Biology subjects

Larsen, A. H.

Publications and source records attributed to Larsen, A. H..

10 recordsLinked to original sources

bioRxiv2025

A proteome-wide screen for membrane-interactions in intrinsically disordered regions of transmembrane proteins reveals a role in disease

Transmembrane proteins mediate essential cellular processes including signaling, transport, and ion flux. Besides their well-characterized structured domains, most contain intrinsically disordered regions, whose biological roles remain poorly understood. Evidence suggests that the functions of intrinsically disordered regions are context-dependent, a trait particularly relevant when anchored to cellular membranes. In this study, we probed peptide arrays with fluorescent liposomes to generate a high-resolution, proteome-wide map of membrane-interaction sites within intrinsically disordered regions of human transmembrane proteins. Screening 4,000 proteins, we identified membrane-interaction sites in [~]60% of cases. Among these, [~]63% represent amphipathic helices, while [~]17% resemble cationic cell-penetrating peptides. We demonstrate that membrane-interaction motifs can influence subcellular localization and may contribute to both physiological and pathological processes. Our findings establish membrane association as a key functional aspect of intrinsically disordered regions and provide a valuable resource for discovering non-canonical regulatory mechanisms in transmembrane proteins. The resource is available at MemRIDRdb.

cell biology↗

bioRxiv2025

Rab2 and Arl8/BORC control retrograde axonal transport of dense core vesicles via Syd/dJIP3/4 and RUFY dynein adaptors

Neuropeptide-containing dense core vesicles (DCVs) are generated in the neuronal cell body and circulate throughout the axonal arbor to supply distal release sites. This circulation depends on the anterograde kinesin-1 and kinesin-3 motors and the retrograde dynein-dynactin motor. While kinesin-3 is recruited to DCVs with the aid of the small GTPase Arl8, it is unclear how dynein and kinesin-1 are recruited and regulated. Here we show that DCV motility in Drosophila (fruit flies) depends on the dynein and kinesin-1 adaptor Sunday Driver/dJIP3/4 (Syd) and the novel dynein adaptor RUFY. Syd and RUFY bind each other; moreover, Syd binds the DCV-located GTPase Rab2 that controls retrograde DCV transport, and RUFY binds Arl8. Disruption of Rab2, Syd, RUFY, dynein, and the Arl8 activator BORC all produce a similar DCV axonal transport phenotype characterized by axonal accumulation of immobile DCVs and a selective reduction in retrograde DCV flux. Our data suggest a model where dynein is recruited and activated by a complex of Syd and RUFY, which is anchored to DCVs by a Rab2- and Arl8-dependent mechanism. Lastly, we show that loss of Rab2 results in missorting of the DCV membrane proteins VMAT and Synaptotagmin-, similar to the reported effect of Rab2 deletion on the sorting of synaptic vesicle and active zone proteins. However, disruption of Syd, RUFY or dynein does not phenocopy the Rab2-specific VMAT sorting defect, suggesting that Rab2 employs separate effectors in DCV biogenesis and motility.

neuroscience↗

bioRxiv2024

Membrane curvature association of amphipathic helix 8 drives constitutive GPCR endocytosis

Cellular signaling relies on the activity of transmembrane receptors and their presentation on the cellular surface. Their continuous insertion in the plasma membrane is balanced by constitutive and activity dependent internalization, which is orchestrated by adaptor proteins recognizing semi-specific motifs within the receptors intracellular regions. Here we describe a complementary and evolutionary conserved and refined trafficking mechanism for G-protein coupled receptors (GPCR). This mechanism relies on the insertion of their amphipathic helix 8 into the inner leaflet of lipid membranes, orthogonal to the transmembrane helices. These amphipathic helices dictate subcellular localization of the receptors and autonomously drive their endocytosis by cooperative assembly and association with areas of high membrane curvature. The strength of helix 8 membrane insertion propensity quantitatively predicts the rate of constitutive internalization of GPCRs. This discovery advances our understanding of membrane protein trafficking and highlights a new principle of receptor-lipid interactions that may have broader implications for cellular signaling and therapeutic targeting. One-Sentence SummaryReceptor proteins navigate cellular membranes by interacting with their curvature using an evolutionary conserved mechanism that relies on amphipathic helices and complements direct coupling to the endocytic protein machinery.

cell biology↗

bioRxiv2023

Recombinant dimeric PDZ protein inhibitors for long-term relief of chronic pain by AAV therapeutics

