Diverse regulation of functional dimerization of a sugar transporter by different interfacial lipids

Domain: physics
Published: 2026-02-23
Editorial outcome: major_revision

Abstract
Endogenous lipids play essential roles in modulating membrane protein structure and function, yet the molecular mechanisms governing lipid-specific regulation remain elusive. Here, we combine solid-state NMR spectroscopy and molecular dynamics simulations to elucidate how distinct lipids regulate the structure and activity of a membrane protein in a native-like membrane environment. Using VsSemiSWEET as a model system, we determine its high-resolution structure with bound lipids, identifying three lipid types: phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CDL). These lipids bind at the monomer-monomer interface, stabilizing the dimeric structure of VsSemiSWEET. Notably, PG and CDL exhibit differential binding modes, with CDL demonstrating a dual interaction mechanism involving both its headgroup and acyl chains that enhances both dimer stability and functional activity. These findings reveal how lipids with different physicochemical properties differentially control membrane protein oligomerization and function, providing a mechanistic framework for lipid-specific regulation.

Introduction
Growing evidence highlights the critical role of specific bound lipid species in modulating the function and structure of membrane proteins. For instance, the KirBac1.1 channel is activated by anionic lipids such as cardiolipin (CDL) and phosphatidylglycerol (PG), but is inactivated by phosphatidylinositol 4,5-bisphosphate (PIP)13. Additionally, the anionic lipid phosphatidic acid (PA) and the zwitterionic lipid phosphatidylethanolamine (PE) have been reported to selectively bind to the two-pore domain potassium channel K2P4.1, with each lipid featuring non-overlapping interaction sites and distinct regulatory mechanisms14. The structural and functional roles of diverse lipid binding were further underscored by recent structural studies of mechanosensitive potassium leak channel TREK114, pentameric ligand-gated ion channels (pLGICs)15,16, and Mycobacterium tuberculosis efflux pump EfpA17. These studies reveal the binding of multiple lipid species within the structures of these membrane proteins, demonstrating that the binding of multiple lipids is not merely incidental but often integral to the structural integrity and biological activity of membrane proteins.

Although diverse lipid–protein interactions have been extensively studied across membrane proteins, the role of bound lipids in regulating protein structure and function are still widely debated. Currently, it is challenging to experimentally investigate the specific interactions between lipids and membrane proteins using conventional structural biology methods such as X-ray crystallography, solution NMR spectroscopy, and mass spectrometry. This is primarily because these approaches typically rely on detergent-containing membrane mimetics, which disrupt native lipid–protein interactions in non-native membrane environments. In X-ray crystallography, lipids frequently fail to adopt ordered arrangements due to their high flexibility, rendering them undetectable in membrane protein crystals6,18. Similarly, in mass spectrometry, the complexity and overlap of lipid fragmentation spectra impede the clear identification of specific lipids and their interactions19. As a result, the specific bound lipid species, their binding sites on proteins, and their functional relevance are often poorly defined for many membrane proteins.

A representative example is the bacterial sugar transporter Vibrio sp. SemiSWEET (VsSemiSWEET), a prototypical member of the major facilitator superfamily (MFS)20. The SWEET family of transporters is responsible for facilitating sugar uptake across cellular membranes, enabling solute permeation down a concentration gradient. These proteins play essential roles in various biological processes, including nectar secretion, pollen nutrition, and susceptibility to pathogens21. Their bacterial homologs, known as SemiSWEETs, are widely distributed among prokaryotes and represent one of the smallest known transporter families. To date, the structures of four distinct SemiSWEETs have been determined by X-ray crystallography in the lipid cubic phase (LCP), revealing three conformational states: outward-open, occluded, and inward-open22–26. All four structures exhibit the same dimeric architecture, suggesting that SemiSWEETs consistently function as dimers and employ an alternating-access mechanism for sugar transport.

However, it is important to note that none of these reported crystal structures showed the presence of bound lipids. In contrast, mass spectrometry analysis of VsSemiSWEET in detergent identified tightly bound lipid species, including CDL, PG, and PE. Among these, only CDL was found to regulate VsSemiSWEET dimerization1,27. The presence of detergent in these experimental conditions may disrupt the native structure of membrane proteins and interfere with protein–lipid interactions. Additionally, the use of different membrane-mimetic environments, such as detergent micelles versus LCP, may further contribute to the discrepancies in the observed results. Consequently, it is still unclear whether VsSemiSWEET harbors tightly bound lipids in its native membrane environment, where they bind, and what specific functional roles they play. Further experimental investigation is required to elucidate the nature of lipid binding and its functional implications in VsSemiSWEET28. In this context, solid-state NMR (ssNMR) has emerged as a powerful technique for determining membrane protein structures and characterizing their lipid interactions at atomic resolution within native-like lipid bilayers29–32.

In this work, we explore the molecular basis for lipid modulation in VsSemiSWEET through an integrated approach incorporating NMR, additional biochemical experimental techniques, and molecular dynamics (MD) simulations. We identify three co-purifying lipid types: PE, PG, and CDL, among which CDL exhibits the highest binding affinity. Through integrated ssNMR and MD analyses, we determine the structure of lipid-bound VsSemiSWEET in native-like lipid bilayers and resolve the distinct binding sites and interaction modes for each lipid. Our results reveal that PG and CDL exert differential effects on dimer stability and function of VsSemiSWEET via their specific acyl tails and headgroups. These findings provide a mechanistic understanding of how lipids with distinct physicochemical properties differentially regulate membrane protein oligomerization and activity.

Results
Identification of bound lipids co-purified with VsSemiSWEET
SsNMR spectral analysis of VsSemiSWEET liposomes confirmed the presence of bound lipids that co-purified with the protein. The purified protein was successfully reconstituted into total E. coli lipid extracts, and resonances for 85 out of 97 residues were assigned using a series of multidimensional ssNMR spectra33. To identify these co-purified bound lipids, we performed a 13C-13C combined R2v -driven (CORD) experiment34 on uniformly-13C, 15N-labeled VsSemiSWEET. Since the co-purified lipids were 13C-labeled, they generated 13C-13C correlation signals in the CORD spectrum. In contrast, the E. coli total lipid extracts used for reconstitution were unlabeled and did not produce such correlations in the spectrum. As shown in Fig. 1a, several cross-peaks were observed in the 13C chemical shift region of 60−75 ppm, corresponding to characteristic chemical shifts of lipid headgroups and glycerol backbone. To rule out the possibility that these signals originated from the protein itself, we employed an unfolding-refolding approach. During this process, 13C-labeled bound lipids were removed during the unfolding procedure, while unlabeled lipids were reintroduced to the protein during the refolding and reconstitution process. Following this treatment, the signals in this region were either absent or significantly weakened, confirming their origin from 13C-labeled bound lipids. Furthermore, Ser and Thr residues are the only amino acids exhibiting intra-residual correlations in this region. The signals of these residues have been unambiguously assigned, confirming that the identified signals arise from co-purified lipids rather than protein-derived signals.

To identify the specific species of the bound lipids, we first measured the 13C chemical shifts corresponding to the headgroups and glycerol backbone of anionic lipid PG, zwitterionic lipids PE, and CDL using 1H-13C HSQC (heteronuclear single quantum coherence) solution NMR spectra of pure lipid standards (Fig. S1 and Table S1)35. We then acquired a 2D 13C-13C CORD spectrum of 13C, 15N-labeled VsSemiSWEET with a 500 ms mixing time, optimized to enhance correlation signal intensities from bound lipids (Fig. S2). Although minor chemical shift differences existed between the free and protein-bound states of the lipids, the characteristic α–β correlation patterns allowed us to assign the glycerol backbone and headgroups signals for PE, PG, and CDL36–38. Specifically, well-resolved and symmetric correlations at 64.8–42.9, 73.4–69.2, and 72.0–69.0 ppm were unambiguously assigned to the headgroups of co-purified PE, PG and CDL, respectively (Fig. 1b). To further verify these assignments, we prepared a 13C-labeled total E. coli lipid extract sample containing no protein. This sample consisted of approximately 57% PE, 15% PG, 10% CDL, and 18% other minor species identified by mass spectroscopy39. Notably, this lipid profile is analogous to the native lipid composition of Vibrio species40. As illustrated in Fig. 1b, the 13C chemical shifts of PE, PG and CDL in the E. coli lipid extract align perfectly with those observed for the lipids bound to VsSemiSWEET. This exact signal match provides direct evidence that these three lipid species co-purify with the protein, demonstrating their stable and high-affinity interaction with VsSemiSWEET.

To further identify the bound lipids, we employed 1D 31P solution NMR, a technique that yields distinct chemical shifts for lipid phosphate groups in each lipid type41–43. In our spectra, PE, CDL, and PG were identified by their characteristic shifts at −0.20 ppm, 0.22 ppm, and 0.30 ppm, respectively (Fig. S3). As shown in Fig. 1c, the 31P NMR spectra of VsSemiSWEET in detergent micelles verified the co‑purification of PE, PG, and CDL. Notably, after a wash with 1% dodecyl-β-D-maltoside (DDM), the 31P signals of PE and PG decreased by 80% approximately, while the CDL signal showed little reduction, demonstrating that CDL binds more tightly than PE or PG. We extended this analysis to SemiSWEET proteins from E. coli, L. biflexa, and Bradyrhizobium species. Although all three lipids co‑purified with these homologs, the binding strength of CDL was consistently lower than that observed for VsSemiSWEET (Fig. S4). This trend agrees with our MD simulations, which also identified CDL binding in these variants but with comparatively weaker interactions (Fig. S5).

Structural characterization of VsSemiSWEET in lipid bilayers using ssNMR
Next, we proceeded to identify the specific lipid-binding sites on VsSemiSWEET. Although the X-ray crystal structure of VsSemiSWEET is available25, we determined a high‑resolution 3D structure of the protein in native‑like lipid bilayers to avoid potential artifacts introduced by the non‑native detergents used in X-ray crystallography. To obtain the necessary distance restraints for structure determination, we conducted 2D 13C-13C CORD experiments on sparsely 13C-labeled samples. During bacterial protein expression, [2-13C] or [1,3-13C] glycerol was used as the carbon source (denoted as 2-VsSemiSWEET and 1,3-VsSemiSWEET), resulting in significant 13C spin dilution44. This dilution greatly improved spectral resolution and suppressed dipolar truncation effect, thereby providing numerous well‑resolved cross‑peaks corresponding to medium‑ and long‑range distance restraints45–47. The “medium-range” and “long-range” correspond to distances between residues where i and j are residue numbers. For medium-range, the distance is defined as 2 ≤ |i–j| < 5, while for long-range, it is |i–j| ≥ 5. Both the 2-VsSemiSWEET and 1,3-VsSemiSWEET samples were prepared using an unfolding-refolding approach to replace the original 13C-labeled co-purified lipids with unlabeled ones, thereby eliminating potential lipid‑derived signal interference during restraint assignment. Comparison of the chemical shifts and signal intensities between the refolded and native VsSemiSWEET samples confirmed that the unfolding‑refolding process did not perturb the protein structure (Figs. S6 and S7).

As shown in Fig. 2a, b and Fig. S8, the CORD spectra with a 500 ms mixing time exhibit hundreds of well-resolved cross-peaks. However, significant spectral overlap complicates their unambiguous assignment. To address this, we employed a structural template to guide distance restraint determination. We constructed an experimental structural model using CS-Rosetta calculations, inputting chemical shift data and initial distance restraints determined solely based on residue-specific chemical shifts (Table S2). The resulting monomeric structure of VsSemiSWEET exhibits high convergence, with a root mean square deviation (RMSD) of 0.9±0.2 Å for backbone heavy atoms among the ten lowest-energy structures, and is consistent with the AlphaFold3-predicted structure (Fig. S9)48. To illustrate how this structural model can resolve assignment ambiguities, we provide an example (Fig. S10). The cross-peak at (70.0, 61.4) ppm in the CORD spectrum of 2-VsSemiSWEET has eight candidate assignments; however, seven possibilities are excluded because their corresponding 13C-13C distances exceed the upper limit (9 Å) for a 500 ms mixing time, leaving only the 4.3 Å correlation between T60 Cα and W72 Cα. Further assignment details regarding the assignment of distance restraints can be found in the Methods Section. In total, we successfully assigned 442 13C-13C intra-monomer distance restraints, including 117 long- and medium-range restraints (Fig. 2a, b and Fig. S11). Structural calculations performed using Xplor-NIH software with these comprehensive intra-monomer restraints yielded an ensemble of ten structures (selected from 200) with a backbone RMSD of 1.5 Å across all α-helices of the protein (Fig. S10c).

VsSemiSWEET exists as a dimer in lipid bilayers, as demonstrated by our cross-linking experiments (Fig. S12). Assignment of inter-monomer distance restraints, which are critical for determining the dimer structure, is challenging due to potential overlap with intra‑monomeric contacts. To address this, we initially assigned inter-monomer distance restraints by excluding intra-monomer contacts based on the monomer structure. All possible intra-monomer distances exceeded the upper limit of 9.0 Å, allowing us to exclude them during distance restraint assignment (Fig. 2c and S13a, b). To further distinguish between intra- and inter-monomer contacts, we prepared a spin-diluted sample by mixing 13C-labeled monomers with natural abundance (NA) analogs at a 1:2 ratio using an unfolding-refolding approach (Fig. S14a). In this setup, intra-monomer signals are reduced to one-third of their original intensity in uniformly labeled samples, while inter-monomer signals were absent (Fig. S14b). By comparing the signal intensities in 2D 13C-13C spectra of 2-VsSemiSWEET and the spin-diluted [(1:2) 2-13C/NA] VsSemiSWEET samples, we unequivocally identified cross‑peaks originating from inter‑molecular contacts (Fig. 2d). The analysis of CORD spectra from a diluted sample revealed six ambiguous inter-monomer distance constraints. These constraints were used to construct an initial dimer structural model using the docking module of Xplor-NIH. Further details on the homodimer construction are provided in the Methods Section. Utilizing this initial model, thirteen unambiguous and eight additional ambiguous inter-monomer contacts were identified. The strategy for resolving intra- versus inter-monomer contact ambiguity is illustrated in Figs. S13c and S15, with examples of assignments for Q29 CG-T48 CA and K32 CA-G43 CA restraints. All inter-monomer long-range distance restraints are listed in Table S3. A total of 21 inter-monomer and 442 intra-monomer distance restraints, along with 78 backbone torsion angles predicted by the TALOS+ program using assigned chemical shifts (Table S4), were employed to calculate 1024 dimer structures of VsSemiSWEET using Xplor-NIH. For further details on the structure calculations, refer to the experimental section. Among the calculated structures, the ten lowest-energy structures exhibited high convergence, with a backbone RMSD of 0.9±0.3 Å (Fig. 2e).

