Biology subjects

Buck, M.

Publications and source records attributed to Buck, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Host membrane cholesterol constrains constitutive signaling of the oncogenic KSHV GPCR ORF74

Cholesterol is a major structural component of the plasma membrane and a key allosteric regulator of G protein-coupled receptors (GPCRs), yet its role in controlling constitutive receptor activity remains poorly understood. Virally encoded GPCRs provide an ideal system to address this question because many exhibit constitutive signaling that promotes viral persistence and pathogenesis, although how excessive receptor activation is restrained remains unknown. Here, we identify a previously unrecognized cholesterol-dependent allosteric mechanism by which the oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) GPCR ORF74 constrains its constitutive activity. CryoEM structural analysis reveals a cholesterol-binding pocket formed by transmembrane helices 3, 5, and 6 that is present only in the inactive receptor. Cholesterol binding restrains the outward movement of transmembrane helix 6, stabilizes the inactive conformation, and suppresses spontaneous receptor activation, as supported by molecular dynamics simulations. Mechanistically, replacement of the canonical DRY motif with a non-canonical VRY motif exposes the conserved R1433.50 residue, creating a membrane-facing cholesterol-binding interface that couples membrane cholesterol to receptor conformational control. Consistent with this model, cellular cholesterol depletion enhances ORF74 signaling, whereas disruption of cholesterol binding impairs receptor stabilization. Together, our findings uncover a previously unrecognized mechanism by which a viral GPCR exploits host membrane cholesterol to regulate its constitutive activity, suggesting that persistent viruses optimize constitutive signaling by coupling receptor activity to host lipid-dependent allosteric regulation.

microbiology↗

bioRxiv2026

A model for PIP2/3 and Rnd1 effects on Plexin-B1 GAP activity on Rap1b GTPase derived from molecular dynamics simulations

Plexin-B1 is a transmembrane receptor that integrates signals from Rho-family and Ras-family (Rap1b) GTPases to regulate cellular processes. While ligand simulated activation of the receptor is largely understood, the role of membrane composition and GTPase allosteric effects on plexin structure, internal protein dynamics, and function is still to be elucidated. Here, we performed multi-replica, 1 s all-atom simulations of Plexin-B1-GTPase complexes on PIP2- and PIP3-containing membranes to investigate the effects of these two signaling lipids, as well as on the GTPases. We found that both Rap1b and Rnd1 stably associate with the membrane, with PIP2 promoting broader lipid engagement and stronger Rap1b-Plexin-B1 interactions, whereas PIP3 enhances Rnd1-Plexin contacts and induces a membrane proximal orientation of Plexins juxtamembrane helix and makes contacts with a previously discovered activation switch loop. Contact map and network analyses revealed lipid-dependent shifts in allosteric communication, with PIP2 favoring Rap1b-centric hotspots and PIP3 favoring Rnd1-centric pathways. These predictions allow us to suggest a model for plexin intracellular region activation where both the identity of phosphoinositides and GTPase context synergistically stabilize Plexin-B1 membrane engagement, alter structural dynamics, and allosteric networks. Thus, we propose that the membrane is an active modulator of plexin receptor signaling.

biophysics↗

bioRxiv2026

Temperature-dependent biofilm and sublancin production immobilise arsenic and antibiotic resistance gene mobility in soil system

