Biology subjects

Merino, F.

Publications and source records attributed to Merino, F..

17 recordsLinked to original sources

bioRxiv2025

Membrane Proteins at Scale: Automated Copolymer Nanodisc Purification for Structure and Function

Membrane proteins remain among the most important yet least accessible classes of drug targets. Conventional detergents can remove native lipids, destabilizing proteins and limiting downstream biochemistry and structural biology. Amphiphilic copolymers offer a powerful alternative, directly extracting membrane proteins in their native lipid environment, but solubilization outcomes remain unpredictable, turning each new target into a slow empirical search. Here, we introduce an automated, plate-based copolymer screening platform that compresses this process from days to hours using millilitre-scale volumes. Lyophilized copolymer libraries combined with magnetic-bead affinity purification enable parallel testing of dozens of copolymers against multiple targets; across 14 diverse human membrane proteins, next-generation copolymers (AASTY, CyclAPol and Cubipol) systematically outperform classical scaffolds. In this work, we show that this automated copolymer screening robustly identifies the right target-copolymer combination, yielding native-like, active, ligand-binding competent protein suitable for structure determination, and establishes a scalable route to systematic exploration of the membrane proteome.

biochemistry↗

bioRxiv2025

Cryo-EM structure of a cell-free synthesized full-length human β1-adrenergic receptor in complex with Gs

The third intracellular loop (ICL3) of the {beta}1-adrenergic receptor ({beta}1AR) plays a critical role in regulating G protein coupling, yet the structural basis has remained unclear due to truncations of ICL3 in all available structures of the {beta}1AR in complex with Gs or a G protein mimetic nanobody. To address this, we used cell-free cotranslational insertion of full-length human {beta}1AR into nanodiscs and determined its cryo-EM structure in complex with Gs. In this structure, ICL3 extends transmembrane helix 5, resulting in enhanced interactions with Gs and in a slight rotation of the engaged G protein. This repositioning enables new polar interactions between Gs, ICL2 and helix 8, while ICL1 and helix 8 form additional contacts with G{beta}. These structural insights, supported by mutational analysis, demonstrate that ICL3 enhances G protein activation and downstream cAMP signaling by promoting more extensive interactions between the receptor and the heterotrimeric G protein.

biochemistry↗

bioRxiv2024

Occurrence of the Humboldt Penguin (Spheniscus humboldti, Meyen, 1834) in El Ferrol Bay, Chimbote, Peru, and strategies for its conservation

The Humboldt penguin is an emblematic species of the Humboldt ecosystem of Peru and Chile, declared as vulnerable in The IUCN Red List of Threatened Species in 2020. By 2023, its population was estimated around 9,900 pairs, which means a decrease of 93% of its population in 70 years. In response to a warning of its occurrence on the Guaneras Islands located in El Ferrol Bay (Chimbote, Peru), the objective of this study was to verify its presence in situ. Between October and November 2024, four expeditions were carried out on the leeward side of the Islote Pena Blanca (White Rock Islet), Isla Blanca (White Island), Isla Ferrol del Norte (Northern Ferrol Island), Isla Ferrol del Centro (Central Ferrol Island), and Isla Ferrol del Sur (Southern Ferrol Island). The presence of 149 individuals, 5 of them juveniles, and one chick accompanied by its parent, was verified on Northern Ferrol Island, Central Ferrol Island, and Southern Ferrol Island. It is estimated that the presence of these organisms is explained by the improvement of oceanographic conditions in this bay. Considering the high vulnerability of the species, the local community must articulate efforts to guarantee the establishment of its population in this particular habitat.

ecology↗

bioRxiv2024

Kinetic and structure-based comparisons of silent and stimulatory flagellin interactions with TLR5

