Biology subjects

Klebl, D. P.

Publications and source records attributed to Klebl, D. P..

7 recordsLinked to original sources

bioRxiv2024

Swinging lever mechanism of myosin directly demonstrated by time-resolved cryoEM

Myosins are essential for producing force and movement in cells through their interactions with F-actin. Generation of movement is proposed to occur through structural changes within the myosin motor domain, fuelled by ATP hydrolysis, that are amplified by a lever swing1, transitioning myosin from a primed (pre-powerstroke) state to a post-powerstroke state. However, the initial, primed actomyosin state, proposed to form prior to lever swing, has never been observed. Nor has the mechanism by which actin catalyses myosin ATPase activity been resolved. To address this, we performed time-resolved cryoEM of a myosin-5 mutant having slow hydrolysis product release. Primed actomyosin was captured 10 ms after mixing primed myosin with F-actin, whereas post-powerstroke actomyosin predominated at 120 ms, with no abundant intermediate structures. The structures were solved to 4.4[A] and 4.2[A] global resolution respectively. The primed motor binds to actin through its lower 50 kDa subdomain, with the actin-binding cleft open and Pi release prohibited. N-terminal actin interactions with myosin promote rotation of the upper 50 kDa subdomain, which closes the actin-binding cleft, and enables Pi release. Formation of upper 50 kDa subdomain interactions with actin creates the strong-binding interface required for effective force production. The myosin-5 lever swings through an angle of 93{degrees}, predominantly along the actin axis, with little twisting, to produce the post-powerstroke state. The magnitude of the lever swing matches the typical step length of myosin-5 walking along actin. These time-resolved structures directly demonstrate the swinging lever mechanism, ending decades of conjecture on how myosin produces force and movement.

biochemistry↗

bioRxiv2023

Exploiting the Affimer platform against influenza A virus

Influenza A virus (IAV) is well known for its pandemic potential. While current surveillance and vaccination strategies are highly effective, therapeutic approaches are short-lived due to the high mutation rates of IAV. Currently, monoclonal antibodies (mAbs) have emerged as a promising approach to tackle future IAV pandemics. Additionally, several antibody-like alternatives exist that aim to improve upon mAbs. Affimers, one such alternative, benefit from a short development time, high expression levels in E. coli, and complete animal-free production. Here we exploited the Affimer platform to isolate and produce specific and potent inhibitors of IAV. Starting from a monomeric version of the IAV trimeric hemagglutinin (HA) fusion protein, we isolated 12 Affimers that inhibit IAV H3 subtype infection in vitro. Two of these Affimers were characterised in detail: they exhibited binding affinities to the target H3 HA protein in the nM range and bound specifically to the HA1 head domain. Cryo-EM employing a novel spray approach to prepare cryo-grids allowed us to image HA-Affimer complexes. Combined with functional assays, we determined that the mode of inhibition of these Affimers is based on blocking the interaction of HA to the host-cell receptor - sialic acid. Additionally, these Affimers inhibited IAV strains closely related to the one employed for Affimer isolation. Overall, these results support the use of Affimers as an alternative to existing targeted therapies for IAV and pave the way for their use as diagnostic reagents. ImportanceInfluenza A virus is one of the few viruses that can cause devastating pandemics. Due to the high mutation rates of this virus, annual vaccination is required and antivirals are short-lived. Monoclonal antibodies present a promising approach to tackle influenza virus infections, but are associated with some limitations. To improve on this strategy, we explored the Affimer platform, which are antibody-like, bacterially made proteins. By performing phage-display against a monomeric version of influenza virus fusion protein, an established viral target, we were able to isolate Affimers that inhibit influenza virus infection in vitro. We characterised the mechanism of inhibition of the Affimers by challenging with related influenza virus strains. We additionally characterised an HA-Affimer complex structure, using a novel approach to prepare samples for cryo-electron microscopy. Overall, these results show that Affimers are a promising tool against influenza virus infection.

microbiology↗

bioRxiv2023

Exploiting cryo-EM structures of actomyosin-5a to reveal the physical properties of its lever