Climate change-induced warming and arsenic soil contamination synergistically threaten agricultural sustainability by restructuring microbial communities and accelerating antibiotic resistance evolution. Here, we demonstrate that Bacillus subtilis 168-derived sublancin, a temperature-optimised glycosylated antimicrobial peptide, simultaneously maximises microbial arsenic sequestration and suppresses antibiotic resistance gene (ARG) horizontal transfer. Temperature-dependent biofilm formation increased progressively from 25-35{degrees}C (absorbance: 0.37-1.2), correlating with enhanced arsenic accumulation within the extracellular polymeric substance matrix (74% increase in weight percentage). Time-of-flight secondary ion mass spectrometry confirmed maximum arsenic sequestration at 35{degrees}C (total counts: 4.725x10). Sublancin production peaked at 30.8{degrees}C (129.72 mg/L) and significantly reduced soil microbial biomass carbon by 33% (p < 0.001), with selective suppression of Gram-positive bacteria (42%-28% survival reduction). Conjugation assays revealed sublancin bio-amendment dramatically suppressed horizontal ARG transfer, reducing HGT frequency by 74.7% (6.64 x 10-3 to 1.68 x 10-3, p < 0.001) through dual mechanisms: decreased donor viability and reduced conjugation efficiency. Gene ontology enrichment identified coordinated upregulation of exopolysaccharide synthesis (FDR ~1.0x10-{superscript 2}), phosphotransferase systems, and arsenic resistance pathways, alongside downregulation of conventional antibiotic resistance genes. These findings establish sublancin as a dual-function soil amendment simultaneously addressing two critical agricultural threats: heavy metal bioaccumulation and antibiotic resistance dissemination under climate warming.

microbiology↗

bioRxiv2025

From Dimerization to Signaling: Molecular Dynamics Insights into EphA1 and EphA2 Isoform Specificity

Ephrin receptors (Ephs) are receptor tyrosine kinases that regulate cellular growth, differentiation, and motility. EphA2, often overexpressed in cancer, is notable for its ligand-independent activation, which drives pro-oncogenic signaling distinct from the canonical, ligand-dependent pathway that restricts cell movement. While ligand binding induces extracellular clustering, kinase activation depends on dimerization within the transmembrane (TM) region. EphA1 and EphA2 differ substantially in their function, likely due to differences in their TM, but more-so in their juxtamembrane (JM), and membrane-proximal fibronectin type III (FN1/FN2) domains. How these latter two regions modulate dimerization has not yet been investigated. To address this, we performed extensive coarse-grained (CG) simulations using Martini 3 in an anionic POPC/PS/PIP2 model of the plasma membrane. Both receptors formed stable TM dimers, though EphA1 favored a symmetric AXXXGXXXG-centered interface, whereas EphA2 favoured an alternate leucine zipper interface. All protein constructs sampled multiple configurations, reflecting substantial intrinsic variability. AlphaFold3 does not yield reliable predictions and cannot account of effects of the lipid bilayer. In the CG simulations, basic residues in the JM region remained membrane-bound, and the EphA2 FN domain displayed sustained PIP2 interactions, consistent with previous observations. Notably, constructs with the FN2 domain alone restricted TM association in both receptors, whereas inclusion of the second FN1 domain restored dimerization but produced receptor-specific extracellular interfaces. These differences arise from distinct FN1-FN2 linker flexibilities, a different level sequence conservation of EphA1 compared to several other Ephs and FN-domain membrane contacts, which together shape TM geometry and lipid engagement. Our results predict how TM and TM-proximal elements cooperatively tune dimerization in Eph receptors. This work offers a molecular rationale into the divergent activation behaviors of EphA1 and EphA2 and provides testable models relevant to cancer biology and Eph-driven signaling.

biophysics↗

bioRxiv2025

Tyrosine phosphorylation and the inhibitory C-terminal SAM domain moderately affect transient interactions in a EphA2 cytoplasmic fragment in solution: A combined experimental and molecular modeling study.

