Biology subjects

Siebold, C.

Publications and source records attributed to Siebold, C..

12 recordsLinked to original sources

bioRxiv2025

Design principles of a membrane-spanning ubiquitin ligase

Receptor-type E3 ubiquitin ligases are membrane-spanning assemblies that enable extracellular signals to directly control ubiquitylation in the cytoplasm. Despite playing widespread roles in tissue patterning and homeostasis, metabolism, and immunity, their structures and mechanisms remain poorly understood. Using cryo-electron microscopy, integrated with biophysical and functional studies, we visualized an E3 complex composed of two transmembrane proteins, MEGF8 and MOSMO, and the intracellular RING-family protein MGRN1. This MEGF8-MOSMO-MGRN1 (MMM) complex regulates left-right patterning of the body axis and the development of multiple organs, partly by attenuating signaling through the Hedgehog pathway. We find that the MMM complex functions like a fishing pole: a long, flexible helix attached to a membrane platform suspends an activated and precisely oriented RING domain--like a fishhook--to ubiquitylate the cytoplasmic surfaces of target receptors. Our structure explains how mutations in MEGF8 cause multi-organ birth defects in humans and defines a paradigm for receptor regulation by ubiquitylation.

biochemistry↗

bioRxiv2025

The E3 ubiquitin ligase MGRN1 targets melanocortin receptors MC1R and MC4R via interactions with transmembrane adapters

E3 ubiquitin ligases play a crucial role in modulating receptor stability and signaling at the cell surface, yet the mechanisms governing their substrate specificity remain incompletely understood. Mahogunin Ring Finger 1 (MGRN1) is a membrane-tethered E3 ligase that fine-tunes signaling sensitivity by targeting surface receptors for ubiquitination and degradation. Unlike cytosolic E3 ligases, membrane-tethered E3s require transmembrane adapters to selectively recognize and regulate surface receptors, yet few such ligases have been studied in detail. While MGRN1 is known to regulate the receptor Smoothened (SMO) within the Hedgehog pathway through its interaction with the transmembrane adapter Multiple Epidermal Growth Factor-like 8 (MEGF8), the broader scope of its regulatory network has been speculative. Here, we identify Attractin (ATRN) and Attractin-like 1 (ATRNL1) as additional transmembrane adapters that recruit MGRN1 and regulate cell surface receptor turnover. Through co-immunoprecipitation, we show that ATRN and ATRNL1 likely interact with the RING domain of MGRN1. Functional assays reveal that MGRN1 requires these transmembrane adapters to ubiquitinate and degrade the melanocortin receptors MC1R and MC4R, in a process analogous to its regulation of SMO. Loss of MGRN1 leads to increased surface and ciliary localization of MC4R in fibroblasts and elevated MC1R levels in melanocytes, with the latter resulting in enhanced eumelanin production. These findings expand the repertoire of MGRN1-regulated receptors and provide new insight into a shared mechanism by which membrane-tethered E3 ligases utilize transmembrane adapters to dictate substrate receptor specificity. By elucidating how MGRN1 selectively engages with surface receptors, this work establishes a broader framework for understanding how this unique class of E3 ligases fine-tunes receptor homeostasis and signaling output.

cell biology↗

bioRxiv2023

Mapping Structural and Dynamic Divergence Across the MBOAT family

Membrane Bound O-acyltransferases (MBOATs) are membrane embedded enzymes which catalyse acyl chain transfer to a diverse group of substrates including lipids, small-molecules and proteins. Recent MBOAT structures reveal a conserved structural core, despite wide-ranging functional specificity across both prokaryotes and eukaryotes. The structural basis of catalytic specificity, regulation and interactions with the surrounding environment remain uncertain, hindering effective therapeutic targeting. Here, we combine comparative molecular dynamics (MD) simulations with bioinformatics to assess molecular and interactional divergence across the family. In simulations, MBOATs differentially distort the bilayer depending on their substrate type. Additionally, we identify specific lipid binding sites surrounding MBOAT reactant gates into the surrounding membrane. We use bioinformatics to reveal a conserved role for re-entrant loop-2 in stabilisation of the MBOAT fold and identify a key hydrogen bond involved in DGAT1 dimerisation. Finally, we predict differences in MBOAT core solvation and water gating properties across the family. These data are pertinent to the design of MBOAT specific inhibitors that encompass dynamic information within cellular mimetic environments.