Hydrogen/deuterium (H/D) exchange experiments revealed a solvent-accessible cavity between the two monomers near the periplasmic region, supporting an outward‑open conformation (Fig. S16). The overall ssNMR structure is similar to the crystal structure (Fig. S17), with a backbone RMSD of 2.0 Å across well-ordered regions (residues 17–39, 46–67, and 70–92). Notably, a key structural feature in the ssNMR structure is the pronounced kink at Pro29 within the conserved PQ-loop motif of TM1, which divides the helix into TM1a and TM1b segments. Importantly, while bound lipids remained physically present in the VsSemiSWEET structural determination samples, their structural constraints were removed by substituting 13C-labeled lipids with natural abundance lipids during the unfolding-refolding process. Consequently, the bound lipids are absent in the determined structures, leaving empty coordinates due to the lack of distance restraints.

Characterization of lipid interactions with VsSemiSWEET in native-like lipid bilayers
To investigate the interactions between VsSemiSWEET and its bound lipids, we performed 2D CORD spectra of uniformly 13C, 15N-labeled VsSemiSWEET using a long mixing time of 500 ms. The chemical shift at 72.8 ppm was uniquely assigned to the G2 nuclei of the central glycerol moiety in lipids. Therefore, any cross-peaks at this chemical shift in the 2D 13C-13C spectrum of lipid-bound VsSemiSWEET must represent either lipid–lipid or lipid–protein correlations. A 13C-13C spectrum of uniformly 13C-labeled E. coli lipid extracts, which contains no protein signals, serves as a reference to distinguish between lipid–lipid and lipid–protein interactions. As shown in Fig. 3a, additional peaks observed at 72.8 ppm in the VsSemiSWEET spectra correspond specifically to lipid–protein correlations. Based on the correlated chemical shifts of these peaks (Fig. S18), we identified residues S22, Q29, K32, H37, E39, H40, C76, and A84 as potential interaction sites for lipid G2 nuclei, as both the main-chain and side-chain 13C atoms of these residues exhibit correlation with the G2 nuclei of lipids. Critically, the observation of these lipid–protein cross-peaks in the dipolar-coupling-based experiment imply lipid binding times in the microsecond range or longer but excludes shorter lifetimes, which is the timescale of the relevant dipolar couplings that drive spin diffusion37.

To assign these lipid–protein correlated peaks, we carried out MD simulations to identify potential lipid-binding sites on VsSemiSWEET. We conducted 5-μs all-atom MD simulations using the ssNMR structure of VsSemiSWEET in lipid bilayers as the starting structure. The average residence time of different lipids at each residue of the SemiSWEETs was calculated to evaluate their binding ability (Fig. 3b and Fig. S19). The results indicated that CDLs exhibited stronger binding ability to VsSemiSWEET compared to PEs, which is consistent with our experimental observations (Fig. 1c). Analysis of the MD simulations highlighted that residues interacting with CDL were predominantly located at the monomer–monomer interface and near transmembrane regions. Notably, basic residues K32, H37, and H40 in the intracellular region demonstrated significantly stronger interactions with CDL compared to other residues (Fig. 3b).

To validate these findings, we mutated the three basic residues K32, H37, and H40 to basic-to-neutral substitutions (K32A, H37A/H40A, and K32A/H37A/H40A). We then assessed the effects of these mutations on lipid binding using 1D 31P solution NMR spectra of the mutated VsSemiSWEET solubilized in micelles. As shown in Fig. 3c, the signal intensities for PE, PG, and CDL lipids all decreased in these mutants, indicating that these lipids bind at the interface between the two monomers. However, while signals from PE and PG persisted, nearly no signals were observed for CDL in the K32A/H37A/H40A mutant. This suggests that CDL exclusively binds at the monomer–monomer interface, whereas PE and PG bind both at the interface and the vicinity of transmembrane regions. These findings align with a recent MD simulation study on VsSemiSWEET, which demonstrated that CDL molecules bind in sufficient numbers to the dimer interface on the cytoplasmic side28.

To further elucidate the nature of the interaction between bound lipids and these basic residues, we constructed a K32R mutant, which involved a basic-to-basic substitution (Fig. 3d). As expected, the lipid signals observed in the 1D 31P spectrum of the K32R mutant were nearly identical to those of the wild-type VsSemiSWEET, indicating that this basic-to-basic mutation does not significantly disrupt the interaction between the residues and lipids. This finding supports the conclusion that their interactions are predominantly mediated by electrostatic forces. Moreover, in contrast to the dipolar-coupling-based signals from glycerol backbones and headgroups of the bound lipids, which indicate that these groups exhibit structural rigidity on a micro- to millisecond timescale, the signals observed in scalar-coupling-based ssNMR spectra suggest a high degree of flexibility on submicrosecond timescales49. As shown in Fig. S20, the tail carbons of lipids in VsSemiSWEET were detected in a 2D 1H-13C INEPT spectrum, indicating the flexibility of the lipid acyl chains. Finally, to generate a composite structural model that incorporates the missing lipid coordinates, we performed molecular docking. This placed two CDL molecules at the dimer interface (Fig. 3e). The docking model revealed that the lipid headgroups establish contacts with basic residues K32, H37, and H40, forming several hydrogen bonds. These findings underscore that lipid binding is predominantly mediated through electrostatic interactions between lipid headgroups and basic residues within the dimer interface.

Bound lipids influence the dimer stability of VsSemiSWEET in lipid bilayers
Dimerization is crucial for enabling VsSemiSWEET to efficiently transport sugars across cellular membranes21. To examine how lipid binding influences dimer stability, we conducted ssNMR, biochemical experiments, and MD simulations. As shown in Fig. S20, VsSemiSWEET maintains its dimeric state in the E. coli cellular membrane, in proteoliposomes, and when affinity-purified in detergent micelles (0.3% DM), as demonstrated by SDS-PAGE analysis (SDS-resistant). However, mutating the key interfacial basic residues K32, H37, and H40 to alanine (single or combined mutants) caused a predominant monomeric migration on SDS-PAGE gels (Fig. 4a), whereas the charge-preserving K32R mutant retained its dimeric form. Furthermore, size-exclusion chromatography of these mutants exhibited a monodisperse peak similar to that of wild-type VsSemiSWEET (Fig. 4b), indicating that these alanine mutants still elute as dimers in solution. The SDS-induced dissociation during SDS-PAGE analysis reflects a lipid-dependent stabilization of the dimer interface. Consistent with this finding, thermal stability assays revealed that lipid binding enhances the structural stability of VsSemiSWEET (Fig. S22). We also acquired ssNMR spectra of the H37A/H40A and K32A/H37A/H40A mutants to examine the conformations of the protein in the absence of lipid binding. As shown in Fig. 4c, d, the signals in the 2D 15N-13Cα NCA spectrum of these mutants exhibited broader linewidths than those of the wild-type, indicating conformational heterogeneity without lipids binding. Moreover, the disappearance of signals from the mutated sites and their neighboring residues, along with the loss of signals from many other residues, suggests increased conformational dynamics and flexibility in the absence of bound lipids. To further evaluate the structural consequences of disrupting lipid interactions, we carried out enhanced sampling simulations to investigate the stability of the H37A/H40A mutant. The resulting free energy landscape (Fig. S23) shows that the mutant dimer is substantially destabilized: it no longer adopts the lowest-energy conformation characteristic of the wild-type protein, and no stable inter-residue interactions persist at the dimer interface. Collectively, these results highlight the importance of lipid binding in sustaining the stability and structural integrity of the VsSemiSWEET dimer, likely through specific interactions at the monomer–monomer interface.

We further employed MD simulations with enhanced sampling to delineate the mechanism by which lipids modulate dimerization. We characterized the free energy landscapes of CDL-bound and lipid-free VsSemiSWEET (Fig. S24). Representative conformations of the lowest free energy state for both systems are shown in Fig. 4e, f, respectively. Compared to the ssNMR structure, the representative lowest-energy conformation of the lipid-free dimer showed fewer interfacial contacts and only rare, weak interprotomer interactions (Fig. S25). In contrast, the CDL-bound dimer adopted a conformation closely matching the ssNMR structure, with stable interactions across the binding interface (Fig. 4g). These results demonstrate that CDL binding enables the stabilization of a robust dimeric interface for VsSemiSWEET.

Differential regulations of interfacial CDL and PG on the structure and function of VsSemiSWEET
Since three distinct lipid types (CDL, PE, and PG) bind to the dimer interface of VsSemiSWEET, we used an unfolding-refolding approach to selectively deplete tightly bound CDL, allowing us to isolate the structural and functional effects of different lipid compositions. As shown in Fig. S26, fully denatured VsSemiSWEET can refold into dimers in the presence of either PE/PG or PE/PG/CDL mixtures. This observation indicates that both PE/PG and PE/PG/CDL lipid combinations support dimer formation, suggesting functional similarity between these lipid types in stabilizing the protein structure. However, MD simulations revealed fundamentally different binding modes for PG and CDL (Fig. 5a). Compared to PG, CDL forms more stable interactions with the protein, particularly through hydrogen bonding (Fig. S27). For example, the hydrogen bond occupancy between CDL and the H40 residue reaches 64%, whereas the maximum hydrogen bond occupancy between PG and H40 is only 23%. This disparity stems from CDL’s structure: its headgroup bears two negatively charged phosphate groups, creating a strong electrostatic surface that interacts tightly with adjacent basic residues (K32, H37, and H40) on the protein. This specific interaction underlies CDL’s high-affinity binding and its critical role in structural stabilization.

Importantly, MD simulations revealed that the tail chains of PG preferentially penetrate the cavity between the two monomers while their headgroups remain spatially confined at the dimer interface of VsSemiSWEET (Fig. 5a, c). By engaging specific residues within this cavity, the PG tails effectively disrupt inter-monomer packing. In contrast, CDL exhibits a distinct binding mode; its bulky hydrophobic tail cannot accommodate the cavity due to steric constraints (Fig. S28). Instead, CDL tails localize to the outward surface of VsSemiSWEET, promoting a tightly packed dimeric interface. The interaction strength between VsSemiSWEET residues and CDL or PG lipids at the interface was quantitatively evaluated based on contact probability differences. As shown in Fig. 5b, PG lipids predominantly interact with residues located on transmembrane helix 1 (TM1), the N-terminal region of TM2, and the C-terminal region of TM3. Notably, key residues (S22, T26, Q29, L33, S36, T48, Y52, and Y82) exhibited higher lipid contact probabilities in the PG-bound state. These residues are predominantly positioned along the inner faces of the transmembrane helices, further supporting the deep penetration of PG lipid tails into the VsSemiSWEET inner cavity. Additionally, we computed the in-plane density of lipid tail carbon atoms in the membrane. As illustrated in Fig. S29, lipid molecules in the PG-bound VsSemiSWEET system penetrate into the dimer interior and occlude the central pore of the protein.

To experimentally validate the distinct binding modes of PG and CDL, we prepared two ssNMR samples: sample I (with a combination of PE, PG, and CDL lipid binding) and sample II (with a combination of PE and PG lipid binding). The preparation procedures for both samples are shown in Fig. S30. Several critical aspects should be noted: First, the lipid bilayers of both samples are composed of PE and PG lipids to exclude the influence of other environmental lipids on the structure of VsSemiSWEET. Second, binding affinities of different lipids to VsSemiSWEET, calculated by MM/GBSA50, indicated that the binding ability of CDL to VsSemiSWEET is ~215 times that of PG and about 3600 times that of PE (Fig. S31). These predicted results align with observations from the above delipidation experiments (Fig. 3e). Third, during the refolding, excess PE/PG or PE/PG/CDL was added to fully refold and reassemble the denatured VsSemiSWEET into a dimeric form, favoring binding of lipids with higher affinities. Together, our results indicate that CDL primarily modulates the structure of VsSemiSWEET in Sample I, while PG exhibits a more pronounced regulatory effect in Sample II.

To probe the distinct structural effects of CDL versus PG binding, we acquired ssNMR spectra for Sample I (PE/PG/CDL) and Sample II (PE/PG only). Chemical shift perturbations (CSPs) greater than 0.3 ppm were considered significant, given the high spectral resolution and reproducibility of the ssNMR spectra (Fig. S6). Figure S32 presents the superimposition of the 2D 15N-13Cα and 13C-13C spectra of VsSemiSWEET in samples I and II. While most peaks showed negligible CSPs, some peaks exhibited significant CSPs (Fig. 5d). As shown in Fig. 5e, multiple transmembrane residues of VsSemiSWEET exhibited significant CSPs in sample II compared to sample I, including S22, T26, Q29, L33, S36, T48, Y52, Y82, V86, and I89. These CSP patterns show strong correlation with our MD simulation results, where the same residues demonstrated the highest differences in lipid contact probability between the PG-bound and CDL-bound states (Fig. 5b). This consistency between experimental CSP data and MD simulation predictions suggests these residues participate in distinct lipid–protein interactions depending on the bound lipid species. The structural differences between CDL and PG likely contribute to their distinct interaction patterns. The larger size of CDL prevents its hydrophobic tails from drilling into the cavity of VsSemiSWEET. In contrast, PG has a smaller steric hindrance, allowing its tails to penetrate deeply into the protein’s cavity. This deeper penetration facilitates direct interactions between PG and inner cavity residues, resulting in significant CSPs in these transmembrane residues. Additionally, Fig. 5f and the bar diagram in Fig. 5g showed that the signal intensities of many residues decreased and, in some cases, became unobservable in the dipolar-coupling-based spectra of sample II. This reduction can be attributed to the enhanced mobility and reduced structural stability of these residues in the absence of CDL binding.

