Membrane Chemistry Tunes the Structure of a Peptide Transporter

Abstract Membrane proteins require lipid bilayers for function. While lipid compositions reach enormous complexities, high‐resolution structures are usually obtained in artificial detergents. To understand whether and how lipids guide membrane protein function, we use single‐molecule FRET to probe the dynamics of DtpA, a member of the proton‐coupled oligopeptide transporter (POT) family, in various lipid environments. We show that detergents trap DtpA in a dynamic ensemble with cytoplasmic opening. Only reconstitutions in more native environments restore cooperativity, allowing an opening to the extracellular side and a sampling of all relevant states. Bilayer compositions tune the abundance of these states. A novel state with an extreme cytoplasmic opening is accessible in bilayers with anionic head groups. Hence, chemical diversity of membranes translates into structural diversity, with the current POT structures only sampling a portion of the full structural space.


Structure and Dynamics of DtpA in Detergent
Thed etergent LMNG (lauryl-maltose-neopentyl-glycol) is commonly used to extract membrane proteins and to determine their structure using x-ray crystallography and cryo-EM. [20,[67][68][69][70][71][72][73][74][75][76][77] We therefore probed the conformation of freely diffusing DtpA in LMNG.The FRET histogram shows two peaks:aminor peak at high FRET (E = 0.89) and amajor peak at lower FRET (E = 0.48) (Figure 2A). Although the FRET efficiency of the major peak agrees with the value computed from the x-ray structure of the inward-open state (0.54) (Methods), its donor fluorescence lifetime deviates from the expected value for asingle donor-acceptor distance (Figure 2A), suggesting ad istribution instead of ad efined structure. [78][79][80] Af it with an empirical model [81] results in adistance distribution width of 1.1 nm (Methods), suggesting variability in the opening of the cytoplasmic side (Figure 2A, inset). Notably,astatic distribution of dye rotamers on arigid x-ray structure would only cause ad istribution width of 0.78 nm (Figure 2A,inset). Yet, since fluorescence anisotropy measurements show that the dyes experience sufficient rotational freedom ( Figure S12) at the timescale of the donor fluorescence lifetime,t he distribution of dye positions only contributes moderately to the donor lifetime.Importantly,the structural variability of DtpA is accompanied by substantial pliability.W ith decreasing temperature,t he FRET efficiency of the major peak shifts to lower values,thus causing an even wider opening ( Figure 2B). Theresult is remarkable because positional shifts of FRET peaks indicate (i)r econfiguration timescales faster than the diffusion of the protein through the confocal spot of our microscope (< 1ms) and (ii)l arge-scale structural adaptations to external conditions,w hich are known for intrinsically disordered and unfolded proteins, [82,83] but not for well-folded proteins such as DtpA. Contrary to the picture sketched by the x-ray structure of DtpA, the results show that the inward-open form of DtpA is af lexible conformational ensemble that is sampled at sub-millisecond timescales.However,what is the structural origin of the minor high-FRET population?
Given our labeling at the cytoplasmic side ( Figure 1B), molecules with high FRET exhibit ac losed cytoplasmic side. Based on the alternate access model, [30,31,33] ac losed cytoplasmic side is indeed expected for outward-open or occluded conformations ( Figure 1A). Unfortunately,t he sensitivity of our FRET-probes (2-8 nm) is insufficient to distinguish between these states and we therefore used ab iochemical approach. Thea ddition of the conformation-specific nanobody (N00) blocks the opening of the periplasmic side ( Figure 2C). If the high-FRET peak represents outwardopen molecules,blocking the periplasmic opening is expected to shift the equilibrium towards the low-FRET peak (inwardopen) due to the mutually exclusive nature of these states.
However,d espite an anomolar affinity for DtpA (Figure 1D,T able S2), N00 neither changes the abundance of the high-FRET peak ( Figure 2C)n or does it alter the temperature adaptation of the inward-open ensemble ( Figure 2D). Tw oscenarios could explain this result:either the high-FRET peak corresponds to an occluded conformation with simultaneously closed cytoplasmic and periplasmic sides,orthe high-FRET peak is not in equilibrium with the open ensemble, suggesting that it represents kinetically trapped misfolded species.T oe xclude the latter,w ec hecked whether the highand low-FRET peaks are in dynamic exchange.