The inadequate state of current pain treatments, the chronic nature of particularly neuropathic pain, and the high impact on quality of life render chronic pain conditions relevant for gene therapy. Here, we describe the development and application of self-assembling dimeric peptide inhibitors of the pain-associated scaffolding protein PICK1 (protein interacting with C-kinase 1) delivered by adeno-associated viral (AAV) vectors. In mice, these peptides prevented mechanical allodynia in inflammatory and neuropathic pain models and reversed neuropathic pain in advanced stages up to one year. Pain relief was obtained by targeting several relays along the somatosensory pain pathways unaccompanied by overt adverse side effects, while selective transduction of peripheral neurons was sufficient for providing full pain relief. We further confirmed PICK1 expression and peptide target engagement in mice and human donor tissue, and we conclude that AAV therapeutics, based on recombinant PICK1 inhibitors, represent a potential clinically meaningful strategy for persistent neuropathic pain conditions. One Sentence SummaryAlleviating neuropathic pain by PICK1-directed gene therapy.

neuroscience↗

bioRxiv2021

Mg2+-dependent conformational equilibria in CorA: an integrated view on transport regulation

The CorA family of proteins regulates the homeostasis of divalent metal ions in many bacteria, archaea, and eukaryotic mitochondria, making it an important target in the investigation of the mechanisms of transport and its functional regulation. Although numerous structures of open and closed channels are now available for the CorA family, the mechanism of the transport regulation remains elusive. Here, we investigated the conformational distribution and associated dynamic behaviour of the pentameric Mg2+ channel CorA at room temperature using small-angle neutron scattering (SANS) in combination with molecular dynamics (MD) simulations and solid-state nuclear magnetic resonance spectroscopy (NMR). We find that neither the Mg2+-bound closed structure nor the Mg2+-free open forms are sufficient to explain the average conformation of CorA. Our data support the presence of conformational equilibria between multiple states, and we further find a variation in the behaviour of the backbone dynamics with and without Mg2+. We propose that CorA must be in a dynamic equilibrium between different non-conducting states, both symmetric and asymmetric, regardless of bound Mg2+ but that conducting states become more populated in Mg2+-free conditions. These properties are regulated by backbone dynamics and are key to understanding the functional regulation of CorA.

biophysics↗

bioRxiv2021

Membrane-binding mechanism of the EEA1 FYVE domain revealed by multi-scale molecular dynamics simulations

Early Endosomal Antigen 1 (EEA1) is a key protein in endosomal trafficking and is implicated in both autoimmune and neurological diseases. The C-terminal FYVE domain of EEA1 binds endosomal membranes, which contain phosphatidylinositol-3-phosphate (PI(3)P). Although it is known that FYVE binds PI(3)P specifically, it has not previously been described of how FYVE attaches and binds to endosomal membranes. In this study, we employed both coarse-grained (CG) and atomistic (AT) molecular dynamics (MD) simulations to determine how FYVE binds to PI(3)P-containing membranes. CG-MD showed that the dominant membrane binding mode resembles the crystal structure of EEA1 FYVE domain in complex with inositol-1,3-diphospate (PDB ID 1JOC). FYVE, which is a homodimer, binds the membrane via a hinge mechanism, where the C-terminus of one monomer first attaches to the membrane, followed by the C-terminus of the other monomer. The total binding energy is 70 kJ/mol, of which 50-60 kJ/mol stems from specific PI(3)P-interactions. By AT-MD, we could partition the binding mode into two types: (i) adhesion by electrostatic FYVE-PI(3)P interaction, and (ii) insertion of amphipathic loops. The AT simulations also demonstrated flexibility within the FYVE homodimer between the C-terminal heads and coiled-coil stem, allowing binding via a mechanism resembling that of a suction cup connected to a locally rigid stem via a flexible hinge.

biophysics↗

bioRxiv2020

Binding of Ca2+-Independent C2 Domains to Lipid Membranes: a Multi-Scale Molecular Dynamics Study