biochemistry↗

bioRxiv2023

The Energetics and Ion Coupling of Cholesterol Transport Through Patched1

Patched1 (PTCH1) is the principal tumour suppressor protein of the mammalian Hedgehog (HH) signalling pathway, implicated in embryogenesis and tissue homeostasis. PTCH1 inhibits the Class F G protein-coupled receptor Smoothened (SMO) via a debated mechanism involving modulating accessible cholesterol levels within ciliary membranes. Using extensive molecular dynamics (MD) simulations and free energy calculations to evaluate cholesterol transport through PTCH1, we find an energetic barrier of ~15-20 kJ mol-1 for cholesterol export. In simulations we identify cation binding sites within the PTCH1 transmembrane domain (TMD) which may provide the energetic impetus for cholesterol transport. In silico data are coupled to in vivo biochemical assays of PTCH1 mutants to probe coupling between transmembrane motions and PTCH1 activity. Using complementary simulations of Dispatched1 (DISP1) we find that transition between inward-open and solvent occluded states is accompanied by Na+ induced pinching of intracellular helical segments. Thus, our findings illuminate the energetics and ion-coupling stoichiometries of PTCH1 transport mechanisms, whereby 1-3 Na+ or 2-3 K+ couple to cholesterol export, and provide the first molecular description of transitions between distinct transport states.

biophysics↗

bioRxiv2022

LipIDens: Simulation assisted interpretation of lipid densities in cryo-EM structures of membrane proteins.

Cryo-electron microscopy (cryo-EM) enables the determination of membrane protein structures in native-like environments. Characterising how membrane proteins interact with the surrounding membrane lipid environment is assisted by resolution of lipid-like densities visible in cryo-EM maps. Nevertheless, establishing the molecular identity of putative lipid and/or detergent densities remains challenging. Here we present LipIDens, a pipeline for molecular dynamics (MD) simulation-assisted interpretation of lipid and lipid-like densities in cryo-EM structures. The pipeline integrates the implementation and analysis of multi-scale MD simulations for identification, ranking and refinement of lipid binding poses which superpose onto cryo-EM map densities. Thus, LipIDens enables direct integration of experimental and computational structural approaches to facilitate the interpretation of lipid-like cryo-EM densities and to reveal the molecular identities of protein-lipid interactions within a bilayer environment. The LipIDens code is open-source and embedded within a notebook format to assist automation and usability.

biophysics↗

bioRxiv2021

Structure and function of the ROR2 cysteine-rich domain in vertebrate noncanonical WNT5A signaling

The receptor tyrosine kinase ROR2 mediates noncanonical WNT5A signaling to orchestrate tissue morphogenetic processes, and dysfunction of the pathway causes Robinow syndrome, Brachydactyly B and metastatic diseases. The domain(s) and mechanisms required for ROR2 function, however, remain unclear. We solved the crystal structure of the extracellular cysteine-rich (CRD) and Kringle (Kr) domains of ROR2 and found that, unlike other CRDs, the ROR2 CRD lacks the signature hydrophobic pocket that binds lipids/lipid-modified proteins, such as WNTs, suggesting a novel mechanism of ligand reception. Functionally, we showed that the ROR2 CRD, but not other domains, is required and minimally sufficient to promote WNT5A signaling, and Robinow mutations in the CRD and the adjacent Kr impair ROR2 secretion and function. Moreover, using function-activating and -perturbing antibodies against the Frizzled (FZ) family of WNT receptors, we demonstrate the involvement of FZ in WNT5A-ROR signaling. Thus, ROR2 acts via its CRD to potentiate the function of a receptor super-complex that includes FZ to transduce WNT5A signals.

biochemistry↗

bioRxiv2021

Structure and Mechanism of Hedgehog Acyl Transferase

The iconic Sonic Hedgehog (SHH) morphogen pathway is a fundamental orchestrator of embryonic development and stem cell maintenance, and is implicated in cancers in various organs. A key step in signalling is transfer of a palmitate group to the N-terminal cysteine residue of SHH, catalysed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT) resident in the endoplasmic reticulum (ER). Here, we present the high-resolution cryo-EM structure of HHAT bound to substrate analogue palmityl-coenzyme A and a SHH mimetic megabody. Surprisingly, we identified a heme group bound to an HHAT cysteine residue and show that this modification is essential for HHAT structure and function. A structure of HHAT bound to potent small molecule inhibitor IMP-1575 revealed conformational changes in the active site which occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the novel mechanism by which HHAT adapts the membrane environment to transfer a long chain fatty acid across the ER membrane from cytosolic acyl-CoA to a luminal protein substrate. This structure of a member of the protein-substrate membrane-bound O-acyltransferase (MBOAT) superfamily provides a blueprint for other protein substrate MBOATs, such as WNT morphogen acyltransferase Porcupine and ghrelin O-acyltransferase GOAT, and a template for future drug discovery.

biophysics↗

bioRxiv2021

Relative Affinities of Protein-Cholesterol Interactions from Equilibrium Molecular Dynamics Simulations

Specific interactions of lipids with membrane proteins contribute to protein stability and function. Multiple lipid interactions surrounding a membrane protein are often identified in molecular dynamics (MD) simulations and are, increasingly, resolved in cryo-EM densities. Determining the relative importance of specific interaction sites is aided by determination of lipid binding affinities by experimental or simulation methods. Here, we develop a method for determining protein-lipid binding affinities from equilibrium coarse-grained MD simulations using binding saturation curves, designed to mimic experimental protocols. We apply this method to directly obtain affinities for cholesterol binding to multiple sites on a range of membrane proteins and compare our results with free energies obtained from density-based equilibrium methods and with potential of mean force calculations, getting good agreement with respect to the ranking of affinities for different sites. Thus, our binding saturation method provides a robust, high-throughput alternative for determining the relative consequence of individual sites seen in e.g. cryo-EM derived membrane protein structures surrounded by a plethora of ancillary lipid densities.