Eph receptors, the largest family of receptor tyrosine kinases (RTKs), orchestrate diverse developmental processes, including axon guidance, tissue patterning, and cell positioning, while also contributing to adult functions such as synaptogenesis and tissue homeostasis. Proteolytic cleavage of Eph receptors by neurodegeneration-associated proteases generates a membrane-detached intracellular region (ICR) fragment whose structural organization and signaling remain poorly understood. Here, we characterize a model for such a fragment from EphA2 and discover that the juxtamembrane region, the kinase domain, and the C-terminally linked sterile alpha motif (SAM) domain comprise a configurational ensemble of domain contacts and orientations which inhibit the intermolecular interactions and tyrosine cross-phosphorylation. Recombinant EphA2 ICR expressed in E. coli retains catalytic activity and undergoes autophosphorylation at multiple tyrosine residues which have previously been identified in eukaryotic systems. Phosphorylation weakens intramolecular interactions, as measured by microscale thermophoresis, indicating a phosphorylation-dependent remodeling of the ICR. While AlphaFold2-Multimer (AF2M) and Alphafold3 (AF3) predictions as well as available crystal structures provide only limited insight into these configurational states, coarse-grained molecular dynamics simulations using the Martini 3 force field reveal highly dynamic and predominantly transient interactions between the kinase and SAM domains that cluster at two discrete regions on the kinase surface. NMR spectroscopy of isotopically labeled EphA2 SAM suggests non-canonical intramolecular contacts that preserve the canonical SAM interaction interface for binding of partner proteins. Addition of the SHIP2 SAM domain promotes formation of the canonical EphA2 and SHIP2 SAM complex while also revealing previously unrecognized interactions of the kinase domain on the SHIP2 SAM domain. Collectively, these findings establish the EphA2 ICR as a dynamic, phosphorylation-responsive signaling module in which transient intramolecular interactions regulate accessibility of several interfaces for binding. Thus, the proteolytically released EphA2 intracellular fragment is likely to have distinct characteristics in cancer and neurodegenerative disease, compared to the membrane bound form in the full receptor.

biophysics↗

bioRxiv2025

Macropinocytosis mediates neurotropism of Cryptococcus neoformans in a human organoid model of the blood-brain barrier.

The opportunistic and neuroinvasive fungus, Cryptococcus neoformans (Cn), causes a life-threatening brain infection that despite treatment can cause long-term cognitive deficiencies. Studies have shown that Cn can infiltrate the central nervous system (CNS) through a transcellular route across the brain endothelium, however, the molecular process that drives brain endothelial cells to internalize Cn remains poorly defined. Here we examine the molecular interactions between fungal cells and the brain endothelium by utilizing a human 3D organoid model of the blood-brain barrier (BBB). We show that Cn exploits the process of macropinocytosis as the mechanism of endocytosis into brain endothelial cells by recruiting CD44 and EphA2 as a molecular complex. We identified two predicted binding sites on EphA2 for CD44, suggesting that the two structurally distinct regions may provide a molecular basis for cooperative signaling in brain endothelial cells that stimulate macropinocytosis as the mode of entry for Cn.

molecular biology↗

bioRxiv2025

Molecular Insights Into PGPR-Biochar-Mediated Arsenic Detoxification and Nutritional Enhancement in Rice Rhizospheres

Arsenic (As) contamination in paddy soils threatens rice productivity and food safety through enhanced As(III) mobility under anaerobic conditions. This study evaluated the synergistic effects of rice biochar (RBC) and plant growth-promoting rhizobacteria (PGPR) on arsenic bioavailability and rice physiological responses under controlled and field conditions. Rice straw biochar pyrolyzed at 400 {degrees}C exhibited high porosity and surface functionality, while Metabacillus indicus AMB4 demonstrated arsenic tolerance ([≤]3500 mg kg-1) and multiple PGPR traits (IAA: 45.2 g mL-1, phosphate solubilization index: 2.8, ACC deaminase: 18.7 nmol -ketobutyrate mg-1 h-1). Combined RBC+PGPR treatment reduced soil arsenic bioavailability by 85% (p<0.001) and plant arsenic accumulation by 78% compared to controls, while significantly enhancing antioxidant enzyme activities (CAT: 4.2-fold, SOD: 3.7-fold, APX: 3.1-fold increase). Metagenomics revealed restructured microbial communities with increased Proteobacteria abundance and upregulated arsenic detoxification genes (arsC, arsB, aioA) coupled with enhanced nutrient cycling pathways (nifH, phoD, soxA). Network analysis demonstrated functional integration between As-transformation and nutrient metabolism, with coordinated expression of 47 arsenic-responsive and 126 nutrient-cycling genes. Field trials confirmed superior plant performance with RBC+PGPR treatment yielding 56% more tillers and 73% more panicles than controls, while maintaining enhanced soil nutrient status. This integrated biochar-PGPR system provides a mechanistically robust approach for simultaneous arsenic remediation and crop productivity enhancement in contaminated rice ecosystems, demonstrating practical applicability for sustainable paddy agriculture.

