Autoregulation of a trimeric transporter involves the cytoplasmic domains of both adjacent subunits

Membrane transporters mediate the passage of molecules across membranes and are essential for cellular function. 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 if they lack a single, defined structure, as they may not be resolved or may be truncated or modified to facilitate crystallization. The betaine permease, BetP, a homotrimer, is a prominent and well-studied example of a membrane protein whose autoregulation depends on cytoplasmic N- and C-terminal segments. These domains sense and transduce changes in K+ concentration and in lipid bilayer properties 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 {micro}s-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 EM data represents a stable global orientation of the C-terminal segment under downregulating conditions. The simulations also allow a detailed molecular description of the C-terminal tail dynamics as well as its interactions with lipids, potassium ions, and the cytoplasmic surface of neighboring transporter subunits. Nevertheless, they do not provide information on the N-terminal segment, whose structure was not resolved by the structural studies. We therefore examined the possible interactions of the N-terminal tail by generating de novo models of its structure in the context of the EM-derived structure. The resultant full-length models of the BetP trimer provide a molecular framework for the arrangement of the terminal domains in the downregulated protein. In this framework, each C-terminal tail contacts the neighboring protomer in the clockwise direction (viewed from the cytoplasm), while the N-terminal tails only contact the protomer in the counterclockwise direction. This framework indicates an intricate interplay between the three protomers of BetP and, specifically, a multi-directionality that may facilitate autoregulation of betaine transport.