Caracterização topológica do complexo de síntese de trealose de Saccharomyces cerevisiae utilizando a espectrometria de massas

Abstract

Trehalose is a disaccharide formed by two glucose residues linked through an α- 1,1 glycosidic bond. It’s found in various organisms, including yeasts, insects, bacteria, fungi, plants, and invertebrates, but the pathway of synthesis isn’t identified in vertebrates. The attributed functions of trehalose were initially described as being a carbon and energy source, a molecule that regulates metabolic pathways, but the function that stands out the most is tolerance to various types of stresses. Trehalose’s function depends on the organism and conditions it finds itself. In pathogens such as C. albicans, trehalose is related to an increased virulence factor, helping the pathogen to adapt during infection in the host. Although the function correlates with the synthesizing organism, the sugar synthesis pathway shows remarkable similarities with other organisms, including the yeast Saccharomyces cerevisiae, where trehalose metabolism is quite elucidated. In the yeast S. cerevisiae sugar is synthesized by four protein subunits, two catalytic Tps1 and Tps2 (56kDa and 102kDa) and two regulatory Tps3 and Tsl1 (both 123 kDa); these enzymes constitute a complex called trehalose-phosphate synthetase (TPS)/trealosephosphatase phosphatase (TPP), or simply TPS/TPP complex, with an estimated molecular mass of 630-800 kDa. However, structural information on the TPS/TPP complex is still scarce. In this regard, the characterization of the complex is critical for the development of targeted drugs to inhibit its synthesis when associated with pathogens, since mammals are unable to synthesize the disaccharide. Thus, goal was to map topology of the structure of the TPS/TPP complex of S. cerevisae after a 40°C heat shock. We present the first visualization of the topology mapping of the TPS/TPP complex structure, which exhibits an intricate topology with inter-links connecting all subunits, indicating that they are spatially close in solution. Data acquired by using the hybrid approach of cross-linking coupled to mass spectrometry (XL/MS). The distance constraints generated from the (XL-MS) methodology allowed us to model the Tps1 and Tps2 catalytic subunits with Rosetta. Our model illustrates the interactions of the C-terminal domain of Tps1 with the N-terminal and phosphatase domains of Tps2. Furthermore, most of the identifications of the covalently modified peptides were validated by TopoLink software, corroborating with our molecular modeling, reinforcing the possible interactions between the catalytic Tps1 and Tps2 subunits of the S. cerevisiae TPS/TPP complex. Thus, the XL-MS dataset made available by this study brings us closer to obtaining a complete structural understanding of the TPS/TPP complex and ultimately paves the way to the development of new drugs aimed at inhibiting trehalose synthesis in pathogens.