microbiology↗

bioRxiv2025

Furrow tillage reduces soil carbon loss and enhances microbial metabolism

With increasing global carbon loss from agricultural fields, agricultural practices should be monitored with proper regulatory measures. To address three problems of minimising carbon loss, optimising soil fertility and enhancing soil microbiota together, this study has developed a new innovative "Furrow tillage" approach that affords a balance between conventional and conservative tillage. Based on two years of trials in rice fields at multiple sites, this practice has justifications from agronomy, geochemistry, crop physiology, and molecular microbiology. Furrow tillage illustrated low carbon loss, similar to no-tillage, while retaining high nutrient bioavailability, similar to deep tillage. Microbial diversity, molecular responses and metabolic activities showed that furrow tillage induced microbial interactions while allowing a better entrapment of carbon dioxide in the soil. The study used CO2 flux, spatial distribution, total and available elemental analysis, plant ultra-structural observations, microbial metagenomics, high-throughput sequencing and molecular modelling to establish an optimal soil-crop quality, microbial maintenance and reduce carbon loss through furrow tillage practice.

ecology↗

bioRxiv2025

Computation model predicts Rho GTPase function with the Plexin Transmembrane receptor GAP activity on Rap1b via dynamic allosteric changes

Plexin-semaphorin signaling regulates key processes such as cell migration, neuronal development, angiogenesis, and immune responses. Plexins stand out because they can directly bind with both Rho- and Ras-family small GTPases through their intracellular domains when these GTPases are in their active, GTP-bound states. This binding occurs via intracellular regions which include a Rho-GTPase Binding Domain (RBD) and a GTPase Activating Protein (GAP) segment. Studies have shown that Rho and Ras GTPases play vital roles in plexin signaling and activation. However, the structural dynamics of plexins and GTPases and how these conformational changes affect interactions when plexin is bound with both Ras and Rho-GTPases or bound to only one specific GTPase has remained unclear. In this study, we conducted molecular dynamics (MD) simulations on six distinct plexin-GTPase bound systems to investigate the differences in conformations and dynamics between Plexin-B1 and three GTPases: Rap1b, Rnd1, and Rac1. Our analysis revealed that dynamics with Rac1 are more altered, compared to Rnd1 depending on whether plexins GAP domain is bound or unbound to Rap1b. In addition, we further investigated alterations in network centralities and compared the network dynamics of the Plexin-GTPases complexes, focusing on the differences when Plexin is bound to both Ras (Rap1b) and Rho-GTPases (Rnd1/Rac1) versus when it is bound to only one GTPase. Our study revealed that Rnd1 exhibits stronger and more stable interactions with Plexin-B1 in the absence of Rap1b, while Rac1 shows fewer and less stable connections in comparison. These computational models have features that broadly agree with experimental results from hydrogen-deuterium exchange detected by mass spectrometry (HDX-MS). Such insights provide a better understanding of the molecular mechanisms underlying Plexin-GTPase interactions and the complexities of signaling mechanisms involving GTPases in general.

biophysics↗

bioRxiv2025

Subunit specialisation in AAA+ proteins and substrate unfolding during transcription complex remodelling