To link the influence of dimer formation of different lipid binding to the functional implications, we analyzed the detailed intermolecular interactions and free energy landscapes of VsSemiSWEET binding with various lipids (Fig. 6a). All-atom MD simulations revealed that while PG binding, unlike CDL, does not hinder VsSemiSWEET monomer dimerization but results in more disordered residue arrangements at the dimeric interface and weaker inter-monomeric interactions compared to CDL-bound VsSemiSWEET (Fig. 6b). Moreover, the RMSD distribution further confirms that the CDL-bound system stabilizes a dimer conformation closely matching the ssNMR-resolved structure of VsSemiSWEET, whereas the PG-bound dimer is more heterogeneous (Fig. S33). These findings align with ssNMR data (Fig. 5f, g), which indicate greater dynamics and heterogeneity in the PG-bound dimer. Furthermore, both MD simulation and experimental findings demonstrate that PE exerts regulatory effects similar to PG, promoting dimerization but also inducing conformational heterogeneity and instability in the resulting dimers (Fig. S34).

The regulatory effects of different types of lipids on the function of VsSemiSWEET was characterized by MD simulations. As proposed by Feng et al., sugar transport by SemiSWEETs follows a “free ride” mechanism23. This mechanism involves spontaneous transitions between outward-open, occluded, and inward-open configurations, facilitating sugar passage from extracellular to intracellular environments. Our analysis of the free energy landscape identified key functional states in CDL-bound VsSemiSWEET: outward-open (states 1 and 2), occluded (states 3 and 4), and inward-open (state 5) structures (Fig. 6c). The outward‑open states correspond to the lowest free‑energy minima and are thus the most populated, whereas the inward‑open state lies at a significantly higher energy and is scarcely populated. This is consistent with our ssNMR findings, wherein inward-open structures were undetected. These results indicate that CDL binding enables VsSemiSWEET to transition between essential functional states, enhancing its activity via the “free ride” mechanism. In stark contrast, the free energy landscape of PG‑bound VsSemiSWEET shows no such conformational transitions (Fig. 6d). The lowest free energy minimum for PG-bound VsSemiSWEET corresponds to a nonfunctional state with an intramembrane cavity filled by PG tails. This suggests that PG creates steric hindrances that not only obstruct ligand passage but also impede the transition cycle between functional states.

The gating mechanism of VsSemiSWEET differs significantly from those observed in its homolog, L. biflexa SemiSWEET (LbSemiSWEET)23,25. In LbSemiSWEET, Feng et al. proposed that TM1 undergoes kinking at the central proline residue, disrupting interactions with TM3 and facilitating conformational transitions between outward-open, occluded, and inward-open states. In VsSemiSWEET, however, TM2 serves as the primary driver of structural transitions between closed and open states at both extracellular and intracellular gates (Fig. 6e). The rocking motion of TM2 in opposite directions across the two protomers of the VsSemiSWEET dimer controls the opening of both the outward and inward gates (Fig. S35). This motion is closely linked to lipid interactions, as shown by our analysis of residue–lipid interactions in all-atom MD simulations. The results reveal extensive interactions between TM2 and CDL (Fig. 6f, g), where the hydrophobic tails of CDL interact with the outer surface of TM2, modulating helical orientational fluctuations. Experimental evidence supports this mechanism: residue L46 in the TM2 region, which exhibits the highest probability of contact with CDL, shows significant CSPs in sample II (lacking CDL) compared to sample I (Fig. 5g). Together, these findings underscore a critical and specific role for CDL in enabling the functional transition cycle of VsSemiSWEET.

Methods
Sample preparation for solid-state NMR
VsSemiSWEET proteoliposome sample. Expression and purification. VsSemiSWEET was expressed in E. coli BL21 (DE3) cells using a pET28a-VsSemiSWEET plasmid with an N-terminal 6×His tag. Colonies of VsSemiSWEET were inoculated into 100 mL of LB media containing kanamycin (100 μg/mL) and grown overnight at 310 K. Then, 20 mL of cell suspension was diluted into 1 L of fresh LB media and allowed to grow at 310 K. When the OD600 reached 1.0−1.1, the cells were harvested by centrifugation and resuspended in 250 mL of M9 media (4 g/L 13C glucose, 1 g/L 15N NH4Cl) and incubated at 310 K for 30 min. Notably, the 2-VsSemiSWEET and 1,3-VsSemiSWEET samples were prepared by substituting glucose in the M9 medium with the corresponding 2-13C– and 1,3-13C–labeled glycerol, respectively. Next, 1 mM IPTG (isopropyl-β-D-thiogalactoside) was added to induce protein expression at 310 K. After 5 h, the cells were collected by centrifugation and resuspended in lysis buffer (20 mM Tris, 100 mM NaCl, pH 8.0), followed by ultrasonication until the solution became clear. The lysate was then subjected to low-speed centrifugation at 4629 × g for 10 min to remove unbroken cells and larger debris. Total cell membranes were collected by high-speed centrifugation at 58,545 × g for 1 h. The membranes were dissolved overnight in 1% (w/v) sodium N-lauroylsarcosinate (NLS) at 277 K. VsSemiSWEET was then purified using 2 mL of Ni-IDA resin, with 10 column volumes (CVs) of wash buffer (20 mM Tris, 100 mM NaCl, 20 mM imidazole, 0.2% (w/v) DM, pH 8.0), followed by 5 CVs of elution buffer (20 mM Tris, 100 mM NaCl, 300 mM imidazole, 0.3% DM, pH 8.0). Approximately 30−35 mg of VsSemiSWEET was purified from 1 L of M9 culture.

Reconstitution. The purified VsSemiSWEET was reconstituted into E. coli liposomes through a dialysis process. A 5 mg/mL stock of E. coli lipids was prepared by hydration and ultrasonication. The lipids were dissolved in 2% (w/v) octyl glucoside (OG) and mixed with the purified VsSemiSWEET at a protein-to-lipid ratio of 1:0.6 (w/w). The mixture was incubated at 291 K for 2 h. Subsequently, the mixture underwent dialysis at 291 K for 6 days against an external solution (20 mM Tris, pH 8.0). The VsSemiSWEET protein−liposomes were collected through ultracentrifugation at 419,832 × g for 3 h, followed by freeze-drying. The dried complexes were then rehydrated with 30% (w/w) water and packed into a 3.2 mm rotor (thin-wall) for solid-state NMR experiments.

VsSemiSWEET cellular membrane sample. Expression of VsSemiSWEET. The method used for VsSemiSWEET expression was similar to that described in the preparation of the VsSemiSWEET proteoliposome sample. However, the OD600 value of the cells before switching from unlabeled to isotopically enriched media was adjusted from 1.0−1.1 to 1.3−1.4, and the incubation time prior to adding IPTG was reduced from 30 to 10 min.

Cellular membrane isolation. The cell membranes obtained from 250 mL of culture of isotope-labeled cells were resuspended in lysis buffer (20 mM Tris, 100 mM NaCl, pH 8.0) and sonicated until the solution became clear. To separate the inner and outer membrane fractions, sucrose gradient centrifugation was employed. Specifically, 2.7 mL of 60% (w/v) sucrose, 3.5 mL of 51% (w/v) sucrose, 3 mL of 35% (w/v) sucrose, and 4 mL of the sample were sequentially added to the buffer system (20 mM Tris, 100 mM NaCl, pH 8.0). The gradients were centrifuged in an SW-40 Ti rotor (Beckman) at 169,818 × g for 16 h. The inner membranes were harvested from the interface between the 35 and 51% sucrose layers using a syringe, while the outer membranes were collected from the interface between the 51 and 60% sucrose layers. For the inner membrane sample, 2 mL was diluted with water to fill an 8.9 mL Beckman centrifuge tube, which was then centrifuged at 419,832 × g for 3 h to collect the inner membranes. After freeze-drying, the inner membranes were rehydrated with 30% (w/w) water and packed into a 3.2 mm thin-wall rotor for solid-state NMR experiments.

13C-labeled total E. coli lipid extract. To assign the chemical shifts of the E. coli lipids, we prepared a sample of 13C-labeled extracts from the total E. coli lipids60. Briefly, E. coli BL21 (DE3) cells were cultured in M9 medium, using ¹³C glucose as the sole carbon source. After an overnight culture, the cells were harvested by centrifugation at 6000 × g for 15 min, yielding a wet mass of 15–20 g from 1 L of M9 culture. The cell pellet (10 mg) was resuspended in 20 mL of chloroform and 40 mL of methanol, followed by filtration. Subsequently, 20 mL of chloroform and 20 mL of distilled water were added. The mixture was transferred to a separator funnel and allowed to stand at 277 K for 24 h. The 13C-labeled E. coli total lipids were collected from the lower chloroform phase.

Unfolding and refolding approach for VsSemiSWEET. Unfolding of VsSemiSWEET. The method used for VsSemiSWEET expression was similar to that described in the preparation of the VsSemiSWEET proteoliposome samples. The obtained 250 mL of cells were collected by centrifugation and resuspended in lysis buffer (20 mM Tris, 100 mM NaCl, pH 8.0), followed by ultrasonication until the solution became clear. The lysate was then subjected to low-speed centrifugation at 4629 × g for 10 min to remove unbroken cells and larger debris. Total cell membranes were collected by high-speed centrifugation at 58,545 × g for 1 h. The membranes were dissolved overnight in 1% (w/v) NLS at 277 K. VsSemiSWEET was subsequently purified using 2 mL of Ni-IDA resin, followed by sequential washing steps with 15 mL of buffer A (20 mM Tris, 100 mM NaCl, 0.2% (w/v) NLS, pH 8.0), and 10 mL of buffer B (8 M Urea, 150 mM NaCl, and lysis buffer in a 1:1 volume ratio) to initiate the unfolding of VsSemiSWEET. Further denaturation of the protein was achieved by treating it with 140 mL of Buffer C (8 M Urea and 150 mM NaCl). Finally, to obtain a fully denatured VsSemiSWEET solution, 10 mL of Buffer D (8 M Urea, 150 mM NaCl, 1% formic acid, and 0.2% SDS) was added, and the mixture was left in a 298 K incubator overnight.

Refolding of VsSemiSWEET. An appropriate volume of 2 M Tris solution (pH 8.0) was added to the denatured VsSemiSWEET solution to achieve a final Tris concentration of 50 mM, enhancing its buffering capacity. Next, NaOH solution was gradually added to raise the pH of the protein solution from 2.8 to ~8.0. The lipids were dissolved in 2% (w/v) OG and mixed with the purified VsSemiSWEET at a protein-to-lipid ratio of 1:1 (w/w). The mixture was then incubated overnight at 310 K.

Delipidation of VsSemiSWEET. Detergent washing was employed to remove lipids loosely bound to VsSemiSWEET. In the purification of VsSemiSWEET, the membranes were dissolved overnight in 1% (w/v) NLS at 277 K. VsSemiSWEET was then purified using 2 mL of Ni-IDA resin and subjected to washing with 15 mL of wash buffer (20 mM Tris, 100 mM NaCl, 20 mM imidazole, 0.2% (w/v) DM, pH 8.0). After washing with buffer containing 1% DDM, VsSemiSWEET was eluted in an elution buffer (20 mM Tris, 100 mM NaCl, 300 mM imidazole, 0.3% DM, pH 8.0), and concentrated to ~0.5 mg/mL.

H/D exchange of VsSemiSWEET proteoliposome. The liposome pellets of VsSemiSWEET were suspended in D2O and incubated with slow shaking at room temperature for 72 h. Subsequently, the pellets were collected through ultracentrifugation at 548,352 × g for 2 h. The pellets were freeze-dried to remove moisture and then rehydrated with 30% D2O for subsequent solid-state NMR experiments.

Solid-state NMR experiments
All solid-state NMR experiments were performed at 272 K on an 800 MHz Bruker Avance spectrometer equipped with a 3.2 mm three-channel E-free T3-HXY MAS probe, at a MAS frequency of 10.5 kHz. The typical 90° pulse length for 1H was 3.1 μs, and the 90° pulse lengths for 13C and 15N were 4.5 and 6.6 μs, respectively. The chemical shifts of 13C and 15N were indirectly calibrated using adamantane as an external referencing standard (40.48 ppm for the downfield carbon). During the acquisition and indirect chemical shift evolution process, SPINAL-64 decoupling on the 1H channel was set to 70−75 kHz. In the 2D and 3D solid-state NMR experiments, SPECIFIC CP61 was employed in polarization transfer between 13C and 15N, and 13C-13C correlations were established through dipolar-assisted rotational resonance (DARR) recoupling62. The 2D 13C-13C CORD experiments of 2-VsSemiSWEET and 1,3-VsSemiSWEET samples were used to acquire 13C-13C distance restraints. The 2D 1H-13C INEPT experiment was performed at 298 K, with experimental conditions were optimized for a 1JCH coupling constant of 225 Hz. All solid-state NMR experimental data were processed using NMRPipe software63 and analyzed using Sparky. Detailed parameters for these ssNMR experiments are compiled in Table S5.

Solution NMR experiments
For the 31P NMR study, approximately 0.15 mM of VsSemiSWEET was solubilized in DM in a pH 8.0 buffer (20 mM Tris, 100 mM NaCl, 30 mM imidazole, 0.3% DM, 10% D2O, pH 8.0). 31P solution-state NMR spectra were collected on a Bruker Avance III spectrometer operating at a magnetic field strength of 11.7 T, corresponding to a proton Larmor frequency of 500 MHz. The spectrometer was equipped with a 5 mm CPPBBO 500 S2 BB-H&F-D-05 Z probe and analyzed by TopSpin 4.0.7. The sample temperature was maintained at 315 K.

