo-ET Reveals Molecular Details of Multi-Megadalton Bacterial Protein Complexes

Cryo-electron tomography (cryo-ET) is a powerful method for investigating the 3D structure of intact cells, organelles, and complex protein macromolecules that cannot be crystallized or are too heterogenous for single-particle cryo-electron microscopy (cryo-EM). However, obtaining high- resolution cryo-ET structures for many biologically important targets is still a challenge. To address this challenge, cryo-ET can be combined with other methods, including X-ray crystallography, single-particle cryo-EM, structure predictions, cross-linking mass spectrometry, biochemistry, and evolutionary analysis to produce integrative models. Recently, with the development of AI-based tools such as AlphaFold2, structure prediction has played an increasingly important role in integrative modeling. The combination of cryo-ET and structure prediction in particular has provided unprecedented insights into the ultrastructure of cellular components. This thesis focuses on two bacterial multi-megadalton protein complexes which are difficult to study by classical structural biology approaches: gas vesicles (GVs) and the Legionella pneumophila Dot/Icm type IV secretion system (T4SS). GVs are gas-filled protein nanostructures that regulate the position of certain microorganisms in water and consequently their access to sunlight and nutrients. Here, we investigate the mechanical properties of GVs and reveal the molecular structure of GVs and its implication for the assembly mechanism. The Dot/Icm T4SS is a macromolecular complex formed by approximately 27 proteins, utilized by L. pneumophila to hijack the host cell's biology for its replication purposes. A nearly-complete integrative model of this complex provides crucial insights into its structural organization and its evolution from conjugation to secretion, as well as the transportation of substrates into the host cell.</p

Synteza receptora dopaminowego D2 w bezkomórkowym systemie ekspresji oraz generacja syntetycznych fragmentów przeciwciał dla różnych wariantów białek G

Receptory z rodziny GPCR należą do największej rodziny białek powierzchniowych zidentyfikowanych u kręgowców. Pełnią one kluczową role w wielu procesach biologicznych a ich niepoprawne funkcjonowanie może prowadzić do wielu zaburzeń. Szacuje się, że od 30 do 50% wszystkich leków dopuszczonych do obrotu celuje w te właśnie receptory. Jednym z przedstawicieli tej rodziny są receptory dopaminowe. Sygnał przekazywany poprzez nie jest odpowiedzialny za funkcje motoryczne, napięcie mięśni, procesy emocjonalne wyższe czynności psychiczne czy też regulację hormonalną. Kaskada sygnalizacyjna receptorów z rodziny GPCR jest następnie przekazywana poprzez heterotrimeryczne białka G. Aktywowany receptor działa jako czynnik wymiany nukleotydu dla białka G co w efekcie prowadzi do dysocjacji podjednostek białka i aktywacji kolejnych białek efektorowych. Pomimo intensywnych badań wiele aspektów funkcjonowania tych receptorów pozostaje niejasne. Spowodowane jest to z trudnościami jakie wynikają z biologicznych właściwości receptorów z rodziny GPCR. W ramach prezentowanej pracy dokonano syntezy ludzkiego receptora dopaminowego D2 wykorzystując bezkomórkowy system ekspresji (ang. cell-free) jako alternatywę dla tradycyjnych metod produkcji białka. W tym celu dokonano optymalizacji warunków syntezy receptora a następnie oczyszczono go przy pomocy chromatografii powinowactwa na złożu niklowym. Oczyszczony receptor został następnie wprowadzony do nanodysków o zdefiniowanym otoczeniu lipidowym tak aby zapewnić jego prawidłowe funkcjonowanie. W drugiej części pracy dokonano generacji syntetycznych fragmentów przeciwciała wiążących antygen przy pomocy techniki ekspresji fagowej. W tym celu dokonano syntezy a następnie oczyszczano różne warianty białek G. Przeprowadzona z sukcesem selekcja przeciwciał pozwoliła na uzyskanie kilkunastu syntetycznych fragmentów przeciwciał specyficznych dla różnych wariantów białek G. Dodatkowo dokonano wpływu uzyskanych przeciwciał na aktywność białka Gs co pozwoliło na zidentyfikowanie przeciwciała posiadającego właściwości aktywujące w stosunku do badanego białka.GPCRs belong to the largest superfamily of the surface receptors in the mammalian genome and play an essential role in the majority of biological processes. Their malfunction lead to many diseases, which make them lucrative drug targets. Therefore, it is not surprising that 30-50% of the currently available drugs on market are targeted to GPCRs. One such representative of the GPCR family are the dopamine receptors, and their signaling is responsible for numerous processes like motor skills, muscle tone, emotional perception, cognition, the reward system, mechanisms of learning and memory, and hormonal regulation. Signaling through GPCRs is transduced by the G-proteins. Activation of the receptor results in dissociation of the G⍺ and Gβ���� subunits from the receptor and regulation of downstream effector proteins. Despite extensive studies many aspects of GPCR functioning remain unclear because of the difficulties related to handling these samples. In the presented work, the human dopamine receptor D2 was synthesized using a cell-free expression system as an alternative for the traditional, cell-based protein production techniques. For this purpose, receptor expression conditions were optimized. Next, the receptor was purified by metal-ion affinity chromatography, and subsequently incorporated into the nanodiscs in well-defined lipid environments. Additionally, synthetic antigen binders were generated for the G-protein variants using phage display technology. Expressed and purified G-protein variants were used for phage display antibody selection. Several dozen synthetic binders were successfully obtained from phage display experiments. The influence of obtained binders on the Gs protein activity was analyzed and it was determined that one binder, Fab G17, significantly increased GTP turnover by the protein. In summary these tools can be used to understand the signaling of the D2 receptor through its cognate G-protein