Bacterial RNA polymerase (RNAP) is a multi-subunit enzyme that copies DNA into RNA in a process known as transcription. Bacteria use {sigma} factors to recruit RNAP to promoter regions of genes that need to be transcribed, with 60% bacteria containing at least one specialized {sigma} factor {sigma}54. {sigma}54 recruits RNAP to promoters of genes associated with stress responses and forms a stable closed complex that does not spontaneously isomerize to the open state where promoter DNA is melted out and competent for transcription. The {sigma}54-mediated open complex formation requires specific AAA+ proteins (ATPases Associated with diverse cellular Activities) known as bacterial enhancer-binding proteins (bEBPs). We have now obtained structures of new intermediate states of bEBP-bound complexes during transcription initiation, which elucidate the mechanism of DNA melting driven by ATPase activity of bEBPs and suggest a mechanistic model that couples the ATP hydrolysis cycle within the bEBP hexamer with {sigma}54 unfolding. Our data reveal that bEBP forms a non-planar hexamer with the hydrolysis-ready subunit located at the furthest/highest point of the spiral hexamer relative to the RNAP. ATP hydrolysis induces conformational changes in bEBP that drives a vectoral transiting of the regulatory N-terminus of {sigma}54 into the bEBP hexamer central pore causing the partial unfolding of {sigma}54, while forming specific bEBP contacts with promoter DNA. Furthermore, our data suggest a mechanism of AAA+ protein that is distinct from the hand-over-hand mechanism proposed for many AAA+ proteins, highlighting the versatile mechanisms utilized by the large protein family.

biochemistry↗

bioRxiv2025

Cholesterol-Dependent Dimerization and Conformational Dynamics of EphA2 Receptors: Insights from Coarse-Grained and All-Atom Simulations

The EphA2 transmembrane receptor regulates cellular growth, differentiation, and motility, and its overexpression in various cancers makes it a potential biomarker for clinical cancer management. EphA2 signaling occurs through ligand-induced dimerization, where the transmembrane (TM) and juxtamembrane (JM) domains play crucial roles in stabilizing the dimer conformations and thereby facilitating signal transduction. Electrostatic interactions between basic JM residues and signaling lipids (PIP2 and PIP3) regulate phosphorylation while Cholesterols potential role in modulating EphA2 activation remains unclear. To investigate this, we modeled the TM-full JM peptide of EphA2 and employed coarse-grain and all-atom simulations to investigate its dimerization in cholesterol-rich and cholesterol-deficient membranes. Our findings reveal that cholesterol stabilizes specific TM dimers and TM-JM interactions with PIP2, highlighting the importance of membrane composition in EphA2 dimerization, oligomerization, and clustering. These insights enhance our understanding of lipid-mediated regulation of EphA2 and its implications in receptor signaling and cancer progression.

biophysics↗

bioRxiv2024

Cholesterol inhibits oncogenic EphA2 activation

The receptor tyrosine kinase EphA2 drives cancer malignancy by facilitating metastasis. EphA2 can be found in different self-assembly states: as a monomer, dimer, and oligomer. However, our understanding remains limited regarding which EphA2 state is responsible for driving pro-metastatic signaling. To address this limitation, we have developed SiMPull-POP, a single-molecule method for accurate quantification of membrane protein self-assembly. Our experiments revealed that a reduction of plasma membrane cholesterol strongly promoted EphA2 self-assembly. Indeed, low cholesterol caused a similar effect to the EphA2 ligand ephrinA1-Fc. These results indicate that cholesterol inhibits EphA2 assembly. Phosphorylation studies in different cell lines revealed that low cholesterol increased phospho-serine levels, the signature of oncogenic signaling. Investigation of the mechanism that cholesterol uses to inhibit the assembly and activity of EphA2 indicate an in-trans effect, where EphA2 is phosphorylated by protein kinase A downstream of beta-adrenergic receptor activity, which cholesterol also inhibits. Our study not only provides new mechanistic insights on EphA2 oncogenic function, but also suggests that cholesterol acts as a molecular safeguard mechanism that prevents uncontrolled self-assembly and activation of EphA2.

biochemistry↗

bioRxiv2024

A mechanistic model of Rtc-regulated RNA repair suggests molecular targets to potentiate antibiotic effects

