(1) Role of Energy-dependent Proteases in Protein Quality Control and Cell Regulation. All cells must be capable of degrading aberrant and foreign proteins that would otherwise pollute them. Programmed degradation of regulatory factors also contributes to controlling the cell cycle and to generating peptides for immune presentation. These activities are all carried out by energy-dependent proteases, which generically consist of two subcomplexes - a peptidase and a chaperone-like ATPase. For several years, we have studied the Clp proteases of E. coli, considered as a model system. We showed that the peptidase ClpP consists of two apposed heptameric rings and the cognate ATPase - either ClpA or ClpX - is a single hexameric ring. ClpA/X stack axially on one or both faces of ClpP in active complexes. We went on to show that substrate proteins bind to distal sites on the ATPase and are then unfolded and translocated axially into the digestion chamber of ClpP. In FY10, we completed and published a cryo-EM study of the ClpAP protease. Specifically, we reconstructed the ClpA hexamer as an integral part of the ClpAP complex. Two segments lining the axial channel were found to exhibit local mobility. By comparing a model for ClpA-ATPgS derived from cryo-EM with one built for ClpA_ADP, we infer that ATP hydrolysis is accompanied by substantial structural changes in the D2 but not the D1 tier of ClpA. Its entire N-domain is rendered invisible by large-scale fluctuations. When deletions of 10 and 15-residues were introduced into the N-domain/D1 linker, N-domain mobility was reduced and changes were observed in enzymatic activities. We also performed cryo-EM reconstructions to compare the structure of ClpP with and without end-mounted ClpA. In the absence of ClpA, the apical region of ClpP is sealed;however, it opens up on ClpA binding, creating an access channel. This region is occupied by the N-terminal loops of ClpP. We were able to model the closed-to-open transition which facilitates the translocation of substrates into the interior of ClpP in terms of movements of these loops. Thus access to the ClpP degradation chamber is controlled by hinged movements of its N-terminal loops, which the binding of ClpA suffices to induce. (2) Membrane Trafficking and Remodelling. These dynamic events In which lipid bilayer structures are remodelled by interacting proteins are central to the functioning and metabolism of cells. We have a long-term interest in elucidating the membrane interactions and remodelling mechanisms of these proteins. (i) The protein clathrin plays a key role in intracellular trafficking by its coating of membranous pits and vesicles (CCVs). In the 1980s, we studied the molecular composition of coated vesicles and the plasticity of the assembly unit, the clathrin triskelion. We returned to this system in FY05, equipped with cryo-EM technology, and compared the structures of coated vesicles with and without binding of the uncoating ATPase, Hsc70. From these observations, we developed a model for uncoating. In FY07, we extended studies initiated during the previous year in which cryo-electron tomography is used to study the structures of individual CCVs isolated from bovine brain. Their polyhedral coats surround cargoes of various shapes and sizes, including vesicles containing neurotransmitters or receptors and viruses. The coated particles reconstructed in the tomograms fall into two sub-populations: 20% contain vesicles and are true CCVs;the remainder lack internal membranes and are termed """"""""clathrin baskets"""""""" (CBs). CCVs range from 80 to 134 nm in diameter, with vesicles of 30 to 68 nm. CBs range from 66 to 120 nm. While many small polyhedral forms of coat are possible in theory, many are not observed, suggesting that they are energetically disfavored. These results are being prepared for publication. (ii) Membrane Tubulation by Alpha-synuclein and Endophilin. This project was initiated in FY10. Synucleins and apolipoproteins have been implicated in several membrane trafficking events. Lipid interaction for both types of proteins is mediated by motifs that form amphipathic helices. We used cryo-electron microscopy to show that when alfa-synuclein is added to large spherical lipid vesicles, it is able to convert them into highly curved tubules and membrane structures with the appearance of nascent budding vesicles. The ability of this protein to function as a minimal machinery for vesicle budding agrees well with recent findings that alfa-synuclein plays a role in vesicle trafficking and enhances endocytosis. Endophilin A1 is a BAR (Bin/Amphiphysin /Rvs) protein abundant in neural synapses that senses and induces membrane curvature, contributing to neck formation in pre-synaptic endocytic vesicles. To investigate its mechanism of action, we used cryoEM to characterize the structural changes induced in lipid vesicles by exposure to endophilin. The vesicles convert rapidly to coated tubules whose morphology reflects the local concentration of endophilin. Their diameters and curvature resemble those of synaptic vesicles in situ. 3D reconstructions of quasi-cylindrical tubes revealed arrays of BAR dimers, flanked by densities that we equate with amphipathic helices whose folding and membrane insertion were attested by EPR. We also observed the compression of bulbous coated tubes into 70-wide cylindrical micelles, which appear to mimic the penultimate (hemi-fission) stage of endocytosis. Our findings suggest that the adaptability of endophilin-lipid interactions underlies dynamic changes of endocytic membranes. 3) In DNA transposition, segments of DNA are rearranged in chromosomes. The lysogenic bacteriophage Mu affords a tractable model system to study transposition. The proteins MuA/B are required for integration and amplification of the phage genome. While MuA plays a direct role, MuB is critical for the selection of target sites on the host chromosome. We have found that MuB forms filamentous polymers in the presence of ATP and filamentation is enhanced in the presence of DNA. We proceeded to study both kinds of filament by electron microscopy and image reconstruction. Both kinds of filament conform to a single-start helix with a pitch of 4.7 nm and the number of MuB monomers per turn varying between 5.6 and 6.2. This indicates that MuB forms a pseudo-hexameric ring-like arrangement with open ends, which extend to form a filament. Taken together with other observations, our data support a transposition model in which MuB-ATP targets the DNA by making a nucleoprotein filament;subsequently, MuB hydrolyzes ATP and the filament partially disassembles, exposing a segment of DNA for the action of MuA.

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National Institute of Arthritis and Musculoskeletal and Skin Diseases
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Tolun, Gökhan; Vijayasarathy, Camasamudram; Huang, Rick et al. (2016) Paired octamer rings of retinoschisin suggest a junctional model for cell-cell adhesion in the retina. Proc Natl Acad Sci U S A 113:5287-92
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Varkey, Jobin; Mizuno, Naoko; Hegde, Balachandra G et al. (2013) ýý-Synuclein oligomers with broken helical conformation form lipoprotein nanoparticles. J Biol Chem 288:17620-30
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Cardone, Giovanni; Heymann, J Bernard; Steven, Alasdair C (2013) One number does not fit all: mapping local variations in resolution in cryo-EM reconstructions. J Struct Biol 184:226-36

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