biophysics↗

bioRxiv2020

Photochemical probe identification of the small-molecule binding site in a mammalian membrane-bound O-acyltransferase

The mammalian membrane-bound O-acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small-molecule inhibition is elusive. This study reports development of a photochemical probe to interrogate the small-molecule binding site in the human MBOAT Hedgehog acyltransferase (HHAT) based on HHAT inhibitor RUSKI-201. Structure-activity relationship investigation identified the improved enantiomeric inhibitor IMP-1575, which is the most potent HHAT inhibitor reported to-date, and guided rational design of a photocrosslinking probe that maintained HHAT-inhibitory potency. Photocrosslinking and proteomic sequencing of HHAT delivered identification of the first small-molecule binding site in a mammalian MBOAT. Topology and homology data suggested a potential mechanism for HHAT inhibition which was confirmed via kinetic analysis. Our results provide an optimal HHAT inhibitor IMP-1575 (Ki = 38 nM) and a strategy for mapping of interaction sites in MBOATs.

biochemistry↗

bioRxiv2019

Sphingomyelin suppresses Hedgehog signaling by restricting cholesterol accessibility at the ciliary membrane

Transmission of the Hedgehog signal across the plasma membrane by Smoothened is proposed to be triggered by its direct interaction with cholesterol. But how is cholesterol, an abundant lipid, regulated tightly enough to control a signaling system that can cause birth defects and cancer? Using toxin-based sensors that distinguish between distinct pools of cholesterol, we find here that Smoothened activation and Hedgehog signaling are driven by a biochemically defined fraction of membrane cholesterol, termed accessible cholesterol. Increasing accessible cholesterol levels by depletion of sphingomyelin, which sequesters cholesterol in complexes, potentiates Hedgehog signaling. By inactivating the transporter-like protein Patched 1, Hedgehog ligands trigger an increase in cholesterol accessibility in the ciliary membrane, the subcellular location for Smoothened signaling. Thus, compartmentalization of Hedgehog signaling in the primary cilium may allow cholesterol accessibility to be used as a second messenger to mediate the communication between Patched 1 and Smoothened, without causing collateral effects on other cellular processes.

cell biology↗

bioRxiv2019

Robin Hood: non-fitting, non-smoothing image detrending for bleaching correction

Recent advances in protein labelling--gene tagging with CRIPSR-Cas9--have made it possible to label proteins of interest endogenously. This represents a major breakthrough in the field of quantitative microscopy, especially when quantifying protein-protein interactions. This is because over-expression of labelled proteins may cause a distortion in localization, function and perhaps artificially force protein-protein interactions due to crowding effects. A microscopy technique that is particularly well suited to detect protein interactions with low photon budgets is number and brightness (N&B). Detrending (removal of global trends in data) is a necessary pre-processing step to N&B calculations, but all current detrending methods perform poorly at low intensities. Here, we present the Robin Hood automatic detrending algorithm which performs well at low intensities, evaluating it with simulated and low photon budget live cell images. RH is available as an ImageJ plugin and as an R package.\n\nSTATEMENT OF SIGNIFICANCEFluorescence microscopy in general and Fluorescence fluctuation methods in particular are very much dependent on detrend algorithms and so far, the user needs to decide an arbitrary number to correct for bleaching when using the box plot or the running average approach. Here, we have developed a tool available to everybody as an ImageJ plugin that is automatic, user-free and able to correct bleached images with very low photon counts.

biophysics↗

bioRxiv2018

Cholesterol interaction sites on the transmembrane domain of the hedgehog signal transducer and Class F G protein-coupled receptor Smoothened

Transduction of hedgehog signals across the plasma membrane is a key process during animal development. This is facilitated by the Class F G-protein-coupled-receptor (GPCR) Smoothened (SMO), a major drug target in the treatment of basal cell carcinomas. Recent studies have suggested that SMO is modulated via interactions of its transmembrane (TM) domain with cholesterol. Long time scale (>0.35 ms of simulation time) molecular dynamics simulations of SMO embedded in two different cholesterol containing lipid bilayers reveal direct interactions of cholesterol with the transmembrane domain at regions distinct from those observed in Class A GPCRs. In particular the extracellular tips of helices TM2 and TM3 form a well-defined cholesterol interaction site, robust to changes in membrane composition and in force field parameters. Potential of mean force calculations for cholesterol interactions yield a free energy landscape for cholesterol binding. Combined with analysis of equilibrium cholesterol occupancy these results reveal the existence of a dynamic greasy patch interaction with the TM domain of SMO, which may be compared to previously identified lipid interaction sites on other membrane proteins. These predictions provide molecular level insights into cholesterol interactions with a biomedically relevant Class F GPCR, suggesting potential druggable sites.

biophysics↗