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Abstract: The L-type neutral amino acid transporter 1 (LAT1/SLC7A5) is one of 7 light chains that can form a heteromeric amino acid transporter (HAT) with the type II single pass glycoprotein CD98hc (SLC3A2). LAT1-CD98hc transports essential amino acids and some of their catabolites, such as tryptophan, methionine and kynurenine, across the plasma membranes of normal and cancer cells. It is also a drug transporter, carrying drugs such as gabapentin and L-DOPA across the blood brain barrier. The atypical heterodimeric nature of LAT1-CD98hc and its role in disease and drug delivery, motivate the structural characterisation of the HAT. Sequence analysis revealed two putative cholesterol binding motifs conserved between dDAT and LAT1 as well as 32 putative CRAC/CARC motifs. The crystal structures of various bacterial homologues of LAT1 were used for structure prediction, in order to visualise these putative cholesterol binding motifs and assess their plausibility. Here is presented the first binding mode analysis of ligands to the inward facing occluded conformation of LAT1. Substrates had lower predicted free energies of binding to the inward facing conformation compared to the outward open. The putative gating residue F252 may play a role in binding to aromatic substrates via p-p stacking in the outward open conformation and with all substrates via p-cation bonding with their amino termini in the inward facing occluded conformation. Based on the docking analysis, inhibitors of LAT1, JPH203 and SKN203 are predicted to transportable substrates of the transporter and KMH233 a non-transportable competitive inhibitor with a unique binding mode. LAT1 was overexpressed in HEK293 cells and co-purified with CD98hc to a sufficient biochemical homogeneity for structural characterisation. The role of cholesterol hemisuccinate in stabilizing detergent solubilized LAT1-CD98hc was established. Detergent solubilized and purified LAT1-CD98hc was subject to structural analysis by single particle electron cryo-microscopy to a resolution of 12 Å. Multibody 3D auto-refinement and principal component analysis revealed flexibility and limited interaction between CD98hc ectodomain and LAT1, contrary to predictions based on homology to LAT2-CD98hc. Docking of CD98hc allowed for visualisation and generation of molecular movies of the structural dynamics of LAT1- CD98hc ectodomain, based on these the ectodomain of CD98hc seems tethered to LAT1 via the inter-subunit disulphide bond and interaction between their transmembrane domains.

Open Access

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Abstract: Solute carriers are a large class of transporters that play key roles in normal and disease physiology. Among the solute carriers, heteromeric amino-acid transporters (HATs) are unique in their quaternary structure. LAT1–CD98hc, a HAT, transports essential amino acids and drugs across the blood–brain barrier and into cancer cells. It is therefore an important target both biologically and therapeutically. During the course of this work, cryo-EM structures of LAT1–CD98hc in the inward-facing conformation and in either the substrate-bound or apo states were reported to 3.3–3.5 Å resolution [Yan et al. (2019), Nature (London), 568, 127–130]. Here, these structures are analyzed together with our lower resolution cryo-EM structure, and multibody 3D auto-refinement against single-particle cryo-EM data was used to characterize the dynamics of the interaction of CD98hc and LAT1. It is shown that the CD98hc ectodomain and the LAT1 extracellular surface share no substantial interface. This allows the CD98hc ectodomain to have a high degree of movement within the extracellular space. The functional implications of these aspects are discussed together with the structure determination.

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Abstract: Requested microscope - using Krios I for now.

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Abstract: Dynein-2 assembles with polymeric intraflagellar transport (IFT) trains to form a transport machinery that is crucial for cilia biogenesis and signaling. Here we recombinantly expressed the ~1.4-MDa human dynein-2 complex and solved its cryo-EM structure to near-atomic resolution. The two identical copies of the dynein-2 heavy chain are contorted into different conformations by a WDR60−WDR34 heterodimer and a block of two RB and six LC8 light chains. One heavy chain is steered into a zig-zag conformation, which matches the periodicity of the anterograde IFT-B train. Contacts between adjacent dyneins along the train indicate a cooperative mode of assembly. Removal of the WDR60−WDR34−light chain subcomplex renders dynein-2 monomeric and relieves autoinhibition of its motility. Our results converge on a model in which an unusual stoichiometry of non-motor subunits controls dynein-2 assembly, asymmetry, and activity, giving mechanistic insight into the interaction of dynein-2 with IFT trains and the origin of diverse functions in the dynein family.

Open Access

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Abstract: In eukaryotes, accurate chromosome segregation in mitosis and meiosis maintains genome stability and prevents aneuploidy. Kinetochores are large protein complexes that, by assembling onto specialized Cenp-A nucleosomes1,2, function to connect centromeric chromatin to microtubules of the mitotic spindle3,4. Whereas the centromeres of vertebrate chromosomes comprise millions of DNA base pairs and attach to multiple microtubules, the simple point centromeres of budding yeast are connected to individual microtubules5,6. All 16 budding yeast chromosomes assemble complete kinetochores using a single Cenp-A nucleosome (Cenp-ANuc), each of which is perfectly centred on its cognate centromere7-9. The inner and outer kinetochore modules are responsible for interacting with centromeric chromatin and microtubules, respectively. Here we describe the cryo-electron microscopy structure of the Saccharomyces cerevisiae inner kinetochore module, the constitutive centromere associated network (CCAN) complex, assembled onto a Cenp-A nucleosome (CCAN-Cenp-ANuc). The structure explains the interdependency of the constituent subcomplexes of CCAN and shows how the Y-shaped opening of CCAN accommodates Cenp-ANuc to enable specific CCAN subunits to contact the nucleosomal DNA and histone subunits. Interactions with the unwrapped DNA duplex at the two termini of Cenp-ANuc are mediated predominantly by a DNA-binding groove in the Cenp-L-Cenp-N subcomplex. Disruption of these interactions impairs assembly of CCAN onto Cenp-ANuc. Our data indicate a mechanism of Cenp-A nucleosome recognition by CCAN and how CCAN acts as a platform for assembly of the outer kinetochore to link centromeres to the mitotic spindle for chromosome segregation.