Francesca Coscia

The low-density lipoprotein receptor-related protein 2 (LRP2) is an endocytic receptor implicated in the homeostasis of multiple organs. While the low-density lipoprotein receptor-related protein-associated protein 1 (LRPAP1) interacts with LRP2, its regulatory role remains elusive. Past studies showed that a single LRPAP1 molecule binds to LRP2 via complement-type repeats. However, many domains of this kind appear unoccupied in LRP2 within the complex. Here, we investigate if multiple LRPAP1 copies could bind the receptor. Using an integrative structural approach, we characterise the human recombinant LRP2 extracellular domain and its complex with LRPAP1, by identifying three additional LRPAP1 binding sites. Notably, two of these sites overlap with ligand-binding regions, suggesting that LRPAP1 may regulate LRP2 ligand-binding activity. Furthermore, we highlight LRPAP1-LRP2 interaction sites unique within the receptor's family and pathogenic LRP2 mutations located at LRP2-LRPAP1 interfaces. Overall, our study redefines the landscape of the LRP2-LRPAP1 interaction, providing insights into its clinical and functional role.

La symétrisation des protéines : un nouvel outil pour la biologie structurale: La détermination de la structure des protéines à une résolution atomique est cruciale pour la compréhension de leur fonction cellulaire. Actuellement, la cristallographie aux rayons X est la méthode la plus efficace pour la détermination à haute résolution de la structure de protéines monomériques allant 40 et 100 kDa. Par contre, elle est limitée par la croissance de cristaux de bonne qualité, qui est problématique pour nombreuses cibles. La cryo-microscopie électronique (cryoME) permet la détermination structurale à résolution quasi-atomique de larges structures protéiques, de préférence symétrique et en solution. Cependant, les images de cryoME sont très bruitées, car une faible dose d'électrons est appliquée de manière à limiter les dommages d'irradiation. En moyennant des dizaines d'images correspondant à la même orientation moléculaire, le rapport signal sur bruit est amélioré. La combinaison des images moyennées de plusieurs orientations permet l'obtention d'une carte de densité électronique 3D de la molécule d'intérêt. Si la taille et la symétrie de la molécule diminuent, l'analyse cryoME devient de moins en moins précise, il est alors impossible d'analyser des protéines monomériques de taille inférieure à 100 kDa. Le but de ce travail a été de développer une nouvelle approche pour réduire cette limite de poids moléculaire. Elle consiste à fusionner la protéine d'intérêt (cible) à une matrice homo-oligomérique, générant une particule symétrique et de taille importante adaptée à l'analyse par cryoME. Dans cette thèse, nous avons cherché à tester et démontrer la faisabilité de cette approche de symétrisation en utilisant des protéines cibles de structure connue.Pour mettre en place notre étude pilote, nous avons choisi différentes combinaisons de cibles et de matrices connectées par des peptides de liaison (linker) de longueur différentes. Nous avons caractérisé les fusions exprimées en bactéries par microscopie électronique après coloration négative et par plusieurs techniques biophysiques. Grace à ces techniques, nous avons trouvé que la meilleure combinaison est la fusion entre la protéine matrice glutamine synthétase (GS), un 12-mer de symétrie D6 et la cible maltose binding protein (Mbp), connectées par un linker contenant trois alanines, que nous avons appelée « Mag ». En jouant sur la longueur du linker nous avons ensuite sélectionné la fusion la plus compacte pour l'analyse cryoME: MagΔ5. Nous avons obtenu la carte cryoME à 10 Å de MagΔ5, qui présente une bonne corrélation avec les modèles atomiques de Mbp et GS. Plus particulièrement, le site catalytique et quelques hélices α sont identifiables. Ces résultats sont confirmés par l'étude cristallographique que nous avons conduite sur MagΔ5. L'ensemble de ce travail souligne que la présence d'une grande interface d'interactions cible-matrice stabilise la fusion et améliore la résolution en cryoME. Pour la symétrisation d'une cible inconnue, nous envisageons la même procédure expérimentale que celle développée pour MagΔ5. La matrice et le linker les plus adaptés devront être identifiés en utilisant les mêmes méthodes biophysiques.En conclusion, ce travail établit la preuve de concept que la méthode de symétrisation des protéines permet la détermination de la structure de protéines de poids moléculaire inférieur à 100 kDa par cryoME. Cette méthode a le potentiel d'être un nouvel outil prometteur, qui faciliterait l'analyse de cibles résistantes à l'analyse structurale conventionnelle.

