Background. Many important cellular functions are performed not by individual molecules but by large 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. We are currently working on four kinds of complexes. 1) Cornified cell envelopes (CEs) are covalently cross-linked layers of protein that line the cytoplasmic surface of terminally differentiated keratinocytes in the epidermis and other squamous stratifying epithelia. CEs are thought to play a major role in conferring the physical resilience and impenetrability of these tissues. We have a long-term interest in the biogenesis of CEs, whose covalently cross-linked character has thwarted conventional biochemical approaches. In previous work, we used immunocytochemistry to demonstrate that loricrin and the small proline-rich proteins (SPRs) are integral components of the CE and that the CE is assembled from dispersed precursors as granulocytes mature into corneocytes. We also developed a mathematical modeling approach for amino acid composition data to estimate the protein composition of CEs, and found that loricrin is the major component (60 ? 80%). Two main lines of enquiry were pursued over the past year. (a) One employs a novel method of mapping of sulfur in thin sections by electron energy loss spectroscopy and element- specific electron imaging. Loricrin has a high content of cysteine (and hence, of sulfur) and thus may be mapped by this technique. Preliminary results, outlined in last year?s report, have been completed and published. Sulfur was detected in round granules in the granulocyte cytoplasm and at the periphery of corneocytes, supporting our proposal that loricrin redistributes from these granules into the nascent CE. The amounts of sulfur detected imply that L-granules consist exclusively of loricrin, or that any additional proteins have an equally high sulfur content. These data also confirmed the large fraction of loricrin in the CE, previously estimated from its amino-acid composition. (b) Loricrin Monolayer Model of the CE. We measured the thickness and density (mass-per-unit-area) of mature CEs isolated from newborn mouse epidermis, forestomach, and footpad, respectively. Their thicknesses were measured from metal-shadowed specimens, and their mass-per-unit-area by dark-field scanning transmission electron microscopy. The CEs were found to be strikingly uniform in both thickness (14.7 nm) and mass-per-unit- area (7.2 kDa/nm2), regardless of source. Based on these data, we formulated a model in which the CE is envisaged to consist of a sheet of elongated (12nm) loricrin molecules, packed side-by- side, with their long axes perpendicular to the plane of the CE. Loricrin is cross-linked by SPRs and attached to a scaffold of involucrin and other minor CE components. This model satisfies all current data and accounts for the remarkable uniformity of the CE, which is otherwise difficult to explain. Our continuing work on the CE applies similar methods to CEs from several sources ? from cultured cells; immature CEs isolated from epidermis; and CEs from loricrin knockout mice created by our colleagues at Baylor. (2) A major portion of intracellular proteolysis in bacteria and eukaryotes alike is carried out by energy-dependent proteases, which generically consist of a proteolytic component and an ATP- hydrolyzing component. We have focused on the ClpAP enzyme of E. coli, an attractive model system. Our earlier work, based on negative staining electron microscopy and two-dimensional image analysis showed that the protease, ClpP, consists of two apposed heptameric rings of 21kDa subunits, and the ATPase, ClpA, is a single hexameric ring of 84-kDa subunits. We found that ClpA stacks axially on one or both faces of ClpP to form active complexes, whose digestion chamber lies inside ClpP. The current paradigm ? to which these observations made a major contribution ? is that ClpA recognizes substrates, unfolds them, and feeds them into the digestion chamber. Our main new results relate to a full three-dimensional account of ClpAP complexes preserved in their native states in vitreous ice. In this analysis, we characterized the symmetry mismatch that occurs when 6-fold ClpA is interfaced with 7-fold ClpP, by identifying the particular angles at which both components were viewed in micrographs of individual complexes. These observations, which made use of the recently solved crystal structure of ClpP, led to a proposal that incremental relative rotation takes place between ClpP and ClpA during processive digestion of substrates. This study has now been prepared for publication and our continuing work is aimed at a more detailed characterization of the conformational changes that we have observed to affect ClpA on binding ATP and substrates. We are also examining other related enzymes, such as Lon, in which both activities occur in the same protein subunit. 3) In a new project, we studied the binding of myosin 1C from Acanthamoeba (AMIC) - a single-headed, non-filamentous, myosin - to actin filaments by cryo-electron microscopy. The goal of this project is to characterize the structural correlates of regulation of AMIC?s ATPase activity by heavy chain phosphorylation and of ADP binding to AMIC. For myosins to function as actin-activated ATPases, a specific residue ? serine- 329, in the case of AMIC - must be phosphorylated. However, constitutively active and inactive AMIC molecules were obtained by substituting this Ser with Glu or Ala, respectively. These mutants were found to be indistinguishable in their mode of binding to actin, implying that the regulatory event affects some other aspect of the force-transducing cycle. We also investigated whether, in the presence of ADP, AMIC assumes a different conformation on the actin filament from its rigor state, as has been reported for two other myosins. We find that any such difference is very slight, a conclusion that correlates with kinetic differences that that have been detected between AMIC and the myosins that exhibit ADP-induced structural changes.
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