Extraction of structural restraints
Dihedral angle restraints. Dihedral angles were predicted using TALOS+ based on the N, CO, Cα, and Cβ chemical shifts64. The backbone chemical shifts were directly adopted from BMRB entry 51331, as the sample preparation protocol and resulting 2D 15N-13Cα and 13C-13C spectra were identical to those in our prior study33. Only angles classified as “good” by TALOS+ were used as dihedral angle restraints, with the program providing error values.

Hydrogen bond restraints. Intrahelical hydrogen bond restraints were applied when both the chemical shift index65 and TALOS+ indicated helical structures for the corresponding residues. The intrahelical hydrogen bond restraint was defined as the distance between the donor oxygen (Oi) and the acceptor nitrogen (Ni+4), and it was set within the range of 1.5 to 3.5 Å.

13C-13C distance restraints. 13C-13C distance restraints were derived from 2D 13C-13C CORD spectra with a mixing time of 500 ms for 2-VsSemiSWEET and 1,3-VsSemiSWEET samples. The signals corresponding to 13C-13C distance restraints were assigned based on the following criteria: (1) The peaks that could be assigned as 13C-13C distance restraints exhibited symmetry, were well-resolved, and had a sufficient signal-to-noise ratio (SNR ≥ 6) in the 2D CORD spectra. (2) All possible assignments of the cross peaks were collected. A tolerance of approximately 0.2 ppm was set for both direct and indirect dimensional 13C chemical shifts, considering the linewidth of the peaks (0.2 to 0.6 ppm) in the CORD spectra. (3) Ambiguity in the assignments was reduced through the following procedures: (i) The ambiguity in the assignment of intra-monomer correlations was notably reduced by utilizing the VsSemiSWEET monomer structure predicted by CS-Rosetta based on the assigned chemical shifts. (ii) If a peak could not be assigned as an intra-monomer correlation, it was classified as a possible inter-monomer correlation. Moreover, the assignments of inter-monomer correlations were confirmed based on the assignment of the long-range inter-monomer correlations.

VsSemiSWEET dimer structure determination in lipid bilayers
Step 1: Monomer VsSemiSWEET structure predicted by CS-Rosetta. The initial monomeric structure of VsSemiSWEET was predicted using the standard POMONA/CS-RosettaCM protocol from the CS-Rosetta package66,67. A set of 80 backbone 15N, 13Cα, 13CO, and 72 13Cβ chemical shifts were utilized as input for the fragment and template selection process. During the subsequent structure generation, only fragments and templates derived from PDB proteins with less than 30% sequence similarity to SemiSWEETs were employed. Additionally, 28 ambiguous long-range intra-monomer distance restraints, determined based on chemical shift specificity, were incorporated for structural prediction. A total of 10,000 all-atom models were generated, from which the ten lowest-energy models were selected as the initial monomeric structures of VsSemiSWEET for further analysis. The RMSD of the backbone heavy atoms for the ten lowest-energy models was 0.9±0.2 Å.

Step 2: Assignments of intra-monomer distance constraints. In the 2D 13C-13C CORD spectra of 2-VsSemiSWEET and 1,3-VsSemiSWEET samples, only isolated peaks with diagonal symmetry, suitable linewidths (0.2 to 0.6 ppm), and sufficient signal-to-noise ratios (SNR ≥ 6) were assigned. To reduce ambiguity in the assignment of intra-monomer distance constraints, the monomer structures of VsSemiSWEET predicted by CS-Rosetta were utilized. In this step, possible assignments involving intra-monomer distances for correlated atoms exceeding an upper limit of 9.0 Å in the monomer structure were excluded.

Step 3: Monomer VsSemiSWEET structure determination by Xplor-NIH. The VsSemiSWEET monomer structures were calculated by using the Xplor-NIH program (version 2.47)68. Standard terms for bond lengths, bond angles, and improper angles were employed to enforce correct covalent geometry. Statistical torsion angle potentials and gyration volume terms were applied, and the hydrogen bond database term HBPot was used to improve hydrogen bond geometry. Folding calculations for full-length single-stranded VsSemiSWEET were derived from first-order sequence extensions of the chain. A total of 522 structures were generated using molecular dynamics simulated annealing in torsion angle space. The calculations involved two consecutive annealing schedules followed by final gradient minimization in Cartesian space. Each VsSemiSWEET structure contained 344 non-redundant 13C-13C distance constraints, 88 hydrogen bond constraints, and 78 torsion angle constraints derived from TALOS-N. The structural calculations began with constant temperature molecular dynamics at 3500 K, running for 800 ps or 10,000 steps, with the time step adjusted to maintain constant energy within tolerance. The temperature was gradually reduced in steps of 25 K. At each temperature, the kinetics were simulated for 2 ps or 500 steps, whichever was shorter. The force constant for the distance constraint was increased from 5 to 50 kcal mol−1 Å−2. During high-temperature kinetics at 3500 K, the dihedral angle constraint was disabled, but was enabled during simulated annealing with a force constant of 200 kcal mol−1 rad−2. The slew volume force constant was geometrically extended from 0.002 to 1. After simulated annealing, the structure underwent energy minimization using Powell’s energy minimization scheme. This methodology resulted in 200 structures, and the first 10 structures with the lowest energy exhibited an RMSD of 1.5±0.4 Å for backbone atoms.

Step 4: Initial dimer structure of VsSemiSWEET generated by Xplor-NIH docking. The initial dimer structure of VsSemiSWEET in lipid bilayers was calculated using the docking module in Xplor-NIH, combined with six ambiguous inter-monomer distance constraints. The assignments of these inter-monomer correlations were confirmed by analyzing the corresponding signal intensities observed in the 2D 13C-13C spectra recorded on the 2-VsSemiSWEET and [(1:2) 2-13C/NA] VsSemiSWEET samples. Furthermore, these correlations could not be assigned as intra-monomer correlations based on the analysis of VsSemiSWEET monomer structure. The PDB Editor tool was employed to create the initial structure for dimer docking, positioning the lowest-energy VsSemiSWEET monomer structure at two fixed points approximately 100 Å apart in a straight line. The ambiguous inter-monomer distance constraints were applied to the inter-monomer interface of the VsSemiSWEET dimer, with force constants ranging from 20 to 100 kcal mol−1 Å−2. Non-crystalline restraints were used to ensure that both monomers in the calculation were identical. This potential allowed the internal coordinates of the VsSemiSWEET monomers to remain fixed during the docking process, enabling changes in the orientation of the monomers while preserving their internal structure. During the annealing process, the hydrogen bond database potential term HBDB was used. Standard bond angles and lengths were set using the terms BOND, ANGL, and IMPR. van der Waals forces were applied with force constants ranging from 0.05 to 5.0 kcal mol−1 Å−2. Torsion angle kinetics were performed for 5000 steps at 3500 K, followed by annealing to 25 K in increments of 12.5 K for 100 steps at each temperature. After annealing, final energy minimization of the torsion angles and Cartesian coordinates was conducted. The resulting structures were sorted by energy, considering all potential energies determined from the calculations. From the final set of 100 structures, the ten structures with the lowest energy were selected for subsequent analysis.

Step 5: Assignment of the inter-monomer distance constraints. The ten initial VsSemiSWEET dimer structures obtained through docking were used to identify inter-monomer distance constraints. The assignment of inter-monomer correlations in the 2D CORD spectra followed the following criteria: (I) Only the peaks that could not be assigned to intra-monomer contacts and that exhibited a noticeable decrease in intensity (>1/3) or even disappearance in the spectra of the [(1:2) 2-13C/NA] VsSemiSWEET sample, compared to the corresponding peaks in the spectra of the 2-VsSemiSWEET sample, were assigned as unambiguous inter-monomer distance constraints. (II) Only peaks with diagonal symmetry, suitable linewidths (0.2 to 0.6 ppm), and sufficient SNR (≥ 6) in the CORD spectra were assigned. (III) If a peak satisfies both intra-monomer and inter-monomer distance constraints, and the signal intensity in the spectrum of the [(1:2) 2-13C/NA] VsSemiSWEET sample is significantly reduced or even disappears, it was considered an ambiguous inter-monomer constraint; otherwise, it was regarded as an intra-monomer constraint.

Step 6: Calculation of the VsSemiSWEET dimer structure by Xplor-NIH. The folding calculations for the full-length single-stranded VsSemiSWEET were extrapolated from first-order sequence extensions of the chain. The two monomers were constrained to maintain identical structures during the annealing process through the non-crystallographic symmetry term PosDiffPot and the translational symmetry term DistSymmPot. A total of 1000 structures were de novo generated through molecular dynamics simulated annealing in torsion angle space with two consecutive annealing schedules, followed by final gradient minimization in Cartesian space. The structure calculation utilized the obtained intra-monomer and inter-monomer distance restraints derived from NMR spectra, dihedral angle restraints, and intrahelical hydrogen bonds.

The structural calculations began with constant temperature molecular dynamics at 3500 K, running for 800 ps or 10,000 steps, with the time step allowed to adjust to maintain constant energy within tolerance. The temperature was gradually reduced in steps of 25 K. At each temperature, the kinetics were simulated for 2 ps or 500 steps, whichever was shorter. The force constant for the distance constraint was increased from 1 to 300 kcal mol−1 Å−2. During high-temperature kinetics at 3500 K, the dihedral angle constraint was disabled but enabled during simulated annealing with a force constant of 300 kcal mol−1 rad−2. The slew volume force constant was geometrically extended from 0.002 to 1. After simulated annealing, the structure underwent energy minimization using Powell’s energy minimization scheme. This methodology resulted in 1024 structures, and the first 10 structures with the lowest energy exhibited an RMSD of 0.9±0.3 Å for backbone atoms.

Molecular dynamics simulations
The initial structure of VsSemiSWEET in MD simulations is based on the solid-state NMR structure reconstituted in E. coli liposome. The membrane bilayer consists of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), and cardiolipin (CDL). The lipid molar ratio is POPE:POPG:CDL = 75:13:12, which mimics the lipid composition found in E. coli membranes69. The total number of lipids in the membrane model was 300, composed of 224 POPE, 40 POPG, and 36 CDL molecules, reflecting a physiologically relevant ratio. For the conventional MD (cMD) simulations used to analyze lipid residence times around VsSemiSWEET, three independent replicas of 3 μs each were carried out per system. The reported lipid residence times are averages derived from these replica simulations. For the PG-bound system, four additional POPG lipids were docked to the binding sites of VsSemiSWEET using MOE70. In the CDL-bound system, two CDL molecules, each containing two phosphate groups, were docked to the binding sites of VsSemiSWEET. The membrane bilayer model and protein insertion were accomplished using the CHARMM-GUI web server71. The CHARMM36 force fields were utilized for both proteins and membranes72, while the TIP3P model73 was adopted for water molecules.

The systems underwent energy minimization for 5000 steps using the steepest descent method. During these minimization steps, proteins and lipids were restrained with force constants of 10 kJ/mol×Å² and 2.5 kJ/mol×Å², respectively. NVT equilibration steps, constrained to the positions of protein-heavy atoms and lipids, were conducted for 500 ps. Subsequently, several 1-ns NPT simulations were performed, during which position constraints on the protein and lipid heavy atoms were gradually reduced to equilibrate the systems. The LINCS algorithm50 was used to constrain the bond lengths involving hydrogen atoms. A semi-isotropic scheme was employed to independently couple the lateral and perpendicular pressures. Long-range electrostatics were calculated using the Particle-Mesh Ewald method74 with a cutoff distance of 12 Å. The temperature was maintained at 300 K using the V-rescale method75, and pressure was controlled by the Parrinello-Rahman barostat76. Production runs were executed with a 2-fs time step using the CUDA-accelerated version of the AMBER suite package77.

