Using site-directed mutagenesis to further the understanding of insulin receptor-insulin like growth factor-1 receptor heterodimer structure.

Type 2 diabetes is characterised by the disruption of insulin and insulin-like growth factor (IGF) signalling. The key hubs of these signalling cascades - the Insulin receptor (IR) and Insulin-like growth factor 1 receptor (IGF1R) - are known to form functional IR-IGF1R hybrid receptors which are insulin resistant. However, the mechanisms underpinning IR-IGF1R hybrid formation are not fully understood, hindering the ability to modulate this for future therapies targeting this receptor. To pinpoint suitable sites for intervention, computational hotspot prediction was utilised to identify promising epitopes for targeting with point mutagenesis. Specific IGF1R point mutations F450A, R391A and D555A show reduced affinity of the hybrid receptor in a BRET based donor-saturation assay, confirming hybrid formation could be modulated at this interface. These data provide the basis for rational design of more effective hybrid receptor modulators, supporting the prospect of identifying a small molecule that specifically interacts with this target.

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 often short-lived due to the high mutation rates of IAV. Recently, monoclonal antibodies (mAbs) have emerged as a promising therapeutic approach, both against current strains and future IAV pandemics. In addition to mAbs, several antibody-like alternatives exist, which aim to improve upon mAbs. Among these, Affimers stand out for their short development time, high expression levels in Escherichia coli, and animal-free production. In this study, we utilized the Affimer platform to isolate and produce specific and potent inhibitors of IAV. Using a monomeric version of the IAV trimeric hemagglutinin (HA) fusion protein, we isolated 12 Affimers that inhibit IAV infection in vitro. Two of these Affimers were characterized in detail and exhibited nanomolar-binding affinities to the target H3 HA protein, specifically binding to the HA1 head domain. Cryo-electron microscopy (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 these Affimers inhibit IAV by blocking the interaction of HA with the host-cell receptor, sialic acid. Furthermore, these Affimers inhibited IAV strains closely related to the one used for their isolation. Overall, our results support the use of Affimers as a viable alternative to existing targeted therapies for IAV and highlight their potential as diagnostic reagents. IMPORTANCE: Influenza 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 proteins made in bacteria. 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 characterized the mechanism of inhibition of the Affimers by using assays targeting different stages of the viral replication cycle. We additionally characterized 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.

CryoEM grid preparation: a closer look at advancements and impact of preparation mode and new approaches.

Sample preparation can present a significant hurdle within single particle cryo-electron microscopy (cryoEM), resulting in issues with reproducibility, data quality or an inability to visualise the sample. There are several factors which can influence this, including sample or buffer composition, grid type, route of sample preparation and interactions with the air-water interface (AWI). Here, we review some of the current routes for sample preparation and the associated challenges. We discuss a range of approaches for overcoming these challenges, such as minimising the grid preparation time, surfactants, grid type and biochemical approaches such as nanomagnetic beads. Finally, we discuss how a set of commercially available protein samples may serve as a benchmark suite for future technologies. This provides a route to compare techniques' abilities not just to generate high-resolution structures but also to overcome the challenges traditionally associated with cryoEM. As the field continues to produce new approaches to sample preparation and we start to better understand the underlying principles behind the behaviour of proteins within a thin film and in response to different environments, especially grid composition, it is hoped that more universal solutions can be provided that make the intractable systems tractable, improve resolution and, importantly, speed up data collection and reduce the currently required dataset sizes.

Structural basis and synergism of ATP and Na+ activation in bacterial K+ uptake system KtrAB.

The K+ uptake system KtrAB is essential for bacterial survival in low K+ environments. The activity of KtrAB is regulated by nucleotides and Na+. Previous studies proposed a putative gating mechanism of KtrB regulated by KtrA upon binding to ATP or ADP. However, how Na+ activates KtrAB and the Na+ binding site remain unknown. Here we present the cryo-EM structures of ATP- and ADP-bound KtrAB from Bacillus subtilis (BsKtrAB) both solved at 2.8 Å. A cryo-EM density at the intra-dimer interface of ATP-KtrA was identified as Na+, as supported by X-ray crystallography and ICP-MS. Thermostability assays and functional studies demonstrated that Na+ binding stabilizes the ATP-bound BsKtrAB complex and enhances its K+ flux activity. Comparing ATP- and ADP-BsKtrAB structures suggests that BsKtrB Arg417 and Phe91 serve as a channel gate. The synergism of ATP and Na+ in activating BsKtrAB is likely applicable to Na+-activated K+ channels in central nervous system.

