This project focuses on the interactions between the insulin-regulated glucose transporter, GLUT4, found in muscle and adipose cells and a recently discovered protein, TUG, that regulates GLUT4 trafficking. Discovered by our collaborator, Dr. Jonathan Bogan, TUG binds directly to GLUT4-containing vesicles and tethers them intracellularly. In response to insulin, TUG releases GLUT4 allowing translocation to the plasma membrane. Like many other proteins, TUG is composed of a modular array of independent protein domains. Our long term goal is to determine the tertiary structures of these TUG domains, to structurally characterize their interactions with each other and with a number of associated proteins, and ultimately to develop a detailed molecular model for TUG-regulated GLUT4 trafficking. A combination of sequence analysis and experimental studies has identified a number ubiquitin-like (UBL) domains in TUG. We have chosen these UBL domains as the initial focus of our structural studies because of (1) their demonstrated functional importance in TUG-mediated GLUT4 tethering and release, (2) the clear delineation of their structural domain boundaries based on sequence alignment, and (3) a pre-existing knowledge base of their potential interactions partners based on the conserved functions of homologous UBL domains in other proteins. The results of these studies will  benefit diabetes research both by contributing to a better understanding of the cellular mechanism for insulin-regulated GLUT4 trafficking, and also by structurally characterizing novel targets for the rational design of pharmaceutical agents with the potential to modulate cellular glucose uptake.

        As part of our ongoing effort to describe the molecular basis for TUG function, we have determined the tertiary structure and characterized the backbone dynamics for an N-terminal ubiquitin-like domain (TUG-UBL1) using NMR spectroscopy. A well-ordered conformation is observed for residues 10 – 83 of full length TUG and confirms a b-grasp or ubiquitin-like topology. Although not required for in vitro association with GLUT4, the functional role of the TUG-UBL1 domain has not yet been described. We undertook a limited literature review of similar N-terminal UBL domains and note that a majority participate in protein-protein interactions, generally functioning as adaptor modules to physically associate the overall activity of the protein with a specific cellular process, such as the ubiquitin-proteasome pathway. In consistent fashion, TUG-UBL1 is not expected to participate in a covalent protein modification reaction as it lacks the characteristic C-terminal di-glycine (“GG”) motif required for conjugation to an acceptor lysine and also lacks the three most common acceptor lysine residues involved in polyubiquitination. Additionally, analysis of the TUG-UBL1 molecular surface reveals a lack of conservation of the “Ile-44 hydrophobic face” typically involved in ubiquitin recognition. Instead, we speculate on the possible significance of a concentrated area of negative electrostatic potential with increased backbone mobility, both of which are features suggestive of a potential protein-protein interaction site.

The figure to the right displays various structural properties of the N-terminal ubiquitin-like domain in TUG. Starting in the upper left and progressing clockwise: Superposed Ca traces for the ensemble of 20 NMR structures, rainbow colored from red at the N-terminus to blue at the C-terminus, prepared using MOLSCRIPT (Kraulis 1991). Backbone ribbon diagram for a representative member of the NMR ensemble demonstrating the b-grasp topology conserved within this protein family, prepared using MOLMOL (Koradi et al. 1996). Electrostatic potential mapped onto the molecular surface of the TUG-UBL1 tertiary structure, with coloring in shades of red representing negative potential (q/d, charge per distance) from -5.5 to -1.5 and shades of blue representing positive potential from 1.5 to 5.5, as calculated using MOLMOL (Koradi et al. 1996). NMR-derived order parameters (S2) are mapped onto a backbone ribbon diagram with coloring from red to light-blue for S2 values from 0.7 to 1.0, respectively. Prolines and degenerate residues are colored gray. Side chains are shown for the four residues requiring conformational exchange (Rex) terms during the Model-free analysis, with coloring according to S2 values.