Free energy simulations were performed using well-tempered metadynamics simulations78,79. Two collective variables (CV), dout and din, were employed to characterize the opening of the extracellular and intracellular gates on VsSemiSWEET. dout calculated the distances between Cα atom on residue Y62 in each protomer, while din calculated the distances between the Cα atoms of residues L42–L44 in each protomer. The bias factor γ, which controls the added bias in a history-dependent manner, was set to 32. The height of the initial bias energy was set at 1.0 kJ mol−1 and the width was set at 0.5 Å. The bias was added every 2 ps. The GROMACS 2022 software80 with the plumed 2.8 plugin was utilized to perform the metadynamics simulations. The simulation details, including the number of atoms, number of water molecules, box sizes, salt concentration, and lipid compositions, are summarized in Table S6. The convergence tests of simulations are analyzed based on the free energy surface deviation along the simulation time and are given in Fig. S37.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Yan et al. employed ssNMR and molecular simulations to investigate how different interfacial lipids regulate SemiSweet. They first identified PG, PE, and CDL as co-purified lipids associated with SemiSweet and characterized their specific binding interactions. Their findings indicate that CDL promotes dimer formation and induces conformational changes, while PG tails bind to the interfacial pore, preventing substrate transport. This study presents an interesting discovery that different lipids regulate distinct functional conformations of SemiSweet. Overall, the manuscript is well-written and well-structured. However, I have some concerns that should be addressed.\nMajor Comments:\n1. The authors identified the co-purified lipids by comparing the chemical shifts of purified 13C-labeled protein with those of de-lipidated reconstituted protein supplemented with 13C-unlabeled lipid extracts. Have the authors attempted to confirm these findings by adding 13C-labeled E. coli lipid extract to the de-lipidated reconstituted protein and checking for the reappearance of peaks?\n2. Binding of CDL and PG: The authors demonstrated that CDL acyl tails do not occupy the interfacial cavity, whereas PG does. Could the authors model a single CDL molecule in place of two PG molecules? A structural figure showing that the hydrophobic tail of CDL is too large to fit into the cavity would be helpful.\n3. Figure 5: The authors performed ssNMR on SemiSweet in PE/PG/CDL and PE/PG environments, showing that CDL binds preferentially to the interfacial cavity. Could CDL and PG bind SemiSweet simultaneously? It is difficult to determine this from Figure 5c,d.\n4. Role of PE: The authors identified PG, PE, and CDL as co-purified lipids, yet they barely discuss the function of PE in the NMR experiments or molecular dynamics (MD) simulations. Could the authors clarify whether PE has a regulatory role?\n5. The authors claim that PG-bound SemiSweet obstructs ligand passage and impedes the functional transition cycle. To support this claim, it would be helpful to include in vitro transport assays comparing PG- and CDL-bound proteins.\nMinor Comments:\n1. The chemical structure of PG is fully saturated, while CDL contains unsaturated tails. Additionally, POPG is more abundant than PSPG.\n2. Figures 5c and 5d: These structural views are shown from different perspectives, making direct comparison difficult. Same or similar viewing angles through the manuscript would improve clarity.\n3. Figure 3f: What does the arrow indicate? A brief explanation in the legend would be helpful.\n4. Figure 3g: The yellow CDL molecule appears to block the cavity, which seems different from the positioning shown in Figure 5d. Could the authors clarify this discrepancy? Using consistent perspectives for SemiSweet structures would aid comparison.\n5. Figure 6e: Helix H2 in the closed state is colored differently from the yellowish box. This inconsistency should be addressed.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "This study by Jun Yang and coworkers is very complete, it presents the structural and dynamics characterization of a bacterial sugar transporter. The authors used numerous biochemical tricks to detect, identify and cross-validate specific lipid-protein interactions. The work is very well executed and the experimental evidence of specific lipid interactions, based on 31P solution NMR and 13C-13C lipid-protein restraints, is solid. Although the 3D structure determination is not de novo but based on a CS-ROSETTA model, it provides compelling evidence of the protein conformation in the relevant lipid environment. The strong point of this study is the methodology, it could be applied to other membrane protein systems of interest to decipher specific lipid-protein interaction using the combined MAS NMR / MD approach.\nI have several comments that need to be addressed:\nMain comments:\n. The main weakness of the study is its relevance to the biological system. What do we learn about sugar transport mechanism?\n. There are no functional assays in the study, so there is a risk that the protein-membrane reconstitution leads to a non- functional system (i.e. not a system able to transport molecules across the membrane). Maybe the authors have further evidence to support the functionality of the protein in this particular membrane environment that has been set up for MAS NMR studies?\nOther points:\n. the description of the membrane protein VcSemiSWEET at the beginning of the manuscript is very short. Please add more on the biological context of these systems, i.e. the role in sugar transport\n. the protein system is from Vibrio. Please provide the lipid composition of native vibrio membranes and a relevant reference to support it. What’s the native molar ratio of cardiolipin compared to PG and PE in Vibrio? Ref 36 is cited but this work was not done on Vibrio species.\n. in the introduction: a short description of previous work on the high-resolution structure, as well as the presence of distinct states (open outside, occluded, open inside) would be necessary. These details only come page 16, please mention them already in the introduction to help the readers to understand the structural basis on this system.\n. in the 1D 31P spectrum (Fig. 1C), what is the contribution observed at 0.11ppm?\n. Line 175: please cite the seminal work of Oschkinat and coworkers (Castellani Nature 2002) when describing the use of 13C spin dilution (both for the use of selectively labeled glycerol carbon sources and the use of a 50/50 diluted sample to discard intermolecular contacts) to obtain distance restraints by MAS NMR.\n. Line 174: replace “truncation” by “dipolar truncation”. A relevant reference (e.g. pubmed.ncbi.nlm.nih.gov/19317544/) should be cited here for non-NMR readers when mentioning the dipolar truncation effect.\n. Line 187: the authors used CS-ROSETTA, why not AF3? The manuscript should provide an AF3 prediction of the protein.\n. Line 189: unclear what means “chemical shift specificity”.\n. Line 207: intramonomeric restraints are excluded because they are >9A in the CS-ROSETTA model? if yes, please add this detail (i.e. the upper distance of 9A) in the main text.\n. Line 241: “but with empty coordinate without distance restraints”: not clear if lipids were used or not in the final Xplor refinement.\n. Supp fig. 13: it might be more relevant to compare the crystal structure to the bundle of NMR structures (10 lowest energy) and not only to one NMR conformer.\n. Line 285: can the authors elaborate on the precise time scale of lipid-protein interactions, based on the fact that lipid-protein contacts are only observed in CP-based experiments?\n. 31P solution NMR experiments were performed at 315K, why this temperature and not 310K?\n. All MAS NMR experiments were performed at 272K, I guess to improve CP-based polarization transfer. However, this relatively low sample temperature might have considerably rigidified lipids, enhancing lipid-protein proximities. Can the authors comment of this aspect?\n. The authors assume that the protein form a homodimer in membranes, is this assumption based on experimental data?\n. There is one Cysteine in the main helical core. Do the authors exclude the presence of intermolecular disulphide bridges?\n. Line 175: please define what is “medium-“ and “long-range” distances for non-NMR readers\n. Line 177: I would suggest to replace “13C-unlabeled lipids” simply by “unlabeled lipids”.\n. Line 137: Long mixing recoupling period (500ms CORD mixing) is used to enhance the signals of bound lipids. Please explain why such long mixing help to observe lipid signals.\n. Fig4e (the contact map) is small and it's difficult to read it, it could move to the SI.", "reviewer_4": "This manuscript, in its current form, is quite difficult to navigate.\nThe significance of the study is not clearly articulated - what makes this research important, and what are the key takeaways?\nAdditionally, the relevance of this transporter's dimerisation needs further clarification.\nThe figures are presented in a way that makes interpretation challenging, leaving the reader uncertain about the core messages they should extract.\nGiven these issues, this work may be better suited for a more specialised journal where the intended audience can fully appreciate its technical depth."}, "author_response": "Response to reviewers\nWe thank the reviewers for carefully reading our manuscript and providing helpful comments on its clarity and presentation. We have addressed each comment as indicated below:\nIn response to reviewer 1:\nIn this manuscript, Yan et al. employed ssNMR and molecular simulations to investigate how different interfacial lipids regulate SemiSweet. They first identified PG, PE, and CDL as co-purified lipids associated with SemiSweet and characterized their specific binding interactions. Their findings indicate that CDL promotes dimer formation and induces conformational changes, while PG tails bind to the interfacial pore, preventing substrate transport. This study presents an interesting discovery that different lipids regulate distinct functional conformations of SemiSweet. Overall, the manuscript is well-written and well-structured. However, I have some concerns that should be addressed.\nMajor Comments:\n1. The authors identified the co-purified lipids by comparing the chemical shifts of purified 13C-labeled protein with those of de-lipidated reconstituted protein supplemented with 13C-unlabeled lipid extracts. Have the authors attempted to confirm these findings by adding 13C-labeled E. coli lipid extract to the de-lipidated reconstituted protein and checking for the reappearance of peaks?\nThank you for the professional suggestion. We indeed considered confirming the co-purified lipids by adding 13C-labeled E. coli lipid extract to the de-lipidated reconstituted protein and checking for the reappearance of peaks. However, this approach would also introduce 13C-labeled lipids into the lipid bilayers, preventing the determination of whether the re-emerging signals originate from lipids in the bilayers or from bound lipids.\nTherefore, we identified the co-purified lipid signals based on the following points:\n1). Both the 13C and 31P chemical shifts of these signals are consistent with the characteristic chemical shifts of lipid headgroup and the glycerol backbone (Figure 1b and 1c). 2). All Ser and Thr residues in VsSemiSWEET, which are the only amino acids exhibiting intra-residual correlations in this region, have been unambiguously assigned (Figure 1a). 3). Following the unfolding-refolding steps, these signals were either entirely absent or significantly weaker, ruling out the possibility that these correlations originated from the protein (Figure 1a). 4). The E. coli total lipid extracts used in the liposomes were unlabeled, meaning their correlated signals cannot appear in the 2D 13C-13C spectrum.\nThese observations collectively confirm that the identified signals arise specifically from co-purified lipids rather than protein or environment-derived lipid signals.\n2. Binding of CDL and PG: The authors demonstrated that CDL acyl tails do not occupy the interfacial cavity, whereas PG does. Could the authors model a single CDL molecule in place of two PG molecules? A structural figure showing that the hydrophobic tail of CDL is too large to fit into the cavity would be helpful.\nAs demonstrated in Figure 5, the tail chains of the two CDLs are arranged beside the two monomers of the protein dimer. The distances between the tail chains of the two molecules are large, so they do not interfere with each other's entry into the interfacial cavity of VsSemiSWEET. This arrangement suggests that even if a single CDL were modeled, its hydrophobic tail would be sterically excluded from occupying the interfacial cavity.\nTo further support this conclusion, we provide Figure S26, which illustrates a direct structural comparison between the CDL tail and the cavity dimensions. The data clearly show that the hydrophobic tail of CDL is too large to fit into the cavity.\nSupplementary Figure 26. The comparison of volumes of CDL and the intracellular cavity of VsSemiSWEET. The maximum lateral and vertical dimensions of both the intracellular cavity and the CDL surface are labeled.\n3. Figure5: The authors performed ssNMR on SemiSweet in PE/PG/CDL and PE/PG environments, showing that CDL binds preferentially to the interfacial cavity. Could CDL and PG bind SemiSweet simultaneously? It is difficult to determine this from Figure 5c,d.\nWe appreciate the thoughtful consideration of this question. Based on experimental results and binding energy analysis, CDL exhibits significantly stronger binding free energy compared to PG and PE near the considered binding sites. In the analysis of MD simulations, the binding ability of CDL to VsSemiSWEET is approximately 215-fold higher than that of PG and about 3600-fold higher than that of PE, indicating that PG can hardly compete with CDL for binding to VsSemiSWEET, and it is difficult for PG to simultaneous binding with CDL. The experimental results validate the MD simulation analysis. Without CDL, the structure of VsSemiSWEET bound with PG and PE differs from that when it binds with CDL, PE, and PG (Figure 5), highlighting the dominant role of CDL in maintaining the structural integrity of VsSemiSWEET.\nThought above results supporting no simultaneous binding of CDL and PG to VsSemiSWEET, we still cannot completely rule out the possibility of simultaneous binding of CDL and PG to VsSemiSWEET. The actual lipid interactions are likely more complex in physiological conditions, as discussed in our previous manuscript:\nWhile we have demonstrated the binding modes of individual lipids, such as PG and CDL, to VsSemiSWEET, the actual binding interactions are likely more complex. Based on both ssNMR and solution NMR experimental results, it is plausible that the binding involves a combination of two or more different types of lipids at the interface of the monomers. This complexity warrants further investigation to fully understand the lipid interactions with VsSemiSWEET.\n4. Role of PE: The authors identified PG, PE, and CDL as co-purified lipids, yet they barely discuss the function of PE in the NMR experiments or molecular dynamics (MD) simulations. Could the authors clarify whether PE has a regulatory role?\nThank you for your comment. As mentioned above, the binding ability of CDL to VsSemiSWEET is approximately 215-fold higher than that of PG and about 3600-fold higher than that of PE. Therefore, we did not consider the regulatory role of PE because of its much weaker binding ability than that of PG in the previous manuscript. To response to the comment of the reviewer, we conducted additional experiments and simulations to delineate functional impact of PE. We prepared an additional sample IV, in which co-purified lipids were removed in the unfolding-refolding procedure, and VsSemiSWEET was bound with PE and reconstituted into pure POPE lipids. In contrast, the co-purified lipids are present in sample III without the unfolding-refolding procedure (Figure S30a).\nSDS-PAGE analysis indicated that PE facilitates the formation of dimers from fully denatured VsSemiSWEET in sample IV (Figure S30b). The superimposition of the 2D 15N-13Cα spectra for VsSemiSWEET in samples IV and III revealed negligible chemical shift perturbations (CSPs), indicating the similar overall structures of VsSemiSWEET in both samples. However, comparison of the 2D 15N-13Cα spectra showed broader linewidths and reduced signal intensities for PE-bound VsSemiSWEET compared to CDL-bound VsSemiSWEET (Figure S30c). These observations indicate that PE induces conformational heterogeneity and instability in VsSemiSWEET dimers, similar to that of PG lipids (Figure 5). As shown in Figure S30d, MD simulations further demonstrate that the tail groups of PE molecules penetrate into the central cavity at the dimer interface, a behavior analogous to PG lipids. This interaction inhibits structural transitions between functional states of VsSemiSWEET.