Here,w em ade use of the fact that ad iffusing molecule may enter and exit the confocal spot numerous times ( Figure 3A). Once amolecule leaves the observation volume, the likelihood of it returning to this volume within as hort time period is larger than the chance of detecting an ew molecule.H ence,c onformational switching events between successive transits can be identified ( Figure 3A), which allows us to probe dynamics in the regime of several millisec-onds. [84,85] To extract the exchange kinetics,w eb inned the photon trace (100 mss teps) and selected all molecules in the high-FRET population (0.7 < E < 1.2). We then constructed FRET histograms of those bins that followed the original set with atime delay.With increasing time delay,the high-FRET population decreases and the low-FRET population increases ( Figure 3B). Thek inetics of forming low-FRET molecules from the high-FRET species follows single-exponential kinetics with ar elaxation time of % 1ms( Figure 3C)t hat clearly demonstrates an exchange between the peaks.M isfolding can therefore be excluded as an origin of the high-FRET peak. Notably,full equilibration is not reached within the accessible time window (Figure 3C), indicating that also slower processes contribute to the exchange.Importantly,the exchange is also observed in the presence of N00 ( Figure 3D), which shows that opening and closing of the cytoplasmic side is independent of locking the periplasmic side.T his loss in cooperativity between sides strongly indicates that the high-FRET peak represents occluded molecules with simultaneously closed cytoplasmic and periplasmic sides.I ndeed, such states have previously been found in detergent-based x-ray structures, [43,46,86] but the timescale of their formation had been elusive.
Occluded molecules are intermediates on the path from outward-open to inward-open states ( Figure 1A). Given the underrepresentation of outward-open x-ray structures, [40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57] we utilized our smFRET approach to identify detergent conditions that stabilize the outward-open conformation. To this end, we screened abroad spectrum of ligands ( Figure 3E and SI Appendix, Figure S13) with the goal to shift the balance between high-and low-FRET peaks.However,none of the ligands and not even changes in the protonation state of DtpA (SI Appendix, Figure S14) altered the balance between high-and low-FRET peaks significantly,s uggesting that detergent does not provide the means to stabilize outwardopen molecules.T his raises the general question of whether outward-open states are sampled at all in the absence of proton-and substrate-gradients.T oaddress this question, we studied DtpA in membrane-like nanoparticles stabilized by the protein Saposin A(SapNPs). [87,88]
While the peaks at low and high FRET efficiency are preserved, the histograms are broader.Still, the fluorescence lifetimes indicate that the peaks correspond to adistribution of conformers (SI Appendix, Figure S16). Since control experiments exclude quenching and as ticking of the FRETdyes to the protein surface (SI Appendix, Figure S12, S17), we conclude that peak broadening is due to as lower sampling within the open and closed ensembles.C ompared to detergent, the abundance of the high-FRET peak has nearly  Surprisingly,the FRET histograms differ strongly among the four membrane environments.Intotal, three populations can be distinguished. Thehigh-and low-FRET peaks are identical to those found in POPE. However,anadditional peak with an extremely low FRET efficiency( E = 0.25 AE 0.02) is found in POPS and POPA( Figure 4D,E) that corresponds to an ensemble with an extreme opening of the cytoplasmic side.In BL-extracts on the contrary,n either high-nor low-FRET peaks are observed and the transporter samples exclusively inward-open conformations ( Figure 4F). Hence,D tpA exhibits as trong sensitivity to the lipid composition and net negatively charged lipids (POPS,P OPA) even cause an extreme opening. Importantly,i rrespective of the membrane composition, we find as trong cooperativity between the cytoplasmic and periplasmic side of the transporter:w hen N00 binds the periplasmic side,a ll populations collapse to predominantly form the inward-open ensemble ( Figure 4B, D-F). Ther esults indicate that it is the membrane environment per se that generates the cooperativity between the hemispheres of DtpA rather than the chemical nature of the lipid head group.H ead group chemistry on the contrary, sensitively tunes the abundance of states.However,this is not the case for ligands.S imilar to our findings in detergent, the addition of ligands to DtpA in POPE SapNPs did not affect the relative abundance of inward-open and outward-open states (SI Appendix, Figure S19). Although the detergentbased inward-open x-ray structure with ap ro-drug bound is also similar to the apo-structure, [48] the lacking structural sensitivity to ligands is surprising.H owever, in cells,l igand concentrations differ significantly between periplasm and cytoplasm, thus generating an asymmetry that is not reproduced in our experiments.I fl igand affinities are similar at each side of DtpA, the isotropic concentration of ligand on both sides in our experiments will cause as tructural invariance of DtpA towards the ligand.

Structural Modeling of DtpA Conformations
Theextreme inward-open population observed in SapNPs containing POPS and POPAremains aconundrum. To obtain an estimate of the domain arrangement, we started from the existing x-ray structure of DtpA and used rigid-body rotations of the N-and C-terminal domains ( Figure 5A-D). Current transport models [33] suggest symmetric motions around the center axis that crosses the substrate-binding site at the domain interface ( Figure 5A). We therefore rotated the domains around this axis and determined the FRET efficiency of the rotamers (see Methods in the Supporting Information). We find that closing the cytoplasmic side by rotating the domains by À408 8 reproduces the experimental FRET efficiency (0.82) of the outward-open ensemble with only introducing 4.8 %atomic clashes [91] (Figure 5B).