Myosin 5a (Myo5a) is a dimeric processive motor protein that transports cellular cargos along actin filaments. Its long lever is responsible for its large powerstroke, step size and load-bearing ability. Little is known about the levers structure and physical properties, and how they contribute to walking mechanics. Using cryo-electron microscopy and molecular dynamics simulations, we resolved the structure of monomeric Myo5a, comprising the motor domain and full-length lever, bound to F-actin. The range of its lever conformations revealed its physical properties, how stiffness varies along its length and predicts a large, 35 nm, working stroke. Thus, the newly released trail head in a dimeric Myo5a would only need to perform a small diffusive search for its new binding site on actin, and stress would only be generated across the dimer once phosphate is released from the lead head, revealing new insight into the walking behaviour of Myo5a.

biophysics↗

bioRxiv2021

The bacterial magnesium transporter MgtA reveals highly selective interaction with specific cardiolipin species

A significant challenge today within protein lipidology is to understand the relationship between cell structure, lipid membrane integrity, ion homeostasis and the embedded membrane proteins. The bacterial magnesium transporter A (MgtA) is a specialized P-type ATPase important for Mg2+ import into the cytoplasm; disrupted magnesium homeostasis is linked to intrinsic ribosome instability and nitro-oxidative stress in Salmonella strains. MgtAs function is highly dependent on anionic lipids, particularly cardiolipin, and further co-localization of cardiolipin with MgtA at the E. coli cell poles has been revealed. Here, we show that MgtA has functional specificity for cardiolipin 18:1, but it reaches maximum activity only in combination with cardiolipin 16:0, equivalent to the major components of native cardiolipin found in E. coli membranes. This is the first time it has been shown experimentally that two different lipid species from the same class, individually promoting low activity, can enhance activity in combination. Native mass spectrometry verifies the presence of two binding sites for cardiolipin and kinetic studies reveal that a cooperative relationship likely exists between the two different cardiolipin variants (cardiolipin 16:0 and cardiolipin 18:1). This is the first experimental evidence of cooperative effects between lipids of the same class, with only minor variations in their acyl chain composition, acting on a membrane protein. In summary, our results reveal that MgtA exhibits a highly complex interaction with one cardiolipin 18:1 and one cardiolipin 16:0, affecting protein activity and stability, and contributing to our understanding of the particular interactions between lipid environment and membrane proteins. Further, a better understanding of Mg2+ homeostasis in bacteria, due to its role as a virulence regulator, will provide further insights into the regulation and mechanism of bacterial infections.

biochemistry↗

bioRxiv2020

Structure of the endocytic adaptor complex reveals the basis for efficient membrane anchoring during clathrin-mediated endocytosis

During clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. Throughout this process, middle coat adaptors, Sla2 and Ent1, must remain attached to the plasma membrane to transmit force from the actin cytoskeleton required for successful membrane invagination. Here, we present a cryoEM structure of a 16-mer complex of membrane binding domains from Sla2 and Ent1 that anchors to the plasma membrane. Detailed mutagenesis in vitro and in vivo of the tetramer interfaces delineate the key interactions for complex formation and deficient cell growth phenotypes demonstrate the biological relevance of these interactions. Finally, time-resolved experiments in solution suggest that adaptors have evolved to achieve a fast subsecond timescale assembly in the presence of PIP2. Together, these findings provide a molecular understanding of an essential piece for the molecular puzzle of clathrin-coated sites.

biophysics↗

bioRxiv2020

Need for speed: Examining protein behaviour during cryoEM grid preparation at different timescales

A host of new technologies are under development to improve the quality and reproducibility of cryoEM grid preparation. Here we have systematically investigated the preparation of three macromolecular complexes using three different vitrification devices (Vitrobot, chameleon and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms, (the fastest reported to date), to interrogate particle behaviour at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimise. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic silver bullet for improving the quality of every cryoEM sample preparation.

biochemistry↗

bioRxiv2019

A Cryo-EM Grid Preparation Device for Time-Resolved Structural Studies

Structural biology generally provides static snapshots of protein conformations that can inform on the functional mechanisms of biological systems. Time-resolved structural biology provides a means to visualise, at near-atomic resolution, the dynamic conformational changes that macromolecules undergo as they function. Recent advances in the resolution obtainable by electron microscopy (EM) and the broad range of samples that can be studied makes it ideally suited to time-resolved studies. Here we describe a cryo-electron microscopy grid preparation device that permits rapid mixing, voltage assisted spraying, and vitrification of samples. We show that the device produces grids of sufficient ice quality to enable data collection from single grids that results in a sub 4 [A] reconstruction. Rapid mixing can be achieved by blot and spray or mix and spray approaches with a delay of ~10 ms, providing greater temporal resolution than previously reported approaches.

biophysics↗