\nIn summary, PE can promote dimerization of VsSemiSWEET, its regulatory role is similar to those of PG but with a weaker binding affinity and a less pronounced regulatory effect. Therefore, our manuscript primarily focuses on the distinct regulatory roles of PG and CDL binding on VsSemiSWEET’s structure and function.\nThese results are now discussed in the revised manuscript:\nFurthermore, our MD simulation and experimental findings demonstrate that the regulatory role of PE is comparable to that of PG. Specifically, PE promotes the dimerization of VsSemiSWEET while inducing conformational heterogeneity and instability in the resulting dimers (Figure S30).\nSupplementary Figure 30. The regulatory role of PE binding in VsSemiSWEET. a, Schematic diagram of the detailed preparation process for sample III and IV. The co-purified lipids were removed from VsSemiSWEET and replaced with PE in sample IV, whereas they remained in sample III. b, SDS-PAGE analysis of the unfolding and refolding of VsSemiSWEET. The presence of VsSemiSWEET monomers formed by unfolding in buffer (pH 2.8), as well as the presence of both VsSemiSWEET monomers and dimers formed by partial refolding and in buffer (pH 8.0), and complete refolding in buffer containing E. coli membrane total extracts and in buffer with POPE lipids, were confirmed by SDS-PAGE. c, Overlay of 2D 15N-13Cα spectra of VsSemiSWEET in sample III (CDL-dominant modulation) and IV (PE-dominant modulation). Typical 1D slices at F54 (highlighted by yellow backgrounds) demonstrate significantly reduced signal intensity and spectral resolution of VsSemiSWEET in Sample IV compared to Sample III, indicating impaired conformational homogeneity and stability of PE-bound VsSemiSWEET. d, FEL of PE-bound VsSemiSWEET as a function of the open sizes of the outward gate (d ) out and inward gate (d ). Representative structures of the states observed on the FEL are shown.\nin\n5. The authors claim that PG-bound SemiSweet obstructs ligand passage and impedes the functional transition cycle. To support this claim, it would be helpful to include in vitro transport assays comparing PG- and CDL-bound proteins.\nWe appreciate the reviewer’s suggestion to include in vitro transport assays comparing PG- and CDL-bound proteins, as this would provide valuable insights into our findings. However, at present, the natural substrate of VsSemiSWEET remains undefined. As reported by Feng et al. (Nature, 2014, 515:448-452), conventional cell-based assays failed to detect significant glucose uptake activity. Structural studies (Feng et al., Cell, 2017, 169:96-107) reveal that SemiSWEET proteins operate via a ‘free-ride’ mechanism, where conformational transitions occur independently of substrate binding. The transporter adopts the same conformations and undergoes the same transitions regardless of substrate presence. Our data demonstrate that PG binding specifically alters the functional transition cycle of VsSemiSWEET. Since ligand passage is directly coupled to these conformational changes, the observed structural perturbations provide compelling evidence that PG binding interferes with the functional transition cycle, thereby disrupting ligand passage in VsSemiSWEET.\nMinor Comments:\n1. The chemical structure of PG is fully saturated, while CDL contains unsaturated tails. Additionally, POPG is more abundant than PSPG.\nThank you for pointing out this oversight. We acknowledge that there was confusion in our previous presentation regarding the chemical structures of PG and CDL. Based on the current experimental data, it is difficult to determine the degree of unsaturation in the lipid tails of bound lipids. Therefore, we have revised the chemical structures shown in the figure by depicting both PG and CDL with saturated acyl chains to avoid any potential misunderstanding and ensure clarity.\n2. Figures 5c and 5d: These structural views are shown from different perspectives, making direct comparison difficult. Same or similar viewing angles through the manuscript would improve clarity.\nWe have revised Figures 5c and 5d as follows:\nFigure 5. The regulatory role of PG and CDL binding in VsSemiSWEET revealed by MD simulations (a, b) and ssNMR experiments (d-g). a, Distinct lipid-binding modes of PG and CDL to VsSemiSWEET analyzed by MD simulations. Left: Residues interacting with lipid headgroups (PG/CDL phosphate groups: orange phosphorus, red oxygen). Right: Residues contacting lipid tails (highlighted in green). Regions with cavity-localized interactions are marked by yellow shading.\n3. Figure 3F: What does the arrow indicate? A brief explanation in the legend would be helpful.\nThe arrow in Figure 3F indicates that the signal intensity of lipid headgroups is much weaker than that of lipid tail carbons in scalar-coupling-based ssNMR spectra. This has been clearly explained in the updated figure legend to enhance clarity and address the reviewer’s comment:\nSuperposition of 1D 1H-13C INEPT spectrum of VsSemiSWEET with bound lipids and that of E. coli lipid extract. The arrow indicates that the signal intensity of lipid headgroups is much weaker than that of lipid tail carbons in scalar-coupling-based ssNMR spectra.\n4. Figure 3g: The yellow CDL molecule appears to block the cavity, which seems different from the positioning shown in Figure 5d. Could you clarify this discrepancy? Using consistent perspectives for SemiSweet structures would aid comparison.\nThe position of CDL in Figure 3g was determined through molecular docking, a computational approach based on the \"lock-and-key principle\" that primarily provides contact information between lipid headgroups and binding sites. However, this method offers limited description of tail chain structures and spatial arrangements.\nIn contrast, MD simulations enable accurate determination of phospholipid molecular conformations and lipid-protein interactions, ultimately yielding the results presented in Figure 5d. We have revised Figure 3g to avoid any potential misunderstanding:\nFigure 3g. Molecular docking results showing the interactions between VsSemiSWEET and CDL molecules.\n5. Figure 6e: Helix H2 in the closed state is colored differently from the yellowish box. This inconsistency should be addressed.\nWe have revised the Figure 6e. Thanks for the valuable suggestion.\nFigure 6e. Representative conformations of the outward-open and inward-open states.\nIn response to reviewer 2:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nThank you for your constructive suggestions.\nIn response to reviewer 3:\nThis study by Jun Yang and coworkers is very complete, it presents the structural and dynamics characterization of a bacterial sugar transporter. The authors used numerous biochemical tricks to detect, identify and cross-validate specific lipid-protein interactions. The work is very well executed and the experimental evidence of specific lipid interactions, based on 31P solution NMR and 13C-13C lipid-protein restraints, is solid. Although the 3D structure determination is not de novo but based on a CS-ROSETTA model, it provides compelling evidence of the protein conformation in the relevant lipid environment. The strong point of this study is the methodology, it could be applied to other membrane protein systems of interest to decipher specific lipid-protein interaction using the combined MAS NMR/MD approach.\nI have several comments that need to be addressed:\nMain comments:\n1. The main weakness of the study is its relevance to the biological system. What do we learn about sugar transport mechanism?\nTo better elucidate the mechanistic insights into sugar transport, we have significantly expanded our discussion on how CDL binding critically regulates both the sugar transport activity and dimerization in VsSemiSWEET.\nHere is the updated text:\nLipid-protein specific interactions play a critical role in regulating the sugar transport activity of SemiSWEETs, which function as sugar transporters by assembling into dimers and cycling through outward-open, occluded, and inward-open conformations. CDL, a unique phospholipid characterized by its dimeric structure with four acyl chains and two phosphate groups, has been shown to play a pivotal role in modulating the oligomerization and conformational dynamics of VsSemiSWEET. The headgroups of CDL molecules bind at the monomer-monomer interface, coordinating the dimerization process of VsSemiSWEET protomers to enable the formation of dimers with a proper and stable conformation that facilitate sugar transport. Furthermore, the four acyl chains of the CDL influence its function in two distinct ways: first, their large size prevents them from entering the transmembrane cavity, thereby avoiding interference with intermolecular interactions; second, the hydrophobic nature of these chains facilitates interaction with transmembrane helix 2, enhancing the transport turnover of VsSemiSWEET. Importantly, various studies have indicated that the interaction between proteins and CDL may depend on specific way on properties of CDL acyl chains. For example, α-synuclein binding to CDL is significantly influenced by the nature of CDL’s acyl side chains: complete saturation drastically reduces affinity, whereas the removal of a double bond increases it. These findings underscore that CDL acts as a regulatory factor in membrane proteins not only through the stabilizing interactions provided by its headgroups but also due to the unique structural characteristics of its four acyl chains. Collectively, this highlights the critical importance of CDL-protein interactions in modulating SemiSWEET-mediated sugar transport activity.\nDimerization is a prerequisite for VsSemiSWEET to achieve transmembrane sugar transport, as it forms the functional unit required for substrate translocation. The two protomers create a central pathway that enables selective sugar movement across the membrane, with the transport mechanism relying on coordinated conformational changes between monomers, generating an alternating-access mechanism. Disruption of dimerization impairs this conformational switching, leading to a loss of transport activity, underscoring the essential role of dimerization in SemiSWEETs function. Notably, the dimer interface is stabilized by interactions with lipids such as CDL, which is critical for maintaining the dimer conformation of VsSemiSWEET. Extensive studies have demonstrated that lipid binding plays a universal regulatory role in membrane protein oligomerization, which is crucial for structural stability and functional integrity. In this study, we found that, similar to CDL, PE and PG lipids promote dimer formation of VsSemiSWEET. Further insights from ssNMR and MD simulations reveal that CDL tails localize to the outer surface of VsSemiSWEET, enhancing intermonomer interactions and stabilizing the dimeric structure while facilitating its functional transition cycle; in contrast, PG tails penetrate deep into the protein cavity, disrupting interprotomer interactions and impeding functional conformational switching. This observation aligns with previous findings on the Na+/H+ antiporter, where CDL was identified as the optimal lipid for promoting dimer assembly, stability, and functional integrity compared to other type of lipids.\n2. There are no functional assays in the study, so there is a risk that the protein-membrane reconstitution leads to a non-functional system (i.e. not a system able to transport molecules across the membrane). Maybe the authors have further evidence to support the functionality of the protein in this particular membrane environment that has been set up for MAS NMR studies?\nYes, experimental support of the functionality of VsSemiSWEET in membrane environment for ssNMR studies is important. However, the substrate specificity of VsSemiSWEET remains unknown at present (Feng et al., Nature, 2014), which makes it challenging to perform transport activity experiments of the protein in the membrane environment for ssNMR studies. In general, SemiSWEETs from other species of E. coli and L. biflexa have been shown to exhibit functional activity when reconstituted into liposomes (Feng et al., Nature, 2014, 515, 448-452; Lee et al., Nat. Commun., 2015, 6, 6112).\nAdditionally, our previous ssNMR studies have shown that the chemical shifts of VsSemiSWEET in native cellular membranes (without purification or reconstitution) closely resemble those observed in synthetic lipid bilayers (Zhang et al., JACS Au, 2023, 3, 3412-3423), indicating the nativeness of the protein structure in synthetic lipid bilayers.\nOther points:\n1. Description of the membrane protein VsSemiSWEET at the beginning of the manuscript is very short. Please add more on the biological context of these systems, i.e. the role in sugar transport\nWe have added the description of the biological context for VsSemiSWEET at the beginning of the revised manuscript as follows:\nThe SWEET family of transporters are responsible for sugar uptake across cellular membranes, enabling solute permeation down a concentration gradient. These proteins play essential roles in various biological processes, including nectar secretion, pollen nutrition, and susceptibility to pathogens. Their bacterial homologues, the SemiSWEETs, are widely distributed among prokaryotes and represent one of the smallest known transporter families identified to date.\n2. The protein system is from Vibrio. Please provide the lipid composition of native vibrio membranes and a relevant reference to support it. What’s the native molar ratio of cardiolipin compared to PG and PE in Vibrio? Ref 36 is cited but this work was not done on Vibrio species.\nYes, Ref 36 provided the lipids composition of E. coli membranes but did not include data for Vibrio species. For accurate Vibrio. membrane composition, we now cite the study by Danevcic et al. (Biochim. Biophys. Acta, 2005, 1712, 1-8), which reports that the native lipid composition of Vibrio species typically includes a mixture of PE, PG, and CDL, which is similar to that found in E. coli membranes. Under optimal growth conditions, the approximate molar ratio of these lipids is as follows: PE, PG and CDL account for approximately 45.4%, 27.5%, and 8.0% of the membrane. These details have been added to the revised manuscript:\nwe prepared a 13C-labeled total E. coli lipid extract sample containing no protein. This sample consisted of approximately 57% PE, 15% PG, 10% CDL, and 18% other minor species identified by mass spectroscopy. Notably, the native lipid composition of Vibrio species is primarily PE, PG, and CDL, which is analogous to the lipid profile observed in E. coli membranes.\n3. In the introduction: a short description of previous work on the high-resolution structure, as well as the presence of distinct states (open outside, occluded, open inside) would be necessary. These details only come page 16, please mention them already in the introduction to help the readers to understand the structural basis on this system.\nWe have added the description for the structural basis of SemiSWEETs in the introduction as follows:\nThe structures of four SemiSWEETs have been resolved via X-ray crystallography in lipid cubic phase (LCP), capturing three distinct states: outward-open, occluded, and inward-open. The same dimeric architecture was observed in all four SemiSWEETs, suggesting that they consistently function in dimer form and exhibit an alternating access mechanism for sugar transport.\n4. In the 1D 31P spectrum (Fig. 1C), what is the contribution observed at 0.11ppm?\nThe signal observed at 0.11 ppm likely arises from other lipid components, as there is an unknown component constituting approximately 18% of E. coli lipids. Identifying specific lipid types based solely on 31P chemical shifts can be challenging, as these shifts often overlap among different lipid classes. In our manuscript, we combined both 13C and 31P chemical shift data to accurately identify lipid types (Figure 1B and 1C). The 13C chemical shifts of the lipid headgroups in VsSemiSWEET align well with published assignments for PE, PG, and CDL lipid headgroups (Markus Weingarth et al., Chem. Eur. J., 2022, 28, e202202472; McDermott et al., Biochimica et Biophysica Acta., 2015, 1848, 260-265). This agreement clearly confirms that the lipids are PE, PG, and CDL. In the 31P spectrum of VsSemiSWEET, there are four distinct signals. Based on literature data (Engberg, O. et al., Sci. Adv. 8, eabq8303; Dror E. et al., Biophys. J., 2018, 114, 1368-1376), the relative chemical shifts for PE, PG, and CDL correspond to signals at -0.20, 0.22, and 0.30 ppm in our spectrum.