Integrative modeling of secondary active transporters
del Alamo, Diego
Secondary active transporters are integral membrane proteins that harness the potential energy stored in electrochemical ion gradients to import and export substrates across cell membranes. Transporters with the LeuT-fold carry out a variety of essential functions in all domains of life including humans, such as importing nutrients and exchanging amino acids, and their structures undergo a diverse range of conformational changes as part of their functional cycles. This investigation focuses on the LeuT-fold glutamate/GABA exchanger GadC, which allows pathogenic bacteria to survive for extended periods of time in low pH environments such as the stomach. Its structure has only been determined at neutral pH in a putatively inactive conformation. To study its conformational dynamics at low pH, we used double electron-electron resonance (DEER) spectroscopy, which measures distance distributions between pairs of strategically placed spin labels attached to the protein backbone. These experimental data were integrated with computational modeling using novel methods developed in the biomolecular modeling program Rosetta. In benchmarks carried out on a wide array of model proteins, including several LeuT-fold transporters, these methods were shown to improve modeling precision and computational efficiency relative to alternative approaches, suggesting that the target conformation can be accurately modeled using experimental DEER data. Models of GadC generated with these methods using DEER data collected at low and neutral pH adopted an inward-facing occluded conformation that deviated from previously published crystallographic data, while closely resembling experimental structures of homologous transporters. The conformational dynamics of GadC were unaffected by the addition of either glutamate or GABA, a finding which contrasted with comparable investigations carried out on homologous LeuT-fold transporters. These results reinforce the structural and mechanistic diversity of LeuT-fold transporters and demonstrate how integrative modeling can reveal structural details in conformationally heterogeneous membrane proteins.