A molecular switch in RCK2 triggers sodium-dependent activation of KNa1.1 (KCNT1) potassium channels.

The Na+-activated K+ channel KNa1.1, encoded by the KCNT1 gene, is an important regulator of neuronal excitability. How intracellular Na+ ions bind and increase channel activity is not well understood. Analysis of KNa1.1 channel structures indicate that there is a large twisting of the ßN-αQ loop in the intracellular RCK2 domain between the inactive and Na+-activated conformations, with a lysine (K885, human subunit numbering) close enough to potentially form a salt bridge with an aspartate (D839) in ßL in the Na+-activated state. Concurrently, an aspartate (D884) adjacent in the same loop adopts a position within a pocket formed by the ßO strand. In carrying out mutagenesis and electrophysiology with human KNa1.1, we found that alanine substitution of selected residues in these regions resulted in almost negligible currents in the presence of up to 40 mM intracellular Na+. The exception was D884A, which resulted in constitutively active channels in both the presence and absence of intracellular Na+. Further mutagenesis of this site revealed an amino acid size-dependent effect. Substitutions at this site by an amino acid smaller than aspartate (D884V) also yielded constitutively active KNa1.1, and D884I had Na+ dependence similar to wild-type KNa1.1, while increasing the side-chain size larger than aspartate (D884E or D884F) yielded channels that could not be activated by up to 40 mM intracellular Na+. We conclude that Na+ binding results in a conformational change that accommodates D884 in the ßO pocket, which triggers further conformational changes in the RCK domains and channel activation.

From TgO/GABA-AT, GABA, and T-263 Mutant to Conception of Toxoplasma.

Toxoplasma gondii causes morbidity, mortality, and disseminates widely via cat sexual stages. Here, we find T. gondii ornithine aminotransferase (OAT) is conserved across phyla. We solve TgO/GABA-AT structures with bound inactivators at 1.55 Å and identify an inactivator selective for TgO/GABA-AT over human OAT and GABA-AT. However, abrogating TgO/GABA-AT genetically does not diminish replication, virulence, cyst-formation, or eliminate cat's oocyst shedding. Increased sporozoite/merozoite TgO/GABA-AT expression led to our study of a mutagenized clone with oocyst formation blocked, arresting after forming male and female gametes, with "Rosetta stone"-like mutations in genes expressed in merozoites. Mutations are similar to those in organisms from plants to mammals, causing defects in conception and zygote formation, affecting merozoite capacitation, pH/ionicity/sodium-GABA concentrations, drawing attention to cyclic AMP/PKA, and genes enhancing energy or substrate formation in TgO/GABA-AT-related-pathways. These candidates potentially influence merozoite's capacity to make gametes that fuse to become zygotes, thereby contaminating environments and causing disease.

Time resolved applications for Cryo-EM; approaches, challenges and future directions.

Developments within the cryo-EM field have allowed us to generate higher-resolution "static" structures and pull out different conformational states which exist at equilibrium within the sample. Moreover, to trap non-equilibrium states and determine conformations that are present after a defined period of time (typically in the ms time frame) new approaches have been developed for the application of time-resolved cryo-EM. Here we give an overview of these different approaches and the limitations and strengths of each whilst identifying some of the current challenges to achieve higher resolutions and trap states within faster time frames. Time-resolved applications may play an important role in the ever-expanding toolkit of cryo-EM and open up new possibilities in both single particle and tomographic studies.

A 2.2 Å cryoEM structure of a quinol-dependent NO Reductase shows close similarity to respiratory oxidases.

Quinol-dependent nitric oxide reductases (qNORs) are considered members of the respiratory heme-copper oxidase superfamily, are unique to bacteria, and are commonly found in pathogenic bacteria where they play a role in combating the host immune response. qNORs are also essential enzymes in the denitrification pathway, catalysing the reduction of nitric oxide to nitrous oxide. Here, we determine a 2.2 Å cryoEM structure of qNOR from Alcaligenes xylosoxidans, an opportunistic pathogen and a denitrifying bacterium of importance in the nitrogen cycle. This high-resolution structure provides insight into electron, substrate, and proton pathways, and provides evidence that the quinol binding site not only contains the conserved His and Asp residues but also possesses a critical Arg (Arg720) observed in cytochrome bo3, a respiratory quinol oxidase.