\nAdditionally, we collected the 31P spectrum of E. coli lipids (consisting of approximately 57% PE, 15% PG, 10% CDL, and 18% other minor species) and compared it with the 31P signals of the lipids bound to the VsSemiSWEET system. Both E.coli lipids and bound lipids in VsSemiSWEET include PE, PG and CDL components. As shown in Figure S3, the signals at -0.20, 0.22, and 0.30 ppm overlap, further confirming that these signals originate from PE, PG, and CDL. Therefore, the signal observed at 0.11 ppm likely arises from other lipid components besides PE, PG, and CDL.\nSupplementary Figure 3. Superimposition of 1D 31P spectra of E.coli lipids and bound lipids in VsSemiSWEET. Both E.coli lipids and bound lipids in VsSemiSWEET include PE, PG and CDL components. he overlapping signals at -0.20, 0.22, and 0.30 ppm demonstrate that these peaks correspond to PE, PG, and CDL.\n5. Line 175: please cite the seminal work of Oschkinat and coworkers (Castellani Nature 2002) when describing the use of 13C spin dilution (both for the use of selectively labeled glycerol carbon sources and the use of a 50/50 diluted sample to discard intermolecular contacts) to obtain distance restraints by MAS NMR.\nThis reference (Castellani et al., Nature, 2002, 420, 98-102) has been included in the revised manuscript.\n6. Line 174: replace “truncation” by “dipolar truncation”. A relevant reference (e.g. pubmed.ncbi.nlm.nih.gov/19317544/) should be cited here for non-NMR readers when mentioning the dipolar truncation effect.\nWe have replaced “truncation” with “dipolar truncation”, and the reference (Griffin et al., J Chem Phys., 2002, 130, 114506) have been included in the revised manuscript.\n7. Line 187: the authors used CS-ROSETTA, why not AF3? The manuscript should provide an AF3 prediction of the protein.\nWe chose to use CS-Rosetta primarily because it has a unique advantage in structure prediction by integrating experimental data (such as residual chemical shifts, distance restraints). We agree that AlphaFold3 (AF3) is a powerful tool for protein structure prediction today and its predictions are highly valuable as a reference. We have included structure predictions of the target protein based on AF3 in the revised manuscript and compare them with the results from CS-Rosetta. As demonstrated in Figure S8, the monomer structure of VsSemiSWEET predicted by CS-Rosetta is consistent with AF3’s prediction, with an RMSD of 1.0 Å. This comparison underscores the compatibility between these two methods and further validates our findings.\nSupplementary Figure 8. Mnomer structure models of VsSemiSWEET in lipid bilayers predicted by CS-Rosetta and AlphaFold3. a, All assigned chemical shifts of VsSemiSWEET, along with the initial two unambiguous and 26 ambiguous intra-monomer distance restraints determined solely based on differences in chemical shifts, were used in the CS-Rosetta calculations. A summary of all initial intra-monomer distance restraints is provided in Table S2. The 10 lowest energy structures among 10000 structures are convergent, with an RMSD of 0.9 ± 0.2 Å for the backbone Cα atoms. b, Comparison of the VsSemiSWEET monomer structures predicted by CS-Rosetta and AlphaFold3.\n8. Line 189: unclear what means “chemical shift specificity”.\nThe term “chemical shift specificity” refers to how initial distance constraints were assigned based solely on residue-specific chemical shifts. For instance, consider a peak at 70.0 and 61.4 ppm: the assignment at 70.0 ± 0.3 ppm had two possibilities (T60CA and T48CB), while the assignment at 61.4 ± 0.2 ppm had four possibilities (W72CA, S36CA, S38CA, and Y34CA). This resulted in eight initial assignments for this peak. In contrast, a peak at 47.5 and 50.1 ppm had only one possibility for both assignments at 47.5 and 50.1 ppm, leading to a single initial assignment (G78CA-P23CD).\nTo clarify this process for readers, we have revised the description of the initial distance constraint assignment as follows:\nWe constructed an experimental structural model using CS-Rosetta calculations, inputting chemical shift data and initial distance restraints determined solely based on residue-specific chemical shifts.\n9. Line 207: intramonomeric restraints are excluded because they are >9 Å in the CS-ROSETTA model? if yes, please add this detail (i.e. the upper distance of 9A) in the main text.\nYes, we excluded intra-monomer contacts with the distance of >9 Å in the CS-Rosetta model. We have revised the description in line 207 as follows:\nTo address this, we initially assigned inter-monomer distance restraints by excluding intra-monomer contacts based on the monomer structure. All possible intra-monomer distances exceeded the upper limit of 9.0 Å, allowing us to exclude them during distance restraint assignment (Figure 2c and S12a, b).\n10. Line 241: “but with empty coordinate without distance restraints”: not clear if lipids were used or not in the final Xplor refinement.\nIn the final Xplor refinement, we did not include any distance restraints from the bound lipids. While bound lipids remained physically present in the VsSemiSWEET samples, we removed their structural constraints to minimize interference from lipid-lipid and lipid-protein correlated signals on intra-residue distance constraints. This was achieved by replacing ¹³C-labeled lipids with natural abundance lipids through our unfolding-refolding protocol, which effectively removed NMR-observable lipid constraints while maintaining native lipid-protein interactions. Furthermore, although the bound lipids are not present in the determined structures but with empty coordinate due to lack of distance restraints, we characterized their binding sites through complementary ssNMR data and MD simulations. The structure of VsSemiSWEET bound with CDL was determined by MD simulations (Figure 3).\nWe have revised the description at line 241 to better clarify whether lipids were included in the final Xplor refinement. The updated text are as follows:\nImportantly, while bound lipids remained physically present in the VsSemiSWEET structural determination samples, their structural constraints were removed by substituting ¹³C-labeled lipids with natural abundance lipids during unfolding-refolding process. Consequently, the bound lipids are absent in the determined structures, leaving empty coordinates due to the lack of distance restraints.\n11. Supp fig. 13: it might be more relevant to compare the crystal structure to the bundle of NMR structures (10 lowest energy) and not only to one NMR conformer.\nWe have revised Figure S13 to compare the crystal structure with the bundle of 10 lowest-energy ssNMR structures.\nSupplementary Figure 16. A comparison of VsSemiSWEET structures derived from ssNMR and X-ray crystallography. a. Superimposition of the 10 lowest-energy ssNMR structures of VsSemiSWEET dimer determined in lipid bilayers (cyan) and the X-ray structure determined in lipid cubic phase (grey). Both structures are shown in cartoon representation. b. Comparison of VsSemiSWEET monomer structures derived from ssNMR and X-ray crystallography, with red and yellow lines indicating the kink angles at position Q29 in TM1.\n12. Line 285: can the authors elaborate on the precise time scale of lipid-protein interactions, based on the fact that lipid-protein contacts are only observed in CP-based experiments?\nIn CP-based experiments, the transfer of the magnetization vector is mediated by the dipole coupling interaction between the spin nuclei, the efficiency of which depends on the spatial distance, the contact time, and the stability of the spatial orientation between the two spin nuclei. The lipid-protein cross-peaks in CP-based experiments imply lipid binding times in the microsecond range or longer but exclude shorter lifetimes, which is the time-scale of the relevant dipolar couplings that drive spin diffusion (Hariharan et al., BMC Biology., 2018, 16, 85; Markus Weingarth et al., J. Am. Chem. Soc., 2013, 135, 3983-3988). Relevant descriptions have been added to the revised result section as follows:\nImportantly, the lipid-protein cross-peaks in the dipolar-coupling-based experiments imply lipid binding times in the microsecond range or longer but exclude shorter lifetimes, which is the time-scale of the relevant dipolar couplings that drive spin diffusion.\nMoreover, in contrast to the dipolar-coupling-based signals from glycerol backbones and headgroups of the bound lipids, which indicate that these groups exhibit structural rigidity on a micro- to millisecond timescale, the signals observed in scalar-coupling-based ssNMR spectra suggest a high degree of flexibility on submicrosecond timescales.\n13. 31P solution NMR experiments were performed at 315K, why this temperature and not 310K?\nThe 31P NMR experiments were conducted at 315 K to optimize signal strength. At lower temperatures, molecular rotation is restricted due to lipid binding with protein and localization within micelles, which weakens the 31P signals. Higher temperatures enhance molecular movement, thereby improving lipid signal intensity in 31P solution NMR experiments. We conducted 1D 31P solution NMR experiment of VsSemiSWEET at 305 K, which confirmed similar chemical shifts at both temperatures (305 K and 315 K) but with significantly lower signal intensities at the lower temperature. Consequently, 315 K was selected to maximize the intensity of 31P NMR signals. This temperature selection aligns with established literature, such as the work by Ladizhansky et al., where similar experimental temperature (313 K) were employed (BBA-Biomembranes, 2020, 1862, 183345).\n14. All MAS NMR experiments were performed at 272K, I guess to improve CP-based polarization transfer. However, this relatively low sample temperature might have considerably rigidified lipids, enhancing lipid-protein proximities. Can the authors comment of this aspect?\nYes, the choice of 272 K for MAS NMR experiments was deliberately selected to optimize cross-polarization (CP) efficiency. Although this temperature increases lipids rigidity, control experiments using proteins without bound lipids (e.g., AqpZ and MscL) were performed. as shown in Figure R1, CP spectra of these control proteins show no detectable lipid signals under identical conditions, confirming that the observed lipid signals in VsSemiSWEET are bound lipids copurified with protein rather than temperature-induced artifacts.\nFigure R1. Validation of specific lipid binding in VsSemiSWEET. 2D 13C-13C correlation spectra of VsSemiSWEET, AqpZ, and MaMscL acquired at 272 K with 50 ms mixing time. Lipid-specific signals (red dotted boxes) are exclusively observed in VsSemiSWEET, demonstrating the presence of bound lipids copurified with VsSemiSWEET.\n15. The authors assume that the protein form a homodimer in membranes, is this assumption based on experimental data?\nWe conducted cross-linking experiments on VsSemiSWEET in native-like lipid bilayers, which demonstrate that the protein indeed forms dimers in membranes. As shown in Figure S11, after boiling, the cross-linked VsSemiSWEET migrated as bands corresponding to double the molecular weight of the monomer band during SDS-PAGE analysis, providing direct evidence for dimer formation.\n16. There is one Cysteine in the main helical core. Do the authors exclude the presence of intermolecular disulphide bridges?\nBased on the ssNMR structure of VsSemiSWEET, the intermonomer distance of residue C76 is approximately 20 Å, which is too far to allow the formation of a disulfide bond between monomers. Furthermore, size-exclusion chromatography experiments confirm that VsSemiSWEET exists in a homogeneous aggregation state (Figure 4b), ruling out any possibility of intermolecular disulfide bridge formation.\n17. Line 175: please define what is “medium”-and “long-range” distances for non-NMR readers.\nWe have added a clear definition of “medium-range” and “long-range” distances for non-NMR readers in line 175 as follows.\nThe ‘medium-range’ and ‘long-range’ correspond to distances between residues where i and j are residue numbers. For medium-range, the distance is defined as 2 ≤ |i – j| <5, while for long-range, it is |i – j| ≥5.\n18. Line 177: I would suggest to replace “13C-unlabeled lipids” simply by “unlabeled lipids”.\nWe thank the reviewer for this suggestion and have changed “13C- unlabeled lipids” to “unlabeled lipids”.\n19. Line 137: Long mixing recoupling period (500ms CORD mixing) is used to enhance the signals of bound lipids. Please explain why such long mixing help to observe lipid signals.\nThe high mobility of lipids slows down magnetization transfer in dipolar-based experiments, necessitating longer mixing times to enhance signal visibility. We systematically tested several mixing times (50, 150, 300, and 500 ms) and found that 300 and 500 ms provided the best results for observing bound lipid signals (Figure S2).\nSupplementary Figure 2. ssNMR 2D 13C-13C CORD spectrum of 13C-labeled VsSemiSWEET with bound lipids(grey) were superimposed to that of pure E. coli lipids without the proteins (pink). The 13C-13C correlation spectra were acquired with different mixing times: 50 ms, 150 ms, 300 ms and 500 ms.\n20. Fig4e (the contact map) is small and it's difficult to read it, it could move to the SI.\nThank you for bringing this to our attention. We have moved Figures 4e and f to the Supplementary Information to improve the readability of the contact map.\nIn response to reviewer 4:\n1. This manuscript, in its current form, is quite difficult to navigate.\nThank you for your feedback. To improve the readability of the manuscript, we have comprehensively restructured and rewritten the article. In particular, we have simplified technical details where possible, added explanatory sections to better guide readers through specialized concepts, and refined the flow of information throughout the manuscript. We have made the following revisions:\n1. In first paragraph of the result section 1, we have added “ssNMR spectral analysis of VsSemiSWEET liposomes confirms the presence of bound lipids that co-purified with the protein” at the beginning to clarify the goal of the signal assignments and better explain their relevance to lipid binding.\n2. In first paragraph of the result section 1, we have revised the sentence “Furthermore, the assignments of Ser and Thr residues could be confidently made, excluding any ambiguity in their resonance assignments.” to “Furthermore, Ser and Thr residues are the only amino acids exhibiting intra-residual correlations in this region. The signals of these residues have been unambiguously assigned, confirming that the identified signals arise from co-purified lipids rather than protein-derived signals.” This revision explicitly explains why it was necessary to exclude these residues during lipid-specific resonance assignments and strengthens the logical flow of the argument.\n3. In second paragraph of the result section 1, we have revised the sentence “The superimposition of 13C-13C CORD spectrum of lipid-bound protein and lipid extract validated the assignments of the co-purified lipids(Figure 1b).” to “As illustrated in Figure 1b, the 13C chemical shifts of PE, PG and CDL in E. coli lipids align perfectly with those of lipids bound to VsSemiSWEET. This exact signal match provides direct evidence that these three lipid species co-purify with the protein, demonstrating their stable and high-affinity interactions with VsSemiSWEET.” The updated version explicitly explains why and how the signal assignment of bound lipids was validated, providing a more direct connection between the data and the conclusion.\n4. In first paragraph of the result section 2, we have added an explanation about medium- and long-range distance restraints in structural characterization: “This dilution greatly improved spectral resolution and suppressed dipolar truncation effect, yielding numerous well-resolved cross-peaks corresponding to medium- and long-range distance restraints. The ‘medium-range’ and ‘long-range’ correspond to distances between residues where i and j are residue numbers. Medium-range distances were defined as 2 ≤ |i–j| < 5, while long-range distances corresponded to |i–j| ≥ 5”. This clarification provides a clearer understanding of the distance definitions used in our analysis.\n5. In third paragraph of the result section 3, we have added an explanation explicitly addresses the distinct timescales of lipid dynamics observed in dipolar-coupling-based and scalar-coupling-based ssNMR experiments: “Furthermore, our ssNMR data revealed distinct dynamics of lipid components bound to the protein. While dipolar-coupling-based signals from glycerol backbones and lipid headgroups suggest structural rigidity on a micro- to millisecond timescale, the signals observed in scalar-coupling-based ssNMR spectra suggest a high degree of flexibility on submicrosecond timescales.” The updated explanation clarifies why lipid headgroups and glycerol backbones exhibit structural rigidity on a micro- to millisecond timescale, while acyl chains show high flexibility on submicrosecond timescales.