Zusammenfassung
Membrane transporters mediate the passage of molecules across membranes and are essential for cellular func-tion. While the transmembrane region of these proteins is responsible for substrate transport, often the cytoplasmic regions are required for modulating their activity. However, it can be difficult to obtain atomic-resolution descriptions of these autoregulatory domains by classical ...
Zusammenfassung
Membrane transporters mediate the passage of molecules across membranes and are essential for cellular func-tion. While the transmembrane region of these proteins is responsible for substrate transport, often the cytoplasmic regions are required for modulating their activity. However, it can be difficult to obtain atomic-resolution descriptions of these autoregulatory domains by classical structural biology techniques, especially if they lack a single, defined structure. The betaine permease, BetP, a homotrimer, is a prominent and well-studied example of a membrane protein whose autoregulation depends on cyto-plasmic N-and C-terminal segments. These domains sense and transduce changes in K thorn concentration and in lipid bilayer prop-erties caused by osmotic stress. However, structural data for these terminal domains is incomplete, which hinders a clear description of the molecular mechanism of autoregulation. Here we used microsecond-scale molecular simulations of the BetP trimer to compare reported conformations of the 45-amino-acid long C-terminal tails. The simulations provide support for the idea that the conformation derived from electron microscopy (EM) data represents a more stable global orientation of the C-terminal segment under downregulating conditions while also providing a detailed molecular description of its dynamics and highlighting specific interactions with lipids, ions, and neighboring transporter subunits. A missing piece of the molecular puzzle is the N-terminal segment, whose dynamic nature has prevented structural characterization. Using Rosetta to generate ensembles of de novo conformations in the context of the EM-derived structure robustly identifies two features of the N-terminal tail, namely 1) short helical elements and 2) an orientation that would confine potential interactions to the protomer in the coun-terclockwise direction (viewed from the cytoplasm). Since each C-terminal tail only contacts the protomer in the clockwise direc-tion, these results indicate an intricate interplay between the three protomers of BetP in the downregulated protein and a multidirectionality that may facilitate autoregulation of transport.
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