PIEZO1 and PECAM1 interact at cell-cell junctions and partner in endothelial force sensing.

Two prominent concepts for the sensing of shear stress by endothelium are the PIEZO1 channel as a mediator of mechanically activated calcium ion entry and the PECAM1 cell adhesion molecule as the apex of a triad with CDH5 and VGFR2. Here, we investigated if there is a relationship. By inserting a non-disruptive tag in native PIEZO1 of mice, we reveal in situ overlap of PIEZO1 with PECAM1. Through reconstitution and high resolution microscopy studies we show that PECAM1 interacts with PIEZO1 and directs it to cell-cell junctions. PECAM1 extracellular N-terminus is critical in this, but a C-terminal intracellular domain linked to shear stress also contributes. CDH5 similarly drives PIEZO1 to junctions but unlike PECAM1 its interaction with PIEZO1 is dynamic, increasing with shear stress. PIEZO1 does not interact with VGFR2. PIEZO1 is required in Ca2+-dependent formation of adherens junctions and associated cytoskeleton, consistent with it conferring force-dependent Ca2+ entry for junctional remodelling. The data suggest a pool of PIEZO1 at cell junctions, the coming together of PIEZO1 and PECAM1 mechanisms and intimate cooperation of PIEZO1 and adhesion molecules in tailoring junctional structure to mechanical requirement.

Maintaining the momentum in cryoEM for biological discovery.

Cryo-electron microscopy (cryoEM) has been transformed over the last decade, with continual new hardware and software tools coming online, pushing the boundaries of what is possible and the nature and complexity of projects that can be undertaken. Here we discuss some recent trends and new tools which are creating opportunities to make more effective use of the resources available within facilities (both staff and equipment). We present approaches for the stratification of projects based on risk and known information about the projects, and the impacts this might have on the allocation of microscope time. We show that allocating different resources (microscope time) based on this information can lead to a significant increase in 'successful' use of the microscope, and reduce lead time by enabling projects to 'fail faster'. This model results in more efficient and sustainable cryoEM facility operation.

Unexpected structures formed by the kinase RET C634R mutant extracellular domain suggest potential oncogenic mechanisms in MEN2A.

The RET receptor tyrosine kinase plays a pivotal role in cell survival, proliferation, and differentiation, and its abnormal activation leads to cancers through receptor fusions or point mutations. Mutations that disrupt the disulfide network in the extracellular domain (ECD) of RET drive multiple endocrine neoplasia type 2A (MEN2A), a hereditary syndrome associated with the development of thyroid cancers. However, structural details of how specific mutations affect RET are unclear. Here, we present the first structural insights into the ECD of the RET(C634R) mutant, the most common mutation in MEN2A. Using electron microscopy, we demonstrate that the C634R mutation causes ligand-independent dimerization of the RET ECD, revealing an unusual tail-to-tail conformation that is distinct from the ligand-induced signaling dimer of WT RET. Additionally, we show that the RETC634R ECD dimer can form complexes with at least two of the canonical RET ligands and that these complexes form very different structures than WT RET ECD upon ligand binding. In conclusion, this structural analysis of cysteine-mutant RET ECD suggests a potential key mechanism of cancer induction in MEN2A, both in the absence and presence of its native ligands, and may offer new targets for therapeutic intervention.

It started with a Cys: Spontaneous cysteine modification during cryo-EM grid preparation.

Advances in single particle cryo-EM data collection and processing have seen a significant rise in its use. However, the influences of the environment generated through grid preparation, by for example interactions of proteins with the air-water interface are poorly understood and can be a major hurdle in structure determination by cryo-EM. Initial interactions of proteins with the air-water interface occur quickly and proteins can adopt preferred orientation or partially unfold within hundreds of milliseconds. It has also been shown previously that thin-film layers create hydroxyl radicals. To investigate the potential this might have in cryo-EM sample preparation, we studied two proteins, HSPD1, and beta-galactosidase, and show that cysteine residues are modified in a time-dependent manner. In the case of both HSPD1 and beta-galactosidase, this putative oxidation is linked to partial protein unfolding, as well as more subtle structural changes. We show these modifications can be alleviated through increasing the speed of grid preparation, the addition of DTT, or by sequestering away from the AWI using continuous support films. We speculate that the modification is oxidation by reactive oxygen species which are formed and act at the air-water interface. Finally, we show grid preparation on a millisecond timescale outruns cysteine modification, showing that the reaction timescale is in the range of 100s to 1,000s milliseconds and offering an alternative approach to prevent spontaneous cysteine modification and its consequences during cryo-EM grid preparation.

