The main focus of the laboratory is in the application of structural genomics to the systematic study of signal transducing proteins. We have recently broadened the scope of this project to include proteins and domains involved in endocytic trafficking and in the regulation of chromatin structure. Trafficking of protein cargo within the cell is a central area of cell biology. Last year we collaborated with Juan Bonifacino?s group in NICHD to determine the high resolution structures of the VHS domain of the novel endocytic trafficking adaptor GGA3 bound to two different acidic-cluster-dileucine signal peptides, from the CI and CD mannose 6-phosphate receptors. Phosphorylation of the cytosolic tails of these receptors regulates their intracellular trafficking. The cytosolic tail of the cation-independent mannose 6-phosphate receptor contains a serine residue within an acidic-cluster-dileucine signal that is important for the function of the receptor in the biosynthetic sorting of lysosomal hydrolases. In a continuation of the collaboration with the Bonifacino group, we showed that phosphorylation of this serine residue enhances interactions of the signal with its recognition module, the VHS domain of the GGA proteins. Crystallographic analyses by postdoctoral fellow Saurav Misra demonstrated that the phosphoserine residue interacts electrostatically with two basic residues on the VHS domain of GGA3, thus providing an additional point of attachment of the acidic-cluster-dileucine signal to its recognition module. Our current attention focuses on the other two domains of the GGA proteins, the GAT and GAE domains. Post-doctoral fellows Silke Suer and Greg Miller, and biologist Layla Saidi have obtained diffraction quality crystals of both of these domains in complex with their relevant binding partners, and expect to solve these structures in the near future, thereby completing the structural analysis of this family of adaptor proteins. In the past year it has become clear that mono-ubiquitination of proteins is a major determinant of their cell sorting, and much attention has focused on the mechanism of ubiquitination and ubiquitin-dependent targeting. This function of ubiquitin is separate from the role of polyubiquitin in proteasomal targeting. Several ubiquitin targeting domains have recently been discovered, including the UBA, UIM, and CUE domains. Saurav Misra and biologist Eudora Jones have determined the first structure of a CUE domain, that of Vps9p, and showed by isothermal titration calorimetry that it binds ubiquitin with moderate affinity. We found an unexpected similarity between the CUE and UBA structures that unites them in a superfamily of small helical ubiquitin binding domains. The structure is more open than the UBA structure and suggests these domains bind ubiquitin by undergoing a large conformational change. Misra, Jones, and postdoctoral fellow Gali Prag have crystallized the CUE:ubiquitin complex and anticipate the solution of this structure in the near future. Protein lysine methylation by SET domain enzymes regulates chromatin structure, gene silencing, transcriptional activation, plant metabolism, and other processes. Postdoctoral fellow Ray Trievel and biologist Bridgette Beach determined the 2.6 ? resolution structure of Rubisco large subunit methyltransferase in a pseudo-bisubstrate complex with S-adenosylhomocysteine and a HEPES ion reveals an all-beta architecture for the SET domain embedded within a larger alpha-helical enzyme fold. We found that conserved regions of the SET domain bind S-adenosylmethionine and substrate lysine at two sites connected by a pore. We initiated a collaboration with Robert Houtz, Univ. of Kentucky, to determine the enzymatic and catalytic mechanism of methyl transfer. We proposed that methyl transfer is catalyzed by a conserved Tyr at a narrow pore connecting the sites. The cofactor enters by a ?back door? on the opposite side of the enzyme from substrate, promoting highly specific protein recognition and allowing addition of multiple methyl groups.