Cryo-electron tomography of the onion cell wall shows bimodally oriented cellulose fibers and reticulated homogalacturonan networks

One hallmark of plant cells is their cell wall. They protect cells against the environment and high turgor and mediate morphogenesis through the dynamics of their mechanical and chemical properties. The walls are a complex polysaccharidic structure. Although their biochemical composition is well known, how the different components organize in the volume of the cell wall and interact with each other is not well understood and yet is key to the wall’s mechanical properties. To investigate the ultrastructure of the plant cell wall, we imaged the walls of onion (Allium cepa) bulbs in a near-native state via cryo-focused ion beam milling (cryo-FIB milling) and cryo-electron tomography (cryo-ET). This allowed the high-resolution visualization of cellulose fibers in situ. We reveal the coexistence of dense fiber fields bathed in a reticulated matrix we termed “meshing,” which is more abundant at the inner surface of the cell wall. The fibers adopted a regular bimodal angular distribution at all depths in the cell wall and bundled according to their orientation, creating layers within the cell wall. Concomitantly, employing homogalacturonan (HG)-specific enzymatic digestion, we observed changes in the meshing, suggesting that it is—at least in part—composed of HG pectins. We propose the following model for the construction of the abaxial epidermal primary cell wall: the cell deposits successive layers of cellulose fibers at −45° and +45° relative to the cell’s long axis and secretes the surrounding HG-rich meshing proximal to the plasma membrane, which then migrates to more distal regions of the cell wall

Isoform- and ligand-specific modulation of the adhesion GPCR ADGRL3/Latrophilin3 by a synthetic binder

Adhesion G protein-coupled receptors (aGPCRs) are cell-surface proteins with large extracellular regions that bind to multiple ligands to regulate key biological functions including neurodevelopment and organogenesis. Modulating a single function of a specific aGPCR isoform while affecting no other function and no other receptor is not trivial. Here, we engineered an antibody, termed LK30, that binds to the extracellular region of the aGPCR ADGRL3, and specifically acts as an agonist for ADGRL3 but not for its isoform, ADGRL1. The LK30/ADGRL3 complex structure revealed that the LK30 binding site on ADGRL3 overlaps with the binding site for an ADGRL3 ligand – teneurin. In cellular-adhesion assays, LK30 specifically broke the trans-cellular interaction of ADGRL3 with teneurin, but not with another ADGRL3 ligand – FLRT3. Our work provides proof of concept for the modulation of isoform- and ligand-specific aGPCR functions using unique tools, and thus establishes a foundation for the development of fine-tuned aGPCR-targeted therapeutics

Morphological remodeling of Coxiella burnetii during its biphasic developmental cycle revealed by cryo-electron tomography

Summary: Coxiella burnetii is an obligate zoonotic bacterium that targets macrophages causing a disease called Q fever. It has a biphasic developmental life cycle where the extracellular and metabolically inactive small cell variant (SCV) transforms inside the host into the vegetative large cell variant (LCV). However, details about the morphological and structural changes of this transition are still lacking. Here, we used cryo-electron tomography to image both SCV and LCV variants grown either under axenic conditions or purified directly from host cells. We show that SCVs are characterized by equidistant stacks of inner membrane that presumably facilitate the transition to LCV, a transition coupled with the expression of the Dot/Icm type IVB secretion system (T4BSS). A class of T4BSS particles were associated with extracellular densities possibly involved in host infection. Also, SCVs contained spherical multilayered membrane structures of different sizes and locations suggesting no connection to sporulation as once assumed