Many important cellular functions are performed by large complexes which operate like macromolecular machines. Complexes also play primarily structural roles as biomaterials in many tissues, including skin and muscle.
Twe aim to elucidate the structures, assembly properties, and interactions of complexes of both kinds, with close attention to the functional connotations. We pursued three main projects over the past year. (1) Energy-dependent Proteases. Protein quality control is essential for eliminating aberrant proteins that would otherwise pollute the cell, for example by amyloid formation. This activity is largely carried out by energy-dependent proteases which generically consist of two subcomplexes - a peptidase and a chaperone-like ATPase. Our studies focus on the Clp proteases of E. coli which offer a tractable model system. Earlier we showed that peptidase ClpP consists of two apposed heptameric rings and the cognate ATPase - either ClpA or ClpX - is a single hexameric ring. The ATPases stack axially on one or both faces of ClpP to form active complexes. We went on to study the interaction of ClpAP and ClpXP with model substrates. In both cases, substrate proteins initially bind to distal sites on the ATPase and are then translocated along an axial pathway into the digestion chamber inside ClpP. Results. (a) In our cryo-EM reconstruction of the ClpA hexamer at 1.2 nm resolution, two hexameric rings corresponding to the ATPase domains (D1, D2) are seen with a cavity between. However, there is little sign of the N-terminal domains although they are quite large, 17 kDa each. Evidently, the N-domains, are highly mobile. Nevertheless, we managed to visualized them by variance mapping of sideviews and difference mapping with averaged sideviews of an N-domain-deleted mutant. We also measured the scale of their mobility by molecular modeling, showing it to involve movements of up to 3.5 nm for each N-domain. (b) ClpP may partner either ATPase ? ClpA or ClpX ? and each ClpP oligomer may bind two ATPases. Can it bind one copy of each ATPase? and if so, are the hybrid complexes functional in substrate binding and internalization? We addressed these questions by statistical analysis of micrographs recorded after initiating translocation of ClpA-spcific and ClpX-specific substrates. The answers to both questions are in the affirmative. (2) Amyloid filament formation by the yeast prion protein, Ure2p. Amyloid is fibrous aggregates of protein(s) in protease-resistant, beta-sheet-rich, non-native conformations. Amyloid accumulates in a number of disease situations including rheumatoid arthritis. Prions (infectious proteins) are transmissible amyloids that have been implicated in certain neuropathies, including the spongiform encephalopathies. To investigate the structure of amyloids and the mechanisms that underlie their formation, we study yeast prions. Unlike mammalian prions, their phenotypes are expressed as lack of metabolic functions rather than cytopathic effects. This greatly simplifies and accelerates their study. We focus on Ure2p, a protein normally involved in nitrogen metabolism. Its prion phenotype presents as an inability to grow on poor nitrogen sources. In earlier work, we demonstrated filament formation by Ure2p in vitro and the presence of filaments in prion-infected cells. Results: We focused on substantiating our ?amyloid backbone model, formulated in 1999. Ure2p has an N-terminal prion domain that is necessary for filament formation and a C-terminal domain that performs in nitrogen regulation. According to the model, in filaments, prion domains form an amyloid backbone that is surrounded by the C-terminal domains, whereas in soluble Ure2p, the prion domain is unfolded. This model successfully predicted that fusions of the prion domain with exogenous proteins should also form filaments. We characterized Ure2p filaments and fusion protein filaments by biochemical and EM experiments. Protease digestion of 25-nm diameter Ure2p filaments trimmed them to 4-nm filaments which mass spectrometry showed to be composed of prion domain fragments. Fusion protein filaments with diameters of 14 to 25 nm were similarly reduced to 4-nm filaments by proteolysis. In each case, the prion domain transforms from the most to the least protease-sensitive part upon filament formation, implying a large conformational change. Filaments imaged by cryo-EM or after vanadate staining by STEM revealed a central 4-nm core with globular appendages. STEM mass-per-unit-length measurements of unstained filaments yielded 1 monomer per 0.45nm in each case. These observations all support the amyloid backbone model. (3) Structure and Assembly of Cornified Cell Envelopes (CEs). The CE is a covalently cross-linked layer of protein that lines the cytoplasmic surface of terminally differentiated keratinocytes. CEs are thought to contribute physical resilience and impenetrability to these tissues. We study their biogenesis, and have applied a variety of EM approaches, both to isolated CEs and in situ. Including compositional inferences based on mathematical modeling of amino acid compositions, we developed a model of CEs as monolayers of molecules of the protein, loricrin, cross-linked both directly and via minor CE proteins. We envisage the CE as a ?composite? biomaterial with a matrix substance (loricrin) and cross-linkers (the minor proteins). Results. By immunogold-EM of cryosections, we found that the cornified envelopes in newborn mouse skin labeled positive for LEPs, as did granules in the stratum granulosum. We have recently extended these observations to loricrin knockout mice. The main difference compared to wildtype is that LEP appears to label from both the outside and the inside of the surrogate (loricrin-less) CEs found in LKO animals.
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