No abstract

SUMMARY Tapasin is a glycoprotein critical for loading Major Histocompatibility Complex (MHC) class I molecules with high affinity peptides. It functions within the multimeric peptide-loading complex (PLC) as a disulfide-linked, stable heterodimer with the thiol oxidoreductase ERp57, and this covalent interaction is required to support optimal PLC activity. Here we present the 2.6 Å resolution structure of the tapasin/ERp57 core of the PLC. The structure reveals the basis for the stable dimerization of tapasin and ERp57 and provides the first example of a protein disulfide isomerase family member interacting with a substrate. Mutational analysis identified a conserved surface on tapasin that interacts with MHC class I molecules and is critical for the peptide loading and editing function of the tapasin-ERp57 heterodimer. By combining the tapasin/ERp57 structure with those of other defined PLC components we present a molecular model that illuminates the processes involved in MHC class I peptide loading.

The exocyst is a large complex that is required for tethering vesicles at the final stages of the exocytic pathway in all eukaryotes. Here we present the structures of the Exo70p subunit of this complex and of the C-terminal domains of Exo84p, at 2.0-A and 2.85-A resolution, respectively. Exo70p forms a 160-A-long rod with a novel fold composed of contiguous alpha-helical bundles. The Exo84p C terminus also forms a long rod (80 A), which unexpectedly has the same fold as the Exo70p N terminus. Our structural results and our experimental observations concerning the interaction between Exo70p and other exocyst subunits or Rho3p GTPase are consistent with an architecture wherein exocyst subunits are composed of mostly helical modules strung together into long rods.

The fine structural organization of the epidermis, dermal/epidermal junction, and dermis from an unexposed site (upper inner arm) of elderly people was compared with the organization of a similar region of young people. Despite an overall thinning of the epidermis and focal areas of cytologic atypia, the characteristic morphological markers associated with the keratinization process are not markedly altered in appearance or amount. A well-formed stratum corneum consisting of flattened, enucleated horny cells enveloped by a thickened membrane, and intracellular spaces filled with electron-dense material provide structural evidence that barrier ability is not compromised in senile skin. The dermal/epidermal changes in aged skin are marked and have significant physiologic implications. The major change is a relatively flat dermal/epidermal junction resulting from the retraction of the epidermal papillae as well as the microprojections of basal cells into the dermis. This flattening results in a more fragile epidermal/dermal interface and, consequently, the epidermis is less resistant to shearing forces. Retraction of the epidermal downgrowths (preferential sites of the putative epidermal stem cell) may also explain the loss in proliferative capacity associated with the aged epidermis. The three-dimensional arrangements of collagen and elastic fibers showed marked alterations with age. Both fibrous components appear more compact because of a decrease in spaces between the fibers. Collagen bundles appear to unravel, and the individual elastic fibers show signs of elastosis. These changes may contribute to the loss of resilience that is one of the salient features of senile skin.

The La protein is a ubiquitous nuclear phosphoprotein that recognizes the 3' uridylates found in all newly synthesized RNA polymerase III transcripts. La binding stabilizes these transcripts from exonucleases and may also assist their folding. Here we present the first structural insights into how the La protein specifically interacts with its RNA substrates. The most conserved region of the La protein is the La motif, a domain also found in several other RNA-binding proteins. We have determined the structure of the La motif from the Trypanosoma brucei La protein to 1.6 A resolution (PDB code 1S29). The La motif adopts a winged helix-turn-helix architecture that has a highly conserved patch of mainly aromatic surface residues. Mutagenesis experiments support a critical role for this patch in RNA binding and show that it partly determines binding specificity for RNAs ending in 3' hydroxyl, a defining characteristic of the La protein. These findings reveal that the La motif is essential for high-affinity binding and also contributes to specificity.

The flagellar pocket (FP) of the pathogen Trypanosoma brucei is an important single copy structure that is formed by the invagination of the pellicular membrane. It is the unique site of endo- and exocytosis and is required for parasite pathogenicity. The FP consists of distinct structural sub-domains with the least explored being the annulus/horseshoe shaped flagellar pocket collar (FPC). To date the only known component of the FPC is the protein BILBO1, a cytoskeleton protein that has a N-terminus that contains an ubiquitin-like fold, two EF-hand domains, plus a large C-terminal coiled-coil domain. BILBO1 has been shown to bind calcium, but in this work we demonstrate that mutating either or both calcium-binding domains prevents calcium binding. The expression of deletion or mutated forms of BILBO1 in trypanosomes and mammalian cells demonstrate that the coiled-coil domain is necessary and sufficient for the formation of BILBO1 polymers. This is supported by Yeast two-hybrid analysis. Expression of full-length BILBO1 in mammalian cells induces the formation of linear polymers with comma and globular shaped termini, whereas mutation of the canonical calcium-binding domain resulted in the formation of helical polymers and mutation in both EF-hand domains prevented the formation of linear polymers. We also demonstrate that in T. brucei the coiled-coil domain is able to target BILBO1 to the FPC and to form polymers whilst the EF-hand domains influence polymers shape. This data indicates that BILBO1 has intrinsic polymer forming properties and that binding calcium can modulate the form of these polymers. We discuss whether these properties can influence the formation of the FPC.

Rab GTPases, the largest subgroup in the superfamily of Ras-like GTPases, play regulatory roles in multiple steps of intracellular vesicle trafficking. They are activated by guanine nucleotide exchange factors (GEFs), which catalyze the interconversion of the GDP-bound, or inactive, form of Rab to the GTP-bound, or active, form. Relatively little is known of the mechanisms by which GEFs activate Rabs. Here, we present the crystal structure of the GEF domain of Sec2p in complex with its Rab partner Sec4p. The Sec2p GEF domain is a 220 Angstroms long coiled coil, striking in its simplicity and in the use of the coiled-coil motif for catalysis. The structure suggests a mechanism whereby Sec2p induces extensive structural rearrangements in the Sec4p switch regions and phosphate-binding loop that are incompatible with nucleotide binding. We show that Sec2p is specific for Sec4p and that specificity determinants reside in the two switch regions of Sec4p.

The trypanosome bilobe is a cytoskeletal structure of unclear function. To date, four proteins have been shown to localize stably to it: TbMORN1, TbLRRP1, TbCentrin2, and TbCentrin4. In this study, a combination of immunofluorescence microscopy and electron microscopy was used to explore the morphology of the bilobe and its relationship to other nearby cytoskeletal structures in the African trypanosome procyclic trypomastigote. The use of detergent/salt-extracted flagellum preparations was found to be an effective way of discerning features of the cytoskeletal ultrastructure that are normally obscured. TbMORN1 and TbCentrin4 together define a hairpin structure comprising an arm of TbCentrin4 and a fishhook of TbMORN1. The two arms flank a specialized microtubule quartet and the flagellum attachment zone filament, with TbMORN1 running alongside the former and TbCentrin4 alongside the latter. The hooked part of TbMORN1 sits atop the flagellar pocket collar marked by TbBILBO1. The TbMORN1 bilobe occasionally exhibits tendrillar extensions that seem to be connected to the basal and probasal bodies. The TbMORN1 molecules present on these tendrils undergo higher rates of turnover than those for molecules on the main bilobe structure. These observations have been integrated with previous detailed descriptions of the cytoskeletal elements in trypanosome cells.