Sortilin is a ubiquitous membrane receptor mediating trafficking of protein cargoes. In the thyroid, sortilin binds thyroglobulin (TG) during its endocytosis, a key process in thyroid homeostasis. Although sortilin has been proposed to recognise highly iodinated TG, the molecular details of this interaction remain unknown. In this work, using an integrative structural biology approach, we reveal that sortilin binds an unstructured TG C-terminal peptide and exhibits a strong preference for the monomeric TG over the commonly known dimeric form. We find that sortilin-TG interaction is independent of the iodination state of TG and instead relies on the conversion to its monomeric state, presumably promoted by extracellular degradation. Furthermore, using AlphaPulldown and sequence analysis, we show that recognition of other reported ligands by sortilin likely relies on similar unstructured peptide motifs, which are not constrained to a single binding orientation within the receptor cavity. Overall, this study reveals the TG-sortilin binding interface and provides insights into the recognition mechanism of other cargoes by sortilin.

Thyroid hormones (the common name for prohormone thyroxine and the bioactive form triiodothyronine) control major developmental and metabolic processes. Release of thyroid hormones from the thyroid gland into the bloodstream and their transport into target cells is facilitated by plasma membrane transporters, including monocarboxylate transporter (MCT)8 and the highly homologous MCT10. However, the molecular mechanism underlying thyroid hormone transport is unknown. The relevance of such transporters is illustrated in patients with MCT8 deficiency, a severe neurodevelopmental and metabolic disorder. Using cryogenic-sample electron microscopy (cryo-EM), we determined the ligand-free and thyroxine-bound human MCT8 structures in the outward-facing state and the thyroxine-bound human MCT10 in the inward-facing state. Our structural analysis revealed a network of conserved gate residues involved in conformational changes upon thyroxine binding, triggering ligand release in the opposite compartment. We then determined the structure of a folded but inactive patient-derived MCT8 mutant, indicating a subtle conformational change which explains its reduced transport activity. Finally, we report a structure of MCT8 bound to its inhibitor silychristin, locked in the outward-facing state, revealing the molecular basis of its action and specificity. Taken together, this study advances mechanistic understanding of normal and disordered thyroid hormone transport.

Thyroid hormones (the common name for prohormone thyroxine and the bioactive form triiodothyronine) control major developmental and metabolic processes. Release of thyroid hormones from the thyroid gland into the bloodstream and their transport into target cells is facilitated by plasma membrane transporters, of which monocarboxylate transporter (MCT)8 and the highly homologous MCT10 are most important. Patients with MCT8 mutations suffer from a severe neurodevelopmental and metabolic disorder, however, the molecular mechanism underlying thyroid hormone transport is unknown. Using cryogenic-sample electron microscopy (cryo-EM), we determined the ligand-free and thyroxine-bound human MCT8 structures in the outward-open state and the thyroxine-bound human MCT10 in the inward-facing state. Our structural analysis revealed a network of conserved gate residues involved in conformational changes upon thyroxine binding, triggering ligand release on the opposite compartment. We then determined the structure of a folded, but inactive patient-derived MCT8 mutant, indicating a subtle conformational change which explains its reduced transport activity. In addition, we determined the structure of MCT8 bound to its inhibitor silychristin, revealing an interaction with residues essential to drive transition to the inward-facing state, thereby locking the protein in the outward-facing state. This study provides molecular and structural insights into normal and disordered thyroid hormone transport.

Cryo-focused ion beam milling has substantially advanced our understanding of molecular processes by opening windows into cells. However, applying this technique to complex samples, such as tissues, has presented considerable technical challenges. Here we introduce an innovative adaptation of the cryo-lift-out technique, serialized on-grid lift-in sectioning for tomography (SOLIST), addressing these limitations. SOLIST enhances throughput, minimizes ice contamination and improves sample stability for cryo-electron tomography. It thereby facilitates the high-resolution imaging of a wide range of specimens. We illustrate these advantages on reconstituted liquid–liquid phase-separated droplets, brain organoids and native tissues from the mouse brain, liver and heart. With SOLIST, cellular processes can now be investigated at molecular resolution directly in native tissue. Furthermore, our method has a throughput high enough to render cryo-lift-out a competitive tool for structural biology. This opens new avenues for unprecedented insights into cellular function and structure in health and disease, a ‘biopsy at the nanoscale’.

EMPIAR, the Electron Microscopy Public Image Archive centered at EMBL-EBI, is a public resource for raw electron microscopy images related to EMDB, contains micrographs, particle sets and tilt-series.

EMPIAR, the Electron Microscopy Public Image Archive centered at EMBL-EBI, is a public resource for raw electron microscopy images related to EMDB, contains micrographs, particle sets and tilt-series.

EMPIAR, the Electron Microscopy Public Image Archive centered at EMBL-EBI, is a public resource for raw electron microscopy images related to EMDB, contains micrographs, particle sets and tilt-series.