Structural Evidence for Functional Lipid Interactions in the Betaine Transporter BetP

Abstract

Bilayer lipids contribute to the stability of membrane transporters and are crucially involved in their proper functioning. However, the molecular knowledge of how surrounding lipids affect membrane transport is surprisingly limited and despite its general importance is rarely considered in the molecular description of a transport mechanism. One reason is that only few atomic resolution structures of channels or transporters reveal a functional interaction with lipids, which are difficult to detect in X-ray structures per se. Overcoming these difficulties, we report here on a new structure of the osmotic stressregulated betaine transporter BetP in complex with anionic lipids. This lipid-associated BetP structure is important in the molecular understanding of osmoregulation due to the strong dependence of activity regulation in BetP on the presence of negatively charged lipids. We detected eight resolved palmitoyl-oleoyl phosphatidyl glycerol (PG) lipids mimicking parts of the membrane leaflets and interacting with key residues in transport and regulation. The lipid-protein interactions observed here in structural detail in BetP provide molecular insights into the role of lipids in osmoregulated secondary transport.

Tat transport in Escherichia coli requires zwitterionic phosphatidylethanolamine but no specific negatively charged phospholipid

“…Note that BKT29 forms no CL and does not accumulate PG, although it still can form some PG (indicated by an asterisk). stimulatory roles that have been established for CL in case of the Sec system [10], anionic lipid interactions can be involved in conformational transitions such as channel opening processes or transporter oligomerizations as shown for the potassium transporter KcsA from Streptomyces lividans [43,44], they can modulate transporter activity as shown for the betaine transporter BetP from Corynebacterium glutamicum [45,46], and CL has been found to function in targeting to cell poles in case of the E. coli proline transporter ProP [47]. The previously postulated importance of anionic phospholipids for Tat function could have been due to any of these roles.…”

Assignment of the resting state of the secondary active transporter BetP by integrating spectroscopic measurements and molecular simulations

“…The range of possible spin-spin distance values was estimated using the multiscale modeling of macromolecular systems (MMM2015.1) software package (21)(22)(23) to model all possible rotamers of R1 probes attached to either cytoplasmic (S140 and K489) or periplasmic (G450 and S516) sides of BetP. To assess the range of distances arising from spin coupling across protomers, we predicted the rotamers of the spin label on the inward-open trimer (PDB entry 4C7R (14) (14)) states. Since the standard spin label rotamer libraries (R1A_175K and R1A_298K) included, in some cases, only a few rotamers, we used the R1A_298K_xray library (23).…”

“…However, activation is not required for the Na + -coupled uptake of betaine, which occurs at a basal level in the absence of potassium or osmotic stimulation. Through X-ray crystallography, BetP has been captured in almost all expected conformations along the transport cycle (6,(14)(15)(16), indicating conformational changes between transmembrane helices (TM) 1 1´, 5´, 6´ and 10´ (16), relative to the helices in the so-called hash domain (TM 3´-4´ and 8´-9´, Figure 1). In spite of the unprecedented insights into the conformational cycle of BetP garnered from these structures, the question remains how the interconversion between states is controlled by ion and substrate binding.…”

Regulatory role of charged clusters in the N-terminal domain of BetP from Corynebacterium glutamicum

“…The negatively charged residues are arranged in clusters of 2-3 residues ( Figure 1A): E13D14 (negatively charged cluster 1, NC1), E24E25 (negatively charged cluster 2, NC2), and E44E45E46 (negatively charged cluster 3, NC3). In fact, 3D crystals of an N-terminally truncated variant BetPΔ29E44A/E45A/E46A (BetPΔ29NC3A) (Ressl et al, 2009), which is missing NC1 and NC2 via truncation and NC3 due to alanine substitution, showed diffraction to 2.7Å (Koshy et al, 2013). These two clusters were identified by SERp (Surface Entropy Reduction prediction) to impede successful crystallization (Goldschmidt et al, 2007).…”

“…Mutations in charged residues or in residues affecting the helical conformation in the C-terminal domain of BetP severely altered the regulation profile, e.g., the mutation Ala564Pro retained BetP at full activity (Becker et al, 2014). Here, especially the betaine coordinating helix TM3 is affected via lipid-protein interactions in its conformational plasticity (Koshy et al, 2013). For instance, the sensed stress signal is transduced further to the transporter core of BetP.…”