Towards sub-millisecond cryo-EM grid preparation.

Sample preparation is still a significant problem for many single particle cryo-EM workflows and our understanding and developments in the area lag behind that of image processing and microscope design. Over the last few years there has been growing evidence that many of the problems which occur during sample preparation are during the time the sample resides within the thin film created during the conventional blotting process. In parallel, faster grid preparation approaches have been developed for time-resolved cryo-EM experiments allowing for non-equilibrium intermediates to be captured on the ms timescale. Therefore, an important question is how fast can we prepare suitable grids for imaging by cryo-EM and how much does this mitigate the problems observed in sample preparation? Here we use a novel approach which has been developed for time-resolved studies to produce grids on an estimated sub-1 ms timescale. While the method comes with its own challenges, a 3.8 Å reconstruction of apoferritin prepared with the ultrafast method shows that good resolutions can be achieved. Although several orders of magnitude faster than conventional approaches we show using a ribosome sample, that interactions with the air-water interface cannot be avoided with preferred orientations still present. Therefore, the work shows that faster reactions can be captured but poses the question whether speed is the answer to problems with sample preparation.

Recent developments in the structural characterisation of the IR and IGF1R: implications for the design of IR-IGF1R hybrid receptor modulators.

The insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) are dimeric disulfide-linked receptor tyrosine kinases, whose actions regulate metabolic and mitogenic signalling pathways inside the cell. It is well documented that in tissues co-expressing the IR and IGF1R, their respective monomers can heterodimerise to form IR-IGF1R hybrid receptors. Increased populations of the IR-IGF1R hybrid receptors are associated with several disease states, including type 2 diabetes and cancer. Recently, progress in the structural biology of IR and IGF1R has given insights into their structure-function relationships and mechanism of action. However, challenges in isolating IR-IGF1R hybrid receptors mean that their structural properties remain relatively unexplored. This review discusses the advances in the structural understanding of the IR and IGF1R, and how these discoveries can inform the design of small-molecule modulators of the IR-IGF1R hybrid receptors to understand their role in cell biology.

Detergent-Free Functionalization of Hybrid Vesicles with Membrane Proteins Using SMALPs.

Hybrid vesicles (HVs) that consist of mixtures of block copolymers and lipids are robust biomimetics of liposomes, providing a valuable building block in bionanotechnology, catalysis, and synthetic biology. However, functionalization of HVs with membrane proteins remains laborious and expensive, creating a significant current challenge in the field. Here, using a new approach of extraction with styrene-maleic acid (SMA), we show that a membrane protein (cytochrome bo 3) directly transfers into HVs with an efficiency of 73.9 ± 13.5% without the requirement of detergent, long incubation times, or mechanical disruption. Direct transfer of membrane proteins using this approach was not possible into liposomes, suggesting that HVs are more amenable than liposomes to membrane protein incorporation from a SMA lipid particle system. Finally, we show that this transfer method is not limited to cytochrome bo 3 and can also be performed with complex membrane protein mixtures.

Tyrosine 288 in the extracellular domain of the human P2X7 receptor is critical for receptor function revealed by structural modeling and site-directed mutagenesis.

The P2X7 receptor (P2X7R) is a calcium-permeable cation channel activated by high concentrations of extracellular ATP. It plays a role in vital physiological processes, particularly in innate immunity, and is dysregulated in pathological conditions such as inflammatory diseases, neurodegenerative diseases, mood disorders, and cancers. Structural modeling of the human P2X7R (hP2X7R) based on the recently available structures of the rat P2X7 receptor (rP2XR) in conjunction with molecular docking predicts the orientation of tyrosine at position 288 (Y288) in the extracellular domain to face ATP. In this short communication, we combined site-directed mutagenesis and whole-cell patch-clamp recording to investigate the role of this residue in the hP2X7R function. Mutation of this extracellular residue to amino acids with different properties massively impaired current responses to both ATP and BzATP, suggesting that Y288 is important for normal receptor function. Such a finding facilitates development of an in-depth understanding of the molecular basis of hP2X7R structure-function relationships.