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Abstract

The Na⁺-coupled symporter BetP catalyzes the uptake of the compatible solute betaine in the soil bacterium Corynebacterium glutamicum. BetP also senses hyperosmotic stress and regulates its own activity in response to stress level. We determined a three-dimensional (3D) map (at 8 Å in-plane resolution) of a constitutively active mutant of BetP in a C. glutamicum membrane environment by electron cryomicroscopy of two-dimensional crystals. The map shows that the constitutively active mutant, which lacks the C-terminal domain involved in osmosensing, is trimeric like wild-type BetP. Recently, we reported the X-ray crystal structure of BetP at 3.35 Å, in which all three protomers displayed a substrate-occluded state. Rigid-body fitting of this trimeric structure to the 3D map identified the periplasmic and cytoplasmic sides of the membrane. Fitting of an X-ray monomer to the individual protomer maps allowed assignment of transmembrane helices and of the substrate pathway, and revealed differences in trimer architecture from the X-ray structure in the tilt angle of each protomer with respect to the membrane. The three protomer maps showed pronounced differences around the substrate pathway, suggesting three different conformations within the same trimer. Two of those protomer maps closely match those of the atomic structures of the outward-facing and inward-facing states of the hydantoin transporter Mhp1, suggesting that the BetP protomer conformations reflect key states of the transport cycle. Thus, the asymmetry in the two-dimensional maps may reflect cooperativity of conformational changes within the BetP trimer, which potentially increases the rate of glycine betaine uptake.