However,i ti si mpossible to match the experimental FRET efficiencyo ft he extremely inward-open ensemble without inacceptable clashes.W et herefore chose an alternative rotation axis on the periplasmic side ( Figure 5C). Here,the extreme inward-opening can indeed be reproduced with only 2.4 %a tomic clashes ( Figure 5D). Notably,w ed id not optimize these structures,w hich shows that the domain architecture in principle allows an extreme cytoplasmic opening of DtpA ( Figure 5E). , and brain lipid extract (F). In eukaryotic brain lipid extract, 58.7 % of the lipid composition is unspecified by the manufacturer but likely contains > 50 %PC. [90] Interestingly,l ike all bacterial POTs, DtpA contains two transmembrane helices (HA and HB) that are missing in eukaryotic homologs. [48] Thei nteraction between these helices and the core of DtpA hinders the formation of the outward-open state via rigid body rotations and we therefore neglected the connecting loop between HA and HB in our rigid-body motion approach ( Figure 5E). Neglecting these restraints is justified by the fact that the interface between both helices and DtpA is rich in hydrophobic residues.W e conjecture that while these helices are strongly attached to DtpA in the more water-rich detergent environment [25] due to hydrophobic effects,t his interaction is weakened in the strongly hydrophobic environment of al ipid bilayer.W eakening contacts between the helices and the DtpA core would release the restraints that hamper an opening of the periplasmic side such that this mechanism explains why the outward-open state is only observed in membrane-mimicking SapNPs ( Figure 4).

Conclusion
POTs have been extensively studied by x-ray crystallography and biochemical transport assays over the past years. [27,36,39,58,61,64,92,93] Numerous structures of various bacterial homologues in the absence and presence of substrates, drugs,a nd prodrugs are available,h ighlighting crucial residues for substrate binding and proton coupling. [40][41][42][43][44][45][46][47][48][49][50] Unfortunately,o nly inward-open and partially inward-open states of POTs have so far been described at atomic resolution in detergent and the influence of the membrane environment on structure and transport has been difficult to access. [40,41,43,44,46,49,54] An umber of reports showed that the activity of membrane proteins is preserved in detergent environments. [94][95][96] Yet, counter examples are also frequent. Fore xample,G -protein coupled receptors (GPCRs) are known to be sensitive to the sterol content of lipid bilayers [97] and their membrane extraction is often cell-type dependent. [98] Structural differences in as cramblase between detergent and membrane-like environments have recently been found using cryo-EM. [99] Hence,t he way lipid compositions affect structure and function of membrane proteins in vitro varies and it is often unclear whether aloss of function in detergent is caused by al oss in stability or by trapping the protein in one out of several functional conformers.While the latter is clearly preferred, it complicates the crystallization of all functionally relevant states.T his is particularly problematic for POTs whose broad substrate profile makes them key for the pharmacokinetics of drugs.U sing DtpA, we showed that the crystallization bias of POTs results from as trong sensitivity towards the lipid environment. Detergents do not allow as ampling of all functional states of DtpA. Although our smFRET experiments show that the inward-open conformation in detergent is similar to that in POPE nanoparticles (Figure 2A and B), the outward-open state is only accessible in the more native environment of SapNPs. Structurally,aweakening of the hydrophobic contacts between the helices HA/HB and the core of DtpA in the low dielectric medium of am embrane explains this sensitivity ( Figure 5E). However,s tructural differences are not only observed between detergent and SapNPs but also between SapNPs with different lipid composition. In fact, since the aliphatic tails of POPA, POPS,a nd POPE are identical, the difference in the abundance of inward-open, extreme inwardopen, and outward-open conformers (Figure 4) is caused by the chemistry of lipid head groups.F or example,t he newly identified extreme inward-open conformation is only observed in POPAa nd POPS,i .e., lipids with net negatively charged head groups.G iven the complexity of natural membranes in which lipid compositions can vary on submicrometer length scales,e .g., in micro-domains, [100] the heterogeneity found among four membrane-like environments in our experiments may suggest as tructurally heterogeneous DtpA ensemble in the cell membrane.
Care has to be taken when interpreting functional and pharmacological aspects based on structures obtained in detergents.W hile these structures suggest models with welldefined states,o ur results sketch DtpA as an ensemble of multiple conformers with rapid dynamics.R ecent studies showed that enzymes obey structural dynamics much faster than the timescales required for turnover. [101,102] Such dynamics are also prevalent in DtpA, irrespective of the lipid environment ( Figure 3C,D and Figure 4C). Not only do inward-open and occluded states interconvert rapidly with arelaxation time of 1ms, but the inward-open state itself is an ensemble of conformers within which the opening of the cytoplasmic side can vary (Figure 2A). Although the role of these dynamics in the substrate transport cycle is currently elusive,f ast domain closures in adenylate kinase have been proposed to optimize the orientation of substrate for catalysis [101] and as imilar mechanism could guide the POTmediated transport of peptides across biological membranes. In summary,o ur results show that DtpA is aflexible and highly dynamic ensemble of structures that responds sensitively to its lipid environment and it will be important to understand whether this sensitivity is ag eneral feature of MFS transporters.