STRUCTURAL BIOLOGY OF MACROMOLECULAR COMPLEXES

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Many important cellular functions are performed by macromolecular complexes. The goal of this project is to elucidate the structures, assembly properties, and interactions of such complexes with emphasis on their functional connotations. (1) Bacteriophage tail-fibers are emerging as a class of oligomeric fibrous proteins with novel conformations rich in b-sheets, saccharide-binding properties, and evident scope for modular protein engineering. We have developed our previously reported triple-b-helix model of T-even fibers by defining the domainal structures of five such phages by computer-enhanced electron microscopy. Individual domains have been expressed in E. coli and induced to trimerize and fold correctly by fusing them with a trimer-forming domain from another protein. (2) Cornified cell envelopes (CEs), consisting of sheets of covalently cross-linked protein, enhance the protective and impenetrability properties of corneocytes in the epidermis and other stratified squamous epithelia. We are studying CE structure by several electron microscopic (EM) approaches and have developed a new model for their assembly. Sulfur maps obtained by electron spectroscopic imaging identify loricrin (a cysteine-rich protein) in cytoplasmic granules and in the CE, confirming earlier immunocytochemical data. They also estimate the loricrin content of CEs in situ as about 75%, consistent with the figure given by mathematical modeling of amino acid compositions of isolated CEs. Measurements of mass-per-unit-area by scanning transmission EM and of thickness by conventional transmission EM gave remarkably uniform values of 7 kDa/nm2 and 14.5 nm, respectively. To account for them, we hypothesize that the outer portion of the CE is only one loricrin molecule thick, and that inter-loricrin cross-linking by other proteins is an important determinant of the CE's biomechanical properties. (3) A major portion of energy-dependent intracellular proteolysis is carried out by the Clp family of proteases, which generically consist of a proteolytic component and an ATP-hydrolyzing component. The latter proteins are thought to recognize substrates, unfold them, and feed them into the proteases which are hollow shells, whose interiors house the active sites. We are studying the structural properties of these molecules with particular attention to rotational symmetry and the interactions involved in the formation of active complexes. In the past year we have found that both the ClpY ATPase and the ClpQ protease are hexamers, so that their association does not invoke the symmetry mismatch that exists between 7-fold ClpP protease and 6-fold ClpA ATPase. A 3-dimensional density map of ClpA clearly visualizes its two hexameric tiers of domains; and images of ClpA complexed with RepA protein indicates that this dimeric substrate binds near the center of the ClpA hexamer.