Structural Biology and Structural Genomics of Traffickin

Diabetes, Digestive, Kidney Diseases

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Abstract

The main focus of the laboratory in 2002-3 was in the application of structural genomics to the systematic study of signal transducing and trafficking domains. GAT and GAE domains of the GGA trafficking proteins Trafficking of protein cargo within the cell is a central area of cell biology. Clathrin coated vesicles (CCVs) transport receptors and other cargo between organelles. There are two major classes of adaptor that load protein cargo into CCVs: the heterotetrameric AP complexes, and the monomeric multimodular GGAs (Golgi-localized, g-adaptin ear containing, ADP-ribosylation factor binding proteins). The GGAs are responsible for loading mannose 6-phosphate receptors (MPRs) and other acidic cluster-dileucine signal containing receptors into CCVs. GGAs contain three modules: VHS, GAT, and GAE, with a long flexible linker between the latter two domains. The acidic cluster-dileucine sorting signal is recognized by the VHS domain. The GAT domain is responsible for ARF binding and contributes secondarily to accessory protein binding. The GAE domain is primarily responsible for accessory protein interactions. In the past two reporting periods, we determined the structural determinants for sequence specific signal binding and phosphoregulation of VHS domain/receptor interactions. In 2002-3, we solved the structure of the GAT domain, which revealed an unexpected similarity to the N-terminal domain of the syntaxin family of SNARE proteins, suggesting a possible function for the GGAs in vesicle docking and fusion. Also in 2002-3, we determined the structure of the GGA GAE domain in complex with a fragment of the accessory protein rabaptin-5, providing a definitive account for the recognition of the accessory protein hydrophobic motif by an unusual constellation of basic residues. Mechanism of high affinity monoubiquitin recognition by the CUE domain from the structure of a CUE:ubiquitin complex Protein ubiquitination is a major regulatory post-translational modification. Polyubiquitination targets proteins for degradation by the proteasome, while monoubiquitination has a variety of functions, including targeting into the endosomal/lysosomal pathway. Downstream recognition of ubiquitin is carried out by a number of conserved binding domains, including the UEV, UBA, UIM, and CUE domains. Despite its importance to many pathways, the mechanism of recognition of ubiquitin in general, and the mechanism of differential recognition of mono- vs. polyubiquitin has been unclear. We determined the structure of the CUE domain of the Rab GTPase exchange factor Vps9p both alone (previous reporting period) and bound to ubiquitin (current reporting period). The CUE domain forms a domain-swapped dimer. We showed that the dimer is capable of high affinity monoubiquitin binding, as compared to a low-affinity monomer analyzed by others. The structure provides not only the first view of a ubiquitin:Ub binding domain complex, but also suggests a hypothesis for high affinity recognition of monoubiquitin by specific monoubiquitin binding domains. Structure of the SET domain and catalytic mechanism of protein lysine methylation Like phosphorylation, acetylation, and ubiquitination, methylation is an important regulatory modification of proteins. Lysine methylation by a family of enzymes containing a conserved SET (Suvar39, Enhancer of Zeste, Trithorax) is responsible for regulating gene silencing and other aspects of chromatin structure, and has come under intense study since 2000. Protein lysine methylation is unique in two respects. First, there is no know lysine demethylase, hence the modification is, to the best of our current awareness, irreversible. Perhaps because of its irreversibility and the profound effects of Lys methylation, SET enzymes are exquisitely specific, each methylating a unique Lys in a unique protein. Second, either one, two, or three methyl groups can be added to the target lysine, with different regulatory consequences. SET domain structural biology has been extraordinarily competitive because of the intense interest in lysine methylation as a mechanism of gene regulation. Seven different laboratories independently reported structures between Oct. 2002 and Mar. 2003. In the previous year, we obtained one of the first of these structures, that of a nearly full-length Rubisco large subunit methyltransferase (LSMT), revealing a novel knot-like b-sheet fold. The structure was in a pseudo-bisubstrate complex and provided the first insights into the mechanism of processive methyl group transfer. In 2003, we established a catalytic mechanism involving a key role for carbon-oxygen hydrogen bonds by carbonyl groups and a conserved Tyr residue.