Rethinking Leucine Zipper : ribonuclease activity and structural dynamics of a ubiquitous oligomerization motif

This dissertation focuses on structural, dynamic and catalytic properties of a Leucine \nZipper (LZ) motif – a family of protein oligomerization domains which belong to the \nstructural class of coiled coil proteins. LZ possess unique stability owing to high abundance of \nleucine residues in the key positions of the oligomerization interface. This allows increased \ncombinatorial flexibility for the sidechains in coiled coil positions defining oligomerization \nspecificity, thus making LZ an ideal protein-protein interaction determinant. This potential is \nreflected in the omnipresence of LZ within protein signalling pathways. Summarized in the \nChapter I, we review the structure, interaction specificity, folding characteristics and functional \ndiversity of LZ motifs, revealing the molecular mechanisms underlying LZ-enabled protein \nsignaling. \nBeyond the widely acknowledged role of a protein oligomerization motif, recently it \nwas shown that LZ motifs from bZIP factors GCN4 and cJun are capable of catalyzing \ndegradation of RNA. Moreover catalytic RNase activity is conserved within full-length bZIP \nfactors. This discovery was made in the laboratory of Prof. Bernd Gutte (University of Zurich) \nand served as a basis for the structural studies of LZ presented in this thesis. The manuscript \npresented as the Chapter II summarizes the results of the initial LZ RNase studies, performed \nin collaboration with Christine Deillon and Stefan Hoffman. \nOur first structural trials on LZ-GCN4 employing solution NMR led to the discovery of \nthe x-form – a novel monomeric folding intermediate of LZ that exists in equilibrium with the \nclassical coiled coil state. Although marginally populated at experimental in vitro conditions, \nx-form might represent a considerable fraction of the LZ structural ensemble in vivo, providing \na transient interface for specific recombination of interaction partners within bZIP networks. \nResults of these studies are presented as Chapter III of this thesis. Finally, our structural NMR studies of LZ–RNA interactions have shown that the \nsubstrate interacts with the coiled coil (dimeric) conformation, while the x-form is incapable of \nbinding RNA molecules. This is supported by the fact that the catalytic site is formed at the \ninterface of two LZ chains, and therefore is only available upon assembly of the coiled coil \ndimer. Experimental data show that LZ from GCN4 and cJun differ in the topology and \ncatalytic properties of the active site, which points to the ability of LZ to provide a general \nscaffold for assembly of catalytic sites with different properties. These results are presented in \nthe Chapter IV of this thesis.