RNA is susceptible to damage from various internal and external factors. Given the integral role of RNA, its repair is essential for maintaining proper cell function. A highly conserved RNA repair system, the Rtc system, maintains core RNA components of the translational apparatus. In E. coli, Rtc expression is induced upon various stresses, including exposure to ribosome-targeting antibiotics. Its expression enables cells to rescue growth and survive treatment by conferring transient resistance to these antibiotics. The mechanisms by which Rtc-induced resistance arises are largely unknown. Here, we develop and analyse a computational model of Rtc-regulated maintenance of long-lived RNAs that form part of the translational apparatus. Our model analysis and experimental validation provide evidence of cell-to-cell heterogeneity in rtc expression and effects on the translational capacity of cells, indicating that individual cell fates determine antibiotic efficacy and thus rtc may induce a form of heteroresistance. Through further analysis and quantification of single-cell ribosome levels, we then identify targets within the Rtc system that can reduce translational capacity of cells, rendering cells more susceptible and so potentiating antibiotic effects. Our results provide novel tools for the systems analysis of Rtc-regulated RNA repair, they reveal a complex response underpinning resistance conferred by the innate repair system, and they suggest novel avenues to address resistance in E. coli and related clinical pathogens.

systems biology↗

bioRxiv2023

The SAM Domain of EphA2 Inhibits Ligand-Independent Clustering and Activation

Eph receptors are the largest family of receptor tyrosine kinases (RTKs). They play a role in the pathogenesis of various diseases including cancer, atherosclerosis, fibrosis, infectious diseases, diseases of the central nervous system and age-related cataract. EphA2 has attracted much attention over the years owing to its dysregulation in many diseases. Previous studies have revealed the unique molecular organizations of Eph receptors, and particularly EphA2, into large clusters of receptor-ligand complexes. One unique feature of Eph receptors is a C-terminal sterile alpha motif (SAM) domain, which has been proposed to alter dimerization and kinase activity in EphA2. However, the precise role of the SAM domain in regulating the function and oligomerization state of EphA2 has not been reported. Here we apply a time-resolve fluorescence spectroscopy, PIE-FCCS, to characterize the oligomerization state of EphA2 in live cells and determine the role of the SAM domain. We deleted the SAM domain in the context of full length EphA2 and an intracellular domain (ICD) construct to assess the effect of the SAM domain on oligomerization state, kinase activity, and cellular behavior. Overall, we find that the SAM domain inhibits ligand-independent clustering and kinase activity in both full-length EphA2 and the isolated ICD construct at the cell membrane. These results are consistent with the autoinhibitory features of the C-terminal tail of EGFR and may help resolve the allosteric regulation of other RTKs.

biophysics↗

bioRxiv2023

Phosphorylation modulates estrogen receptor disorder by altering long-range hydrophobic interactions

Protein intrinsic disorder is coupled to a range of biological phenomena, from gene regulation to cancer progression. Phosphorylation of the estrogen receptor (ER) at Ser118 through its disordered N-terminal domain (NTD) activates its transcriptional function, but it is challenging to rationalize how this modification regulates ER activity. Using biophysical approaches of small-angle X-ray scattering and nuclear magnetic resonance spectroscopy, we demonstrate that Ser118 phosphorylation triggers long-range conformational changes in ER-NTD, particularly between two hydrophobic clusters of residual structures. Alanine substitution of hydrophobic amino acids near Ser118 produces similar conformational alterations and rescues impaired ER activity caused by a phosphorylation-deficient mutant. These findings establish a direct link between phosphorylation-induced conformational changes and the activation function of this disordered protein as a promising avenue to block ER transcriptional activation.

biophysics↗

bioRxiv2023

Geographic population structure and distinct population dynamics of globally abundant freshwater bacteria

Geographic separation is a principal factor for structuring populations of macroorganisms, with important consequences for evolution, by means of processes such as allopatric speciation. For free-living prokaryotes, implications of geographic separation on their evolution are more unclear. The limited phylogenetic resolution of commonly used markers such as 16S rRNA gene sequences have since long impeded prokaryotic population genetics. However, the vast amount of metagenome sequencing data generated during the last decades from various habitats around the world, now provides an excellent opportunity for such investigations. Here we exploited publicly available and new freshwater metagenomes in combination with genomes of abundant freshwater bacteria to study the impact of geographic separation on population structure. We focused on species that were detected across broad geographic ranges at high enough sequence coverage for meaningful population genomic analyses, i.e. members of the predominant freshwater taxa acI, LD12, Polynucleobacter and Ca. Methylopumilus. Population differentiation increased significantly with spatial distance in all species, but notable dispersal barriers (e.g. oceanic) were not apparent. Yet, the different species showed contrasting rates of geographic divergence and strikingly different population dynamics in time series within individual lakes. While certain populations hardly diverged over several years, others displayed high divergence after merely a few months, similar in scale to populations separated by thousands of kilometers. We speculate that populations with higher strain diversity evolve more monotonously, while low strain diversity enables more drastic clonal expansion of genotypes which will be reflected in strong but transient differentiation between temporally or spatially adjacent populations.

evolutionary biology↗

bioRxiv2023

Structural Basis of σ54 Displacement and Promoter Escape in Bacterial Transcription

Gene transcription is a fundamental cellular process carried out by RNA polymerase (RNAP). Transcription initiation is highly regulated and in bacteria, transcription initiation is mediated by {sigma} factors, which recruit RNA polymerase (RNAP) to the promoter DNA region and facilitate open complex formation, where double stranded DNA is opening up into a transcription bubble and template strand DNA is in position for initial RNA synthesis. During initial transcription, DNA downstream of the transcription start site is fed into the active site of RNAP, whilst the upstream promoter DNA remains tethered to the RNAP via the {sigma} factor, resulting in a build-up of tension. In order to progress to the processive elongation state, RNAP must escape from the promoter, and displace or dissociate the {sigma} factor. Bacteria s factors can be broadly separated into two classes with the majority belong to the s70 class, represented by the {sigma}70 that regulate housekeeping genes. {sigma}54 forms a class on its own and regulate stress response genes. Extensive studies on {sigma}70 have revealed the molecular mechanisms of {sigma}70 promoter escape while how {sigma}54 transitions from initial transcription to elongation is unknown. Here we present a series of cryo electron microscopy structures of the {sigma}54 factor with progressively longer RNA, revealing the molecular mechanism of {sigma}54 displacement and promoter escape. Our data show that the initial instability is driven by DNA scrunching, and the final displacement steps are driven by both RNA extension and DNA scrunching.

biophysics↗

bioRxiv2022

Allosteric inhibition of the epidermal growth factor receptor through disruption of transmembrane interactions

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) commonly targeted for inhibition by anti-cancer therapeutics. Current therapeutics target EGFRs kinase domain or extracellular region. However, these types of inhibitors are not specific for tumors over healthy tissue and therefore cause undesirable side effects. Our lab has recently developed a new strategy to regulate RTK activity by designing a peptide that specifically binds to the transmembrane (TM) region of the RTK to allosterically modify kinase activity. These peptides are acidity-responsive, allowing them to preferentially target acidic environments like tumors. We have applied this strategy to EGFR and created the PET1 peptide. We observed that PET1 behaves as a pH-responsive peptide that modulates the configuration of the EGFR TM through a direct interaction. Our data indicated that PET1 inhibits EGFR-mediated cell migration. Finally, we investigated the mechanism of inhibition through molecular dynamics simulations, which showed that PET1 sits between the two EGFR TM helices; this molecular mechanism was additionally supported by AlphaFold-Multimer predictions. We propose that the PET1-induced disruption of native TM interactions disturbs the conformation of the kinase domain in such a way that it inhibits EGFRs ability to send migratory cell signals. This study is a proof-of-concept that acidity-responsive membrane peptide ligands can be generally applied to RTKs. In addition, PET1 constitutes a viable approach to therapeutically target the TM of EGFR.

cell biology↗