The bacterial protein flagellin is the sole ligand of the innate immune receptor Toll- like receptor 5 (TLR5). Flagellins with strong agonism bind TLR5 at their D1 and D0 domains, while poor agonist "silent" flagellins bind with D1 only. However, D0-TLR5 interactions insufficiently explain the silent phenotype. Here, we characterize the D1 domain binding kinetics of the silent flagellin RhFlaB compared to the strong canonical agonist StFliC. Using Surface Plasmon Resonance, we show that the RhFlaB-D1 binds more strongly than StFliC-D1, but forms shorter-lived complexes. Cryo-EM analysis of RhFlaB showed its D1 primary interface (PI) contains two distinct hydrophobic pockets, which should facilitate rapid association, while its D1s secondary interface (SI) is dominated by negatively charged residues, which likely impede residue-residue interactions. Comparisons with an existing StFliC-D1 structure suggest that strong agonism requires strong binding at both the PI and SI of the D1, and silent flagellins evade detection partly through weaker SI interactions. These findings offer a foundation for designing targeted interventions that can modulate TLR5 activity to influence immunity, inflammation, or even gut microbiome composition.

biochemistry↗

bioRxiv2023

A computational model for lipid-anchored polysaccharide export by the outer-membrane protein GfcD

Many bacteria are protected by different types of polysaccharide capsules, structures formed of long repetitive glycan chains that are sometimes free and sometimes anchored to the outer membrane via lipid tails. One type, called group 4 capsule, results from expression of the gfcABCDE-etp-etk operon in Escherichia coli. Of the proteins encoded in this operon, GfcE is thought to provide the export pore for free polysaccharide chains, but none of the proteins has been implicated in the export of chains carrying a lipid anchor. For this function, GfcD has been a focus of attention as the only outer-membrane {beta}-barrel encoded in the operon. AlphaFold predicts two {beta}-barrel domains in GfcD, a canonical N-terminal one of 12 strands and an unusual C-terminal one of 13 strands, which features a large lateral aperture between strands {beta}1 and {beta}13. This immediately suggests a lateral exit gate for hydrophobic molecules into the membrane, analogous to the one proposed for the lipopolysaccharide export pore LptD. Here, we report an unsteered molecular dynamics study of GfcD embedded in the bacterial outer membrane, with the common polysaccharide anchor, lipid A, inserted in the pore of the C-terminal barrel. Our results show that the lateral aperture does not collapse during simulations, that membrane lipids nevertheless do not penetrate the barrel, but that the lipid chains of the lipid A molecule readily exit into the membrane. Statement of SignificanceDespite the essential role polysaccharide capsules play in the resilience of bacteria to hostile environments, many aspects of their biogenesis are still poorly understood. One aspect concerns the export of capsular polysaccharides carrying a lipid anchor, for which even the proteins mediating the process are unknown. Here we propose that one of the largest families of {beta}-barrel proteins in the bacterial outer membrane is a key agent of this process and show by biophysical simulation that it allows the exit of lipid anchors into the membrane through a lateral opening. More generally, our model illuminates the lateral exit mechanisms proposed for the export of hydrophobic macromolecules into the bacterial outer membrane. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=170 SRC="FIGDIR/small/565983v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@9b2feorg.highwire.dtl.DTLVardef@c668b2org.highwire.dtl.DTLVardef@386b8org.highwire.dtl.DTLVardef@181c418_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Transient structural properties of the Rho GDP-dissociation inhibitor

Rho GTPases are master spatial regulators of the cytoskeleton that control a wide range of cellular processes. Their inactivation by removal from cellular membranes involves the stepwise formation of a stable complex with guanine nucleotide dissociation inhibitors (RhoGDIs), for which process the RhoGDI N-terminus is indispensable. The formation of this interface has been thought to emerge from an intrinsically disordered state of RhoGDI in its free, apo form. Here we use tailored solution NMR analyses, molecular dynamics simulations, and biochemical essays to pinpoint the site-specific structural features of full-length RhoGDI1 before and after binding its GTPase client Cdc42. In contrast to the current mechanistic understanding, a diverse set of NMR data unequivocally shows that the structural properties of the GDI N-terminus seen in crystal structures of the complex with GTPases already exist as largely preformed features in free, apo GDI. Even more interestingly, the required structural properties are imposed onto the terminus context-specifically by modulating interactions with the surface of the folded C-terminal domain. Lastly, upon Cdc42 binding, the flexibility of the N-terminus and its secondary-structural propensities are not largely abrogated. These observations change the textbook picture of the mechanism of membrane extraction of the GTPase. Rather than a disorder-to-order transition upon binding, an active role of the N-terminus with differentially preformed structural properties, suitably modulated by the specific surrounding along the multi-step process, seems required to leverage the intricate and highly selective extraction process.

biophysics↗

bioRxiv2023

The bigger picture: global analysis of solubilization performance of classical detergents versus new synthetic polymers utilizing shotgun proteomics

Integral membrane proteins are critical for many cellular functions. Roughly 25% of all human genes code for membrane proteins, and about 70% of all approved drugs target them. Despite their importance, laborious and harsh purification conditions often hinder their characterization. Traditionally, they are removed from the membrane using detergents, thereby taking the proteins out of their native environment, affecting their function. Recently, a variety of synthetic polymers have been introduced, which can extract membrane proteins together with their native lipids into a so-called native nanodisc. However, they usually show lesser solubilization capacity than detergents, and their general applicability for membrane protein biochemistry is poorly understood. Here, we used Hek293 cell membrane extracts and LC-MS-based proteomics to compare the ability of nanodisc-forming polymers against state-of-the- art detergents to solubilize the membrane proteome. Our data demonstrates the general ability of synthetic co-polymers to extract membrane proteins, rivaling the efficacy of commonly used detergents. Interestingly, each class of solubilization agent presents specific solubilization profiles. We found no correlation between efficiency and number of transmembrane domains, isoelectric point, or GRAVY score for any compound. Our data shows that these polymers are a versatile alternative to detergents for the biochemical and structural study of membrane proteins, functional proteomics, or as components of native lysis/solubilization buffers. Our work here represents the first attempt at a proteome-scale comparison of the efficacy of nanodisc-forming polymers. These data should serve as starting reference for researchers looking to purify membrane proteins in near native conditions.

biochemistry↗

bioRxiv2021

Structure of the RZZ complex and molecular basis of Spindly-driven corona assembly at human kinetochores

In metazoans, a {asymp}1 megadalton (MDa) super-complex comprising the Dynein-Dynactin adaptor Spindly and the ROD-Zwilch-ZW10 (RZZ) complex is the building block of a fibrous biopolymer, the kinetochore fibrous corona. The corona assembles on mitotic kinetochores to promote microtubule capture and spindle assembly checkpoint (SAC) signaling. We report here a high-resolution cryo-EM structure that captures the essential features of the RZZ complex, including a farnesyl binding site required for Spindly binding. Using a highly predictive in vitro assay, we demonstrate that the SAC kinase MPS1 is necessary and sufficient for corona assembly at supercritical concentrations of the RZZ-Spindly (RZZS) complex, and describe the molecular mechanism of phosphorylation-dependent filament nucleation. We identify several structural requirements for RZZS polymerization in rings and sheets. Finally, we identify determinants of kinetochore localization and corona assembly of Spindly. Our results describe a framework for the long-sought-for molecular basis of corona assembly on metazoan kinetochores.

biochemistry↗

bioRxiv2021

Mechanism of actin-dependent activation of nucleotidyl cyclase toxins from bacterial human pathogens

Several bacterial human pathogens secrete nucleotidyl cyclase toxins, that are activated by interaction with actin of the eukaryotic host cells. However, the underlying molecular mechanism of this process which protects bacteria from self-intoxication is unclear. Here, we report structures of ExoY from Pseudomonas aeruginosa and Vibrio vulnificus in complex with their corresponding activators F-actin and profilin-G-actin. The structures reveal that in contrast to the apo state, two flexible regions become ordered and interact strongly with actin. The specific stabilization of these regions results in an allosteric stabilization of the distant nucleotide binding pocket and thereby to an activation of the enzyme. Differences in the sequence and conformation of the actin-binding regions are responsible for the selective binding to either F- or G-actin. This specificity can be biotechnologically modulated by exchanging these regions from one toxin to the other. Other bacterial nucleotidyl cyclases, such as the anthrax edema factor and CyaA from Bortedella pertussis, that bind to calmodulin undergo a similar disordered-to-ordered transition during activation, suggesting that the allosteric activation-by-stabilization mechanism of ExoY is paradigmatic for all bacterial nucleotidyl cyclase toxins.

biochemistry↗

bioRxiv2021

A structure-derived mechanism reveals how capping protein promotes nucleation in branched actin networks

Heterodimeric capping protein (CP/CapZ) is an essential factor for the assembly of branched actin networks, which push against cellular membranes to drive a large variety of cellular processes. Aside from terminating filament growth, CP stimulates the nucleation of actin filaments by the Arp2/3 complex in branched actin networks through an unclear mechanism. Here, we report the structure of capped actin filament barbed ends, which reveals how CP not only prevents filament elongation, but also controls access to both terminal filament subunits. In addition to its primary binding site that blocks the penultimate subunit, we find that the CP sterically occludes the central interaction site of the terminal actin protomer through one of its C-terminal "tentacle" extensions. Deletion of this {beta} tentacle only modestly impairs capping. However in the context of a growing branched actin network, its removal potently inhibits nucleation promoting factors (NPFs) by tethering them to capped filament ends. End tethering of NPFs prevents their loading with actin monomers required for activation of the Arp2/3 complex and thus strongly inhibits branched network assembly both in cells and reconstituted motility assays. Our results mechanistically explain how CP couples two opposed processes -capping and nucleation- in branched actin network assembly.

biochemistry↗

bioRxiv2020

Structural basis of TRPC4 regulation by calmodulin and pharmacological agents

ABSTRACTCanonical transient receptor potential channels (TRPC) are involved in receptor-operated and/or store-operated Ca2+ signaling. Inhibition of TRPCs by small molecules was shown to be promising in treating renal diseases. In cells, the channels are regulated by calmodulin. Molecular details of both calmodulin and drug binding have remained elusive so far. Here we report structures of TRPC4 in complex with a pyridazinone-based inhibitor and a pyridazinone-based activator and calmodulin. The structures reveal that both activator and inhibitor bind to the same cavity of the voltage-sensing-like domain and allow us to describe how structural changes from the ligand binding site can be transmitted to the central ion-conducting pore of TRPC4. Calmodulin binds to the rib helix of TRPC4, which results in the ordering of a previously disordered region, fixing the channel in its closed conformation. This represents a novel calmodulin-induced regulatory mechanism of canonical TRP channels.Competing Interest StatementThe authors declare no competing interests. M.Y., M.W.L, and G.M. are or were shareholders of Goldfinch Bio.View Full Text

biochemistry↗

bioRxiv2020

Structure of the Lifeact F-actin complex

Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The molecular basis for such artefacts is poorly understood. Here, we determined the high-resolution structure of the Lifeact-F-actin complex using electron cryo-microscopy. The structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and stretches over two adjacent actin subunits, stabilizing the DNase I-binding loop of actin in the closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding proteins, such as cofilin and myosin and actin-binding toxins, such as TccC3HVR from Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays and activity measurements demonstrate that Lifeact indeed competes with these proteins, providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations into the peptide, laying the foundation for designing improved actin probes for live cell imaging.

molecular biology↗

bioRxiv2019

Structural effects and functional implications of phalloidin and jasplakinolide binding to actin filaments

Actin undergoes structural transitions during polymerization, ATP hydrolysis and subsequent release of inorganic phosphate. Several actin binding proteins sense specific states during this transition and can thus target different regions of the actin filament. Here we show in atomic detail that phalloidin, a mushroom toxin that is routinely used to stabilize and label actin filaments, suspends the structural changes in actin, likely influencing its interaction with actin binding proteins. Furthermore, high-resolution cryo-EM structures reveal structural rearrangements in F-actin upon inorganic phosphate release in phalloidin-stabilized filaments. We find that the effect of the sponge toxin jasplakinolide differs from the one of phalloidin, despite their overlapping binding site and similar interactions with the actin filament. Analysis of structural conformations of F-actin suggests that stabilizing agents trap states within the natural conformational space of actin.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC=\"FIGDIR/small/794495v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (47K):\norg.highwire.dtl.DTLVardef@1a81b98org.highwire.dtl.DTLVardef@1aba96borg.highwire.dtl.DTLVardef@ffc218org.highwire.dtl.DTLVardef@11fe454_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIFive high-resolution cryo-EM structures of stabilized filamentous actin\nC_LIO_LIPhalloidin traps different structural states depending on when it is added\nC_LIO_LIThe effect of phalloidin and jasplakinolide on filamentous actin is not identical\nC_LIO_LIBoth toxins likely interfere with the binding of proteins sensing F-actins nucleotide state\nC_LI

biochemistry↗

bioRxiv2019

Profilin and formin constitute a pacemaker system for robust actin filament growth