C2 domains facilitate protein-lipid interaction in cellular recognition and signalling processes. They possess a {beta}-sandwich structure, with either type I or type II topology. C2 domains can interact with anionic lipid bilayers in either a Ca2+-dependent or a Ca2+-independent manner. The mechanism of recognition of anionic lipids by Ca2+-independent C2 domains is incompletely understood. We have used molecular dynamics (MD) simulations to explore the membrane interactions of six Ca2+- independent C2 domains, from KIBRA, PI3KC2, RIM2, PTEN, SHIP2, and Smurf2. In coarse grained MD simulations these C2 domains bound to lipid bilayers, forming transient interactions with zwitterionic (phosphatidylcholine, PC) bilayers compared to long lived interactions with anionic bilayers also containing either phosphatidylserine (PS) or PS and phosphatidylinositol bisphosphate (PIP2). Type I C2 domains bound non-canonically via the front, back or side of the {beta} sandwich, whereas type II C2 domains bound canonically, via the top loops (as is typically the case for Ca2+-dependent C2 domains). C2 domains interacted strongly (up to 120 kJ/mol) with membranes containing PIP2 causing the bound anionic lipids to clustered around the protein. The C2 domains bound less strongly to anionic membranes without PIP2 (<50 kJ/mol), and most weakly to neutral membranes (<33 kJ/mol). Productive binding modes were identified and further analysed in atomistic simulations. For PTEN and SHIP2, CG simulations were also performed of the intact enzymes (i.e. phosphatase domain plus C2 domain) with PIP2-contating bilayers and the roles of the two domains in membrane localization were compared. From a methodological perspective, these studies establish a multiscale simulation protocol for studying membrane binding/recognition proteins, capable of revealing binding modes alongside details of lipid binding affinity and specificity.

biophysics↗

bioRxiv2020

Lipid-bound ApoE3 self-assemble into elliptical disc-shaped particles

Apolipoproteins are vital to lipid metabolism and cholesterol transport in the human body. Here we present a structural study of the lipid-bound particles formed by ApoE3 in a full-length and a truncated version. The particles are formed with, respectively, POPC and DMPC and investigated by small-angle X-ray scattering and negative stain electron microscopy. We find that lipid-bound ApoE3 particles are elliptical, disc-shaped particles composed of a central lipid bilayer encircled by two amphipathic ApoE3 proteins. We went on to investigate a truncated form of ApoE3 containing only residue 80 to 255 (ApoE380-255), which is the central helical repeat segment of ApoE3. The lipid-bound ApoE380-255 particles are found to have the same morphology as the particles with full-length ApoE3. However, they are larger, and form more heterogeneous discoidal structures with four proteins per particle. This behavior is in contrast to ApoA1 where the highly similar helical repeat domain determines the size and stoichiometry of the formed particles both in the case of full-length and truncated ApoA1. Our data hence points towards different mechanisms for lipid bilayer structural modulation by ApoA1 and ApoE3 due to different roles of the non-repeat segments.

biochemistry↗

bioRxiv2019

Combining molecular dynamics simulations with small-angle X-ray and neutron scattering data to study multi-domain proteins in solution

Many proteins contain multiple folded domains separated by flexible linkers, and the ability to describe the structure and conformational heterogeneity of such flexible systems pushes the limits of structural biology. Using the three-domain protein TIA-1 as an example, we here combine coarse-grained molecular dynamics simulations with previously measured small-angle scattering data to study the conformation of TIA-1 in solution. We show that while the coarse-grained potential (Martini) in itself leads to too compact conformations, increasing the strength of protein-water interactions results in ensembles that are in very good agreement with experiments. We show how these ensembles can be refined further using a Bayesian/Maximum Entropy approach, and examine the robustness to errors in the energy function. In particular we find that as long as the initial simulation is relatively good, reweighting against experiments is very robust. We also study the relative information in X-ray and neutron scattering experiments and find that refining against the SAXS experiments leads to improvement in the SANS data. Our results suggest a general strategy for studying the conformation of multi-domain proteins in solution that combines coarse-grained simulations with small-angle X-ray scattering data that are generally most easy to obtain. These results may in turn be used to design further small-angle neutron scattering experiments that exploit contrast variation through 1H/2H isotope substitutions.

biophysics↗

bioRxiv2018

Structure and dynamics of the central lipid pool and protein components of the bacterial holo-translocon

The bacterial Sec translocon, SecYEG, associates with accessory proteins YidC and the SecDF-YajC subcomplex to form the bacterial holo-translocon (HTL). The HTL is a dynamic and flexible protein transport machine capable of coordinating protein secretion across the membrane, and efficient lateral insertion of nascent membrane proteins. It has been hypothesized that a central lipid core facilitates the controlled passage of membrane proteins into the bilayer, ensuring efficient formation of their native state. By performing small-angle neutron scattering (SANS) on protein solubilized in match-out deuterated detergent, we have been able to interrogate a naked HTL complex, with the scattering contribution of the surrounding detergent micelle rendered invisible. Such an approach has allowed the confirmation of a lipid core within the HTL, which accommodates between 8 and 29 lipids. Coarse-grained molecular dynamics simulations of the HTL also demonstrate a dynamic, central pool of lipids. An opening at this lipid rich region between YidC and the SecY lateral gate may provide an exit gateway for newly synthesized, correctly oriented, membrane protein helices to emerge from the HTL.

biophysics↗