Doctor of Philosophy

dissertationThis dissertation describes structural and biochemical studies on proteasome assembly chaperones and proteasome activator complexes. Chapters 1 and 2 provide a detailed introduction to proteasome structure and function and related, original research is described in Chapters 3-5. Proteasomes are large multi-subunit proteases that are required to regulate diverse cellular processes. The 20S proteasome is comprised of four heptameric rings, two ? type surrounding two ? type, with proteolytic sites sequestered at its center. Eukaryotic cells assemble the 20S proteasome using a highly orchestrated process during which they employ intermolecular assembly chaperones. Included among 20S assembly chaperones is the Pba3-Pba4 complex. Chapter 3 reports the crystal structure of the Pba3-Pba4 complex from Saccharomyces cerevisiae and associated biochemical studies. Collectively this research supports a model in which a yeast Pba3-Pba4 heterodimer interacts directly with the ?5 subunit to facilitate the incorporation of neighboring ? subunits during early stages of 20S assembly. Three structurally and functionally distinct proteasome activators can bind to 20S ? rings and open an entrance/exit pore, consequently regulating substrate access to the proteolytic sites. The PA26 activator uses its C-terminal tails to bind conserved ? subunit residues and uses an internal activation loop to contact and reposition the proteasome Pro17 reverse turn resulting in gate opening. The unrelated PAN/19S activators can activate the proteasome using only their C-terminal tail residues, which contain a conserved penultimate Tyr motif and have been reported to cause gate opening by inducing a rocking motion of ? subunits rather than by directly contacting the Pro17 turn. Chapter 4 of this dissertation reports crystal structures and binding studies of proteasome complexes with PA26 constructs that display modified C-terminal residues, including those corresponding to PAN. These data suggest that PA26 and PAN/19S Cterminal residues bind superimposably but in the case of PAN/19S use their penultimate Tyr side chain to contact the proteasome Gly19 carbonyl oxygen and stabilize the Pro17 turn in the open conformation. This finding, and data from the recently reported Blm10 20S complex crystal structure, support a unified model for proteasome gate opening in which three structurally and functionally distinct activator complexes induce gate opening by directly contacting and repositioning the proteasome's Pro17 turn. A recently published report challenges this model and is discussed and evaluated in Chapter 5. We favor the model in which all three proteasome activators trigger gate opening by directly interacting with and repositioning the 20S Pro17 reverse turn, although additional analysis and experiments will be needed to resolve differences between existing models

The structures of secretory and dimeric immunoglobulin A

Secretory (S) Immunoglobulin (Ig) A is the predominant mucosal antibody, which binds pathogens and commensal microbes. SIgA is a polymeric antibody, typically containing two copies of IgA that assemble with one joining-chain (JC) to form dimeric (d) IgA that is bound by the polymeric Ig-receptor ectodomain, called secretory component (SC). Here, we report the cryo-electron microscopy structures of murine SIgA and dIgA. Structures reveal two IgAs conjoined through four heavy-chain tailpieces and the JC that together form a β-sandwich-like fold. The two IgAs are bent and tilted with respect to each other, forming distinct concave and convex surfaces. In SIgA, SC is bound to one face, asymmetrically contacting both IgAs and JC. The bent and tilted arrangement of complex components limits the possible positions of both sets of antigen-binding fragments (Fabs) and preserves steric accessibility to receptor-binding sites, likely influencing antigen binding and effector functions