The actin cytoskeleton drives many essential biological processes, from cell morphogenesis to motility. Assembly of functional actin networks requires control over the speed at which actin filaments grow. How this can be achieved at the high and variable levels of soluble actin subunits found in cells is unclear. Here we reconstitute assembly of mammalian, non-muscle actin filaments from physiological concentrations of profilin-actin. We discover that under these conditions, filament growth is limited by profilin dissociating from the filament end and the speed of elongation becomes insensitive to the concentration of soluble subunits. Profilin release can be directly promoted by formin actin polymerases even at saturating profilin-actin concentrations. We demonstrate that mammalian cells indeed operate at the limit to actin filament growth imposed by profilin and formins. Our results reveal how synergy between profilin and formins generates robust filament growth rates that are resilient to changes in the soluble subunit concentration.

biochemistry↗

bioRxiv2019

Cryo-EM structure of the ClpXP protein degradation machinery

The ClpXP machinery is a two component protease complex performing targeted protein degradation in bacteria and eukaryotes. The complex consists of the AAA+ chaperone ClpX and the peptidase ClpP. The hexameric ClpX utilizes the energy of ATP binding and hydrolysis to engage, unfold and translocate substrates into the catalytic chamber of tetradecameric ClpP where they are degraded. Formation of the complex involves a symmetry mismatch, since hexameric AAA+ rings bind axially to the opposing stacked heptameric rings of the tetradecameric ClpP. Here we present the first high-resolution cryo-EM structure of ClpXP from Listeria monocytogenes. We unravel the heptamer-hexamer binding interface and provide novel insights into the ClpX-ClpP crosstalk and activation mechanism. The comparison with available crystal structures of ClpP and ClpX in different states allows us to understand important aspects of ClpXPs complex mode of action and provides a structural framework for future pharmacological applications.

molecular biology↗

bioRxiv2018

SPHIRE-crYOLO: A fast and well-centering automated particle picker for cryo-EM

Selecting particles from digital micrographs is an essential step in single particle electron cryomicroscopy (cryo-EM). Since manual selection is a tedious and time-consuming process, many automatic particle pickers have been developed. However, they have problems especially with non-ideal datasets. Here, we present a novel automated particle picking software called crYOLO, which is based on the deep learning object detection system \"You Only Look Once\" (YOLO). After training the network with 500 - 2,500 particles per dataset, it automatically selects the particles with high accuracy and precision reaching a speed of up to six micrographs per second. Importantly, we show that crYOLO can be trained to select previously unseen datasets paving the way for completely automated \"on-the-fly\" cryo-EM data pre-processing during data acquisition. CrYOLO is available as standalone program under http://sphire.mpg.de/ and will be part of the image processing workflow in SPHIRE.

biophysics↗

bioRxiv2018

Electron cryo-microscopy structure of the canonical TRPC4 ion channel

Canonical transient receptor channels (TRPC) are non-selective cation channels. They are involved in receptor-operated Ca2+ signaling and have been proposed to act as store-operated channels (SOC). Their malfunction is related to cardiomyopathies and their modulation by small molecules has been shown to be effective against renal cancer cells. The molecular mechanism underlying the complex activation and regulation is poorly understood. Here, we report the electron cryo-microscopy structure of zebrafish TRPC4 in its unliganded (apo), closed state at an overall resolution of 3.6 [A]. The structure reveals the molecular architecture of the cation conducting pore, including the selectivity filter and lower gate. The cytoplasmic domain contains two key hubs that have been shown to interact with modulating proteins. Structural comparisons with other TRP channels give novel insights into the general architecture and domain organization of this superfamily of channels and help to understand their function and pharmacology.

biochemistry↗