\n6. We have changed the title of the results section to the following: 1) Identification of Bound Lipids Co-Purified with VsSemiSWEET. 2) Structural Characterization of VsSemiSWEET in Lipid Bilayers Using ssNMR. 3) Characterization of Lipid Interactions with VsSemiSWEET in Lipid Bilayers. 4) Bound Lipids Influence Dimer Stability of VsSemiSWEET in Lipid Bilayers. 5) Differential Regulations of Interfacial CDL and PG on the Structure and Function of VsSemiSWEET. The revised title more intuitively reflects the conclusions of each section, making it easier for individuals without an NMR background to understand.\n7. We added Figure S15a and b to explain the principles of H/D exchange experiments in solid-state NMR.\nFigure S15. Water accessibilities of VsSemiSWEET residues in lipid bilayers measured by H/D exchange experiments. a, The principle of H/D exchange in solid-state NMR: After H/D exchange, the 1H of the amide group on the main chain of water-accessible residues is deuterated, leading to a decrease in signal intensity in the 1H-15N CP-based spectra. b, Comparison of VsSemiSWEET 2D 15N-13Cα spectra before and after H/D exchange, with peaks showing significant signal intensity reduction marked by dashed boxes following the exchange.\n8. We added Figure S17 to illustrate schematic diagram of the detailed preparation process for spin-diluted sample and how a spin-diluted sample helps distinguish between intra- and inter-monomer contacts.\nFigure S17. Analysis of spin-diluted sample in structural calculation. a, Schematic diagram illustrating the detailed preparation process for the spin-diluted sample. b, Schematic representation showing intra- and inter-monomer distance restraints in fully labeled and spin-diluted samples. Blue indicates distance restraints that exhibit observable NMR signals, while gray represents distance restraints with unobservable signals. In a spin-diluted sample, one monomer of the VsSemiSWEET dimer is at natural abundance; therefore, intermonomer distance restraints cannot exhibit NMR signals.\n9. We have added concluding descriptions at the end of key paragraph in the results section to summarize findings and provide context. For example: 1) “These findings underscore that lipid binding is predominantly mediated through electrostatic interactions between lipid headgroups and basic residues within the dimer interface.” 2) “Collectively, these results highlight the importance of lipid binding in sustaining the stability and structural integrity of the VsSemiSWEET dimer, possibly via specific interactions at the dimer interface.” 3) “This suggests that PG creates steric hindrances that not only obstruct ligand passage but also impede the transition cycle between functional states.” 4) “Together, these findings highlight a critical role of CDL in modulating functional transition cycle of VsSemiSWEET.” These additions aim to improve readability by summarizing key points.\n10. To better convey the key findings of the results section, we have incorporated simplified schematic diagrams into revised Figures 4g and 5c. These additions provide a visual framework that helps readers understand the main conclusions of our study.\nFigure 4g. Schematic illustration of the stability changes of VsSemiSWEET after mutations of lipids binding sites. Figure 5c. Schematic diagram of the different interaction modes of PG and CDL with VsSemiSWEET.\n2. The significance of the study is not clearly articulated-what makes this research important, and what are the key takeaways?\nWe have added a paragraph in the revised discussion section to highlight the significance of our work and our main contributions:\nIn summary, our study provides structural and mechanistic insights into a fundamental question of membrane protein biology: how lipid diversity regulates protein behavior through specific interactions. Through a systematic investigation of the interactions between VsSemiSWEET and its specific bound lipids (PE, PG and CDL), Our findings reveal that CDL, with its unique dual phosphates and four acyl chains, modulates the behavior of membrane proteins through interactions involving both the head group and the fatty acid. We also demonstrate that the physicochemical properties of the bound lipids play a crucial role in regulating protein conformational stability and functionality. As shown in Figure 7, the acyl tails of PG insert into the interfacial cavity of VsSemiSWEET, disrupting both the dimer’s structure and its functional transition cycle. In contrast, the larger hydrophobic tails of CDL block access to the interfacial pore, thus avoiding disruption of the dimeric architecture. Additionally, the acyl tails of CDL interact with TM2, thereby facilitating a functional conformational transition in VsSemiSWEET. These insights highlight the complexity of specific lipid-protein interactions and emphasize how variations in the acyl tails and head groups of bound lipids can regulate membrane protein oligomerization and functionality in different ways. Future studies could investigate whether similar lipid-mediated regulatory mechanisms exist in other membrane proteins and explore their roles under physiological or pathological conditions. Such investigations would not only expand our understanding of lipid-protein interactions but also elucidate the molecular mechanisms underlying membrane protein behavior in diverse biological contexts.\n3. Additionally, the relevance of this transporter's dimerisation needs further clarification.\nThe reviewer may refer to functional relevance. The dimerization of VsSemiSWEET is a prerequisite for its function, and lipid binding to the protein facilitates its dimerization. To clarify the functional relevance of SemiSWEET dimerization, we have expanded the discussion in the revised manuscript to emphasize how lipid binding facilitates the dimerization of VsSemiSWEET:\nDimerization is a prerequisite for VsSemiSWEET to achieve transmembrane sugar transport, as it forms the functional unit required for substrate translocation. The two protomers create a central pathway that enables selective sugar movement across the membrane, with the transport mechanism relying on coordinated conformational changes between monomers, generating an alternating-access mechanism. Disruption of dimerization impairs this conformational switching, leading to a loss of transport activity, underscoring the essential role of dimerization in SemiSWEETs function. Notably, the dimer interface is stabilized by interactions with lipids such as CDL, which is critical for maintaining the dimer conformation of VsSemiSWEET.\n4. The figures are presented in a way that makes interpretation challenging, leaving the reader uncertain about the core messages they should extract.\nThank you for your valuable suggestions. We have carefully revised the figures and their legends to address the issues raised, ensuring that they are now more intuitive and better aligned with the text.\n1. To provide clearer guidance, the titles of Figure 1-7 have been revised as follows: 1) Identification of co-purified bound lipids in VsSemiSWEET. 2) Three-dimensional structure of VsSemiSWEET in lipid bilayers determined by ssNMR. 3) Analysis of interactions between bound lipids and VsSemiSWEET. 4) Lipids binding modulates the oligomeric state and conformation of VsSemiSWEET. 5) Regulatory role of PG and CDL binding in VsSemiSWEET, revealed by MD simulations (a, b) and ssNMR experiments (d-g). 6) Functional regulation of VsSemiSWEET by different lipid binding. 7) Distinct lipid-protein interactions and regulatory mechanisms of CDL and PG in VsSemiSWEET.\n2. The changes in Figure 3: 1) changed “G2” to “Lipids” to clearly indicate that 72.8 ppm corresponds to the characteristic chemical shift of lipids. This adjustment ensures that readers can easily identify this key feature. 2) Merged Figure 3c into Figure 3b to improve clarity and facilitate a direct comparison of the relative positions of K32, H37, and H40 in VsSemiSWEET. This change strengthens the visual coherence of the figure. 3) Downward arrows have been added to Figure 3c to visually emphasize the significant reduction in the characteristic 31P NMR signal of lipids after mutating the lipid-binding sites, making this observation more intuitive for readers. 4) In Figure 3e, we have adjusted the perspective of the docking structure between CDL and VsSemiSWEET to improve its visual presentation and ensure that the interactions are clearly depicted. 5) Introduced concise subheading-like descriptions for each panel: a. potential lipid-binding sites identified by ssNMR; b. MD simulation-derived lipid-binding sites; c. validation of lipid-binding sites by solution NMR; d. Solution NMR analysis of protein-lipid interaction type.\n3. The changes in Figure 4: 1) updated the color scheme in Figures 4b, 4c and 4d to distinguish the elution profiles and NMR spectra of the mutants more effectively, using blue for H37A/H40A and green for K32A/H37A/H40A. This change enhances clarity and correspondence between the data. 2) moved Figure 4e (the contact map) to the Supporting Information section to enhance the readability of the contact map. 3) A new panel, Figure 4g, has been added to provide a schematic illustration of the stability and conformational changes in VsSemiSWEET after mutations of the lipid-binding sites. This addition helps readers better understand the relationship between the lipids binding and protein stability. 4) Introduced concise subheading-like descriptions for each panel: a. Oligomeric stability analysis; b. Oligomeric state analysis; c and d. Conformational analysis; e. MD simulation (lipid-free state) versus ssNMR structure; f. MD simulation (CDL-bound state) versus ssNMR structure.\n4. The changes in Figure 5: 1) The presentation of structural images in Figure 5a has been revised to highlight the differences in binding modes between PG and CDL, ensuring that these distinctions are clearly visible. 2) Moved the SDS-PAGE image showing the refolding process of VsSemiSWEET induced by different lipids to the Supporting Information section to maintain a focused presentation in Figure 5. 3) A new panel, Figure 5c, has been added as an illustrative diagram to summarize the distinct binding modes of PG and CDL with VsSemiSWEET. This addition provides readers with a concise visual summary of the key findings. 4) Introduced concise subheading-like descriptions for each panel: a. Distinct lipid-binding modes of PG and CDL to VsSemiSWEET analyzed by MD simulations; b. Contact probability differences of residues with lipids between PG-bound and CDL-bound VsSemiSWEET studied by MD simulations. c. Schematic comparison of PG and CDL binding mechanisms. d. SsNMR validation of lipid-binding modes. e. Quantitative analysis of the CSPs of residues induced by different lipids binding. g. Quantitative analysis of changes in signal intensity of residues induced by different lipids binding.\n5. We have modified our original schematic as Figure 7 in the revised manuscript to better illustrate our central mechanistic finding: the diverse regulation of functional dimerization of a sugar transporter by different interfacial lipids.\nFigure 7. Distinct lipid-protein interactions and regulatory mechanisms of CDL and PG in VsSemiSWEET. a, Molecular topology comparison: PG (single phosphatidylglycerol headgroup with two acyl chains) versus CDL (dimeric phosphatidylglycerol containing four acyl chains). b, Regulatory mechanisms: In the absence of lipids, VsSemiSWEET forms unstable dimers due to repulsion between interfacial basic residues. PG acyl tails insert into the interfacial cavity of VsSemiSWEET, disrupting interprotomer interactions and inhibiting the functional transition cycle. In contrast, the acyl tails of CDL interact with TM2, stabilizing the dimeric structure and facilitating functional transitions.\nWe hope that these revisions can enhance the clarity and presentation of our figures. Thank you again for your constructive feedback.\n5. Given these issues, this work may be better suited for a more specialised journal where the intended audience can fully appreciate its technical depth.\nThank you for your insightful feedback regarding the suitability of our work for this journal. While solid-state NMR is a specialized technique, the findings presented in this study address a universal theme in membrane protein biology: how bound lipids with distinct physicochemical properties regulate the oligomerization and functionality of membrane proteins differentially (e.g., Gupta et al., Nature., 2017, 541, 421-424; Landreh et al., Nat. Commun., 2016, 8, 13993; Xiao et al., Nat. Commun., 2017, 8, 2203; Lyu et al., Nat. Commun., 2022, 13, 7291; Schmidpeter et al., Nat. Commun., 2023, 14, 1077; Thakur et al., Nat. Commun., 2023, 14, 794).\nThe implications of this work span multiple fields: 1) Regulation of membrane proteins: Our findings demonstrate how lipid-protein specific interactions influence the structural stability and functionality of membrane proteins, providing critical insights into how physicochemical properties of bound lipids can be harnessed to control membrane protein behavior across diverse biological contexts. 2) Modulation of important biological processes: The ability of lipids to modulate protein activity opens new avenues for studying important biological processes. This has direct relevance to understanding diseases where lipids binding plays a role, such as neurodegenerative disorders or diabetes. 3) Target identification in drug development: Insights into how membrane proteins interact with lipids can inform the design of drugs targeting these proteins. By considering lipid-protein interactions, researchers can develop more effective therapeutic strategies that account for the cellular environment. 4) Strategies for studying protein-lipid interactions: as highlighted by reviewer 3, the strong point of this study is the methodology, it could be applied to other membrane protein systems of interest to decipher specific lipid-protein interaction using the combined MAS NMR and MD approach.\nThe results presented here are thus of broad interest to readers in structural biology, membrane biophysics, and functional studies of membrane proteins. Furthermore, the findings hold significant potential for applications in drug discovery targeting membrane-associated processes. Therefore, we believe that Nat. Commun. provides an ideal platform for disseminating our work to a broader scientific audience."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed most of my initial concerns. However, I remain concerned about the lack of a suitable transport assay to support their claims. While the author noted that conventional cell-based transporter assay failed for VsSemi-sweet, cell-based or proteoliposome reconstitution assay methods demonstrated sugar transport for SemiSweet from E. coli and L. biflexa. I wonder whether the conclusion in this manuscript is still valid for SemiSweet from E. coli and L. biflexa and if so, the authors could validate it using the established transporter assays to strengthen their study.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The authors provided a very solid response letter to all reviewer's questions.", "reviewer_4": "Regrettably, my assessment of the manuscript remains consistent with the concerns outlined in my previous review. In particular, the NMR data continues to present significant challenges in terms of interpretability. The spectra are highly complex, and without clearer annotation or supporting evidence, it is difficult to independently verify the authors' structural assignments. As such, my evaluation is ultimately dependent on the authors' own interpretation and description of the data, which limits the robustness of the review process. I maintain that this article is better suited to a more specialised journal.", "reviewer_5": "It is well established that the composition of membrane bilayers plays a pivotal role in the regulation of multiple processes. However, how distinct lipid species can affect the activity of membrane proteins is still poorly understood in most cases. In this paper, Yang and co-workers use solid-state NMR and molecular dynamics simulations to study the role of membrane lipids, PG and CDL, in the dimerization and functional activity of a bacterial sugar transporter, VsSemiSWEET.\nThis manuscript makes use of multiple solid-state NMR techniques to resolve the protein structure and identify bound lipids. While the use of these methods is explained with high technical detail, which can make the manuscript difficult to navigate for non-expert readers, the use of MD simulations to investigate the role of the bound lipids is confusing and requires further analysis and clarification, as explained below.\nFirst, they have carried out MD simulations of the VsSemiSWEET dimer in lipid bilayers with no initially bound lipids. The results from these simulations show a preference for CDL over PE and identