Biology subjects

Muench, S. P.

Publications and source records attributed to Muench, S. P..

14 recordsLinked to original sources

bioRxiv2026

High resolution structure of plant Light-Harvesting Complex II (LHCII) provides insight into lutein conformations and energy quenching

Plant Light Harvesting Complex II (LHCII) is found in the thylakoid membranes of chloroplasts and balances two roles: energy collection for photosynthesis and energy dissipation to prevent photo-damage when there is excessive sunlight. The mechanism for LHCII to switch between two energetic states has been debated but may involve a pH-triggered conformational change. Here we present single-particle cryo-electron microscopy (EM) structures of "light-harvesting" LHCII in detergent at pH 7.5 and pH 4.5. The high resolution (2.48 [A]) maps provide clear placements for all bound pigments, giving high confidence in the models. Surprisingly, we find that there is little conformational change to the polypeptide between these new light harvesting structures and previously published crystals structures, thought to be energy dissipating. The crossing angles of helix A/B and the Lutein 1-Chlorophyll 612 separation distances are similar. This contrasts with other recent analyses of LHCII by single-particle EM that suggested a change to the helix A/B angle and a reduction in Lutein 1-Chlorophyll 612 separation may trigger quenching and a photoprotective state. The high resolution of our structures also allowed us to investigate small conformational changes of the lutein within L1/L2 binding sites of LHCII, revealing rotations and distortions in the pigment that could lead to changes in energy transfer. In addition, we find that low pH causes LHCII to form a destabilised structure where pigment loss from the V1 binding site (usually violaxanthin or zeaxanthin) correlated with a disordered C-terminus, often for just one LHCII monomer with an LHCII trimer. Overall, our findings have important implications for the molecular mechanism of photoprotection.

biochemistry↗

bioRxiv2025

Large-Scale Structure-Based Virtual Screening Identifies Diverse KNa1.1 (KCNT1) Potassium Channel Inhibitors

Severe drug-resistant childhood epilepsy is caused by KCNT1 gain-of-function genetic variants, resulting in increased KNa1.1 channel activity. KCNT1-associated epilepsy is thought to affect around 1 in 300,000 births worldwide. Current treatment for KCNT1 epilepsy only provides mild symptomatic relief and uses a cocktail of experimental medications which must be personalised for the individual and are often poorly tolerated. Critically, with many patients, no therapeutic benefit is achieved. We sought to address this by using large-scale virtual screening to accelerate the development of a molecule which binds directly to KCNT1 to supress overactivity. We purchased a total of 71 compounds and using a combination of fluorescent thallium flux assays and patch clamp electrophysiology, identified a series of eight structurally diverse, novel inhibitors of the KNa1.1 channel with potency in the low micromolar range. These provide potential starting points for further development of drugs to treat KCNT1-associated epilepsy. HighlightsO_LIWe have discovered a range of structurally distinct inhibitors of the KCNT1 ion channel using large-scale virtual screening C_LIO_LIThese compound exhibit selectivity for the KCNT1 channel over other related ion channels C_LIO_LIThese compounds could provide starting points for new treatments for KCNT1 related Epilepsy. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/679465v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@481813org.highwire.dtl.DTLVardef@12494dforg.highwire.dtl.DTLVardef@a9f787org.highwire.dtl.DTLVardef@b9f4b9_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2025

(-)-Englerin A binds a conserved lipid site of TRPC5 and exposes a Met-aromatic motif in channel activation

TRPC4/5 cation channels are polymodal cellular sensors that play key roles in signal transduction/integration and have been implicated in various human pathologies, including anxiety, pain and cardiometabolic disease1-3. The plant natural product (-)-englerin A (EA)4 is a potent, selective TRPC4/5 agonist5,6 that has transformed fundamental and translational research on TRPC4/5 channels. However, the structural basis of interactions between EA and TRPC4/5 proteins has remained elusive, limiting our ability to fully understand and exploit mechanisms of TRPC4/5 channel activation by this intriguing natural product. Here, we present nine high-resolution cryo-EM structures (2.4-3.2 [A]) of human TRPC5 - representing different states and ligand occupancies - which show that EA binds to a conserved lipid binding site between transmembrane domains of adjacent TRPC5 subunits. Our structural models are consistent with the effects of mutagenesis of nearby residues on EAs potency, efficacy and activation kinetics, and allow us to rationalise competitive inhibition by other TRPC4/5 modulators as well as EAs selectivity profile within the TRPC family. Comparison of structures containing various TRPC5:EA stoichiometries revealed key structural and molecular determinants of EA-mediated TRPC5 activation - most notably the aromatic interaction network around Phe520 - underscoring the critical function of Met-aromatic motifs in ion channel structure and function. Binding of EA causes conformational changes of nearby amino acid residues, resulting in rearrangement of the pore helices into a pre-open state. Collectively, we provide structural insight into the mode-of-action of the most widely used TRPC4/5 agonist, which will underpin fundamental TRPC4/5 channel research as well as ongoing drug discovery programmes.

molecular biology↗

bioRxiv2025

Identification of new KCNT1-epilepsy drugs by in silico, cell and Drosophila modelling.

ObjectiveHyperactive KCNT1 potassium channels, caused by gain-of-function mutations, are associated with a range of epilepsy disorders. Patients typically experience drug-resistant seizures and in cases with infantile onset, developmental regression can follow. KCNT1-related disorders include epilepsy of infancy with migrating focal seizures and sleep related hypermotor epilepsy. There are currently no effective treatments for KCNT1-epilepsies, but suppressing over-active channels poses a potential strategy. MethodsUsing KCNT1 channel structural data, we in silico screened a library of known drugs for those predicted to block the channel pore to reduce the current amplitude and inhibit channel activity. ResultsEight known drugs were investigated in vitro for their effects on patient-specific mutant KCNT1 channels, with four drugs showing significant reduction of K+ current amplitudes. The action of the four drugs was then analyzed in vivo and two were found to reduce the seizure phenotype in humanized Drosophila KCNT1-epilepsy models. InterpretationThis study identified two known drugs, antrafenine and nelfinavir mesylate, that reduce KCNT1 channel activity and reduce seizure activity in whole animals, suggesting their potential use as new treatments for KCNT1-epilepsy. The sequential in silico, in vitro and in vivo mechanism-based drug selection strategy used here may have broader application for other human disorders where a disease mechanism has been identified.

pharmacology and toxicology↗

bioRxiv2025

Structural elucidation of the haptoglobin-hemoglobin clearance mechanism by macrophage scavenger receptor CD163

Intravascular hemolysis releases hemoglobin into the bloodstream, which can damage vascular and renal tissues due to its oxidative nature. Circulating haptoglobin acts as a primary defense by binding to free hemoglobin, forming a haptoglobin-hemoglobin (HpHb) complex that is then recognized and cleared by the CD163 scavenger receptor on macrophages. While the function and structure of HpHb complex are mostly well-defined, the molecular mechanism underlying its interaction with CD163 remains unclear. Here we report the cryo-electron microscopy structures of CD163 in its unliganded state and in its complex with HpHb. These structures reveal that CD163 functions as a trimer, forming a composite binding site at its center for one protomer of the dimeric HpHb, resulting in a 3:1 binding stoichiometry. In the unliganded state, CD163 can also form a trimer, but in an autoinhibitory configuration that occludes the ligand binding site. Widespread electrostatic interactions mediated by calcium ions are pivotal in both pre-ligand and ligand-bound receptor assemblies. This calcium-dependent mechanism enables CD163/HpHb complexes to assemble and, once internalized, disassemble into individual components upon reaching the endosome, where low calcium and lower pH conditions prevail. Collectively, this study elucidates the molecular mechanism by which CD163-mediated endocytosis efficiently clears different isoforms of HpHb.

biophysics↗

bioRxiv2024

Ideal efficacy photoswitches for TRPC4/5 channels harness high potency for spatiotemporally-resolved control of TRPC function in live tissues

Directly probing the endogenous biological roles of target proteins with high spatial and temporal resolution, as non-invasively and reproducibly as possible, is a shared conceptual goal for research across many fields, as well as for targeted therapies. Here we describe the rational conceptual design and test-case practical implementation of a photopharmacological paradigm to empower high-performance photomodulation studies in vivo. TRPC4/5 ion channels are involved in many spatiotemporally resolved circuits, from pain and anxiety, to reproductive signaling, digestion, and obesity. To unpick their biology requires spatiotemporally precise tools, which were lacking. We developed "ideal efficacy photoswitch" ligands to control their diverse functions in situ. These E{leftrightarrows}Z-photoswitchable ligands bias TRPC[4]/5 channel activity with exquisite photocontrol, from strong agonism under 360 nm, to low agonism at 385 nm, to strong antagonism at 410-460 nm. Cryo-EM structures of both TRPC4 and TRPC5 with both Z-agonists and E-antagonists support the rationale for efficacy switching through competitive E/Z isomer binding. Crucially, since the E/Z ratio is exclusively determined by the light wavelength applied, their channel photocontrol is exclusively wavelength-dependent, yet drug-concentration-independent: so is reproducible from cell culture to >millimetre-depth tissues. Indeed, we were able to photocontrol both direct and downstream TRPC4/5 biology in cell lines or primary cells in culture, from calcium flux, to primary neuron excitability and adrenaline release; and even in tissues, photoswitching small intestine motility and peristalsis. The TRPC4/5 ligands we develop will thus unlock a range of high-precision investigations in TRP biology. More broadly, we propose that the success of this efficacy photoswitch program, from concept to tissue level translation, is mainly a consequence of how biology has evolved proteins for efficacy control. We therefore foresee that a variety of functionally responsive protein targets, not only sensory and signaling ion channels and receptors, will be amenable to similarly high-performance photocontrol even in vivo, if a new generation of reagent development adopts this paradigm of ideal efficacy photoswitching. Table of Contents Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/602451v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1f8e527org.highwire.dtl.DTLVardef@18c72d0org.highwire.dtl.DTLVardef@1c5d648org.highwire.dtl.DTLVardef@177203d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

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.

molecular biology↗

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

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 {beta}N-Q loop in the intracellular RCK2 domain between the inactive and Na+-activated conformations, with a lysine (K885, human subunit numbering) close enough to form a salt bridge with aspartate (D839) in the Na+-activated state. Concurrently, an aspartate (D884) adjacent in the same loop adopts a position within 4 [A] of several acidic or polar residues. In carrying out mutagenesis and electrophysiology with human KNa1.1, we found alanine substitution of each of these residues 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, D884I had Na+- dependence similar to wild-type KNa1.1, whilst 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 acid-rich pocket, which triggers further conformational changes in the RCK domains and channel activation. Statement of SignificanceSodium-activated potassium channels regulate neuronal excitability, and their dysfunction causes severe childhood disorders. Here, we identify a structural determinant in the intracellular domains that is responsible for triggering channel activation in response to sodium ion binding. An increase in the size of a particular amino acid renders the channel sodium-insensitive, whilst a decrease in size enables the channel to activate in the absence of sodium. This enhances our understanding of how this subclass of potassium channels respond to changes in the intracellular ionic environment. Furthermore, this may also further our understanding of the basis of human neurological disorders and their treatment.

biophysics↗

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↗

bioRxiv2020

Cryo-EM structures of human TRPC5 reveal interaction of a xanthine-based TRPC1/4/5 inhibitor with a conserved lipid binding site.

TRPC1/4/5 channels are non-specific cation channels implicated in a wide variety of diseases, and TRPC1/4/5 inhibitors have recently entered the first clinical trials. However, fundamental and translational studies require a better understanding of TRPC1/4/5 channel regulation by endogenous and exogenous factors. Although several potent and selective TRPC1/4/5 modulators have been reported, the paucity of mechanistic insights into their modes-of-action remains a barrier to the development of new chemical probes and drug candidates. The xanthine class of modulators includes the most potent and selective TRPC1/4/5 inhibitors described to date, as well as TRPC5 activators. Our previous studies suggest that xanthines interact with a, so far, elusive pocket of TRPC1/4/5 channels that is essential to channel gating. Targeting this pocket may be a promising strategy for TRPC1/4/5 drug discovery. Here we report the first structure of a small molecule-bound TRPC1/4/5 channel - human TRPC5 in complex with the xanthine Pico145 - to 3.0 [A]. We found that Pico145 binds to a conserved lipid binding site of TRPC5, where it displaces a bound phospholipid. Our findings explain the mode-of-action of xanthine-based TRPC1/4/5 modulators, and suggest a structural basis for TRPC1/4/5 modulation by endogenous factors such as (phospho)lipids and Zn2+ ions. These studies lay the foundations for the structure-based design of new generations of TRPC1/4/5 modulators.

molecular biology↗

bioRxiv2019

Structure-based identification and characterisation of novel inhibitors of KNa1.1 potassium channels, a stratified target for KCNT1-related epilepsy.

Several types of drug-resistant epileptic encephalopathies of infancy have been associated with mutations in the KCNT1 gene, which encodes the sodium-activated potassium channel subunit KNa1.1. These mutations are commonly gain-of-function, increasing channel activity, therefore inhibition by drugs is proposed as a stratified approach to treat disorders. To date, quinidine therapy has been trialled with several patients, but mostly with unsuccessful outcomes, which has been linked to its low potency and lack of specificity. Here we describe the use of a cryo-electron microscopy-derived KNa1.1 structure and mutational analysis to identify the quinidine biding site and identified novel inhibitors that target this site using computational methods. We describe six compounds that inhibit KNa1.1 channels with low- and sub-micromolar potencies, likely through binding in the intracellular pore vestibule. In preliminary hERG inhibition and cytotoxicity assays, two compounds showed little effect. These compounds may provide starting points for the development of novel pharmacophores for KNa1.1 inhibition, with the view to treating KCNT1-associated epilepsy and, with their potencies higher than quinidine, could become key tool compounds to further study this channel. Furthermore, this study illustrates the potential for utilising cryo-electron microscopy in ion channel drug discovery.

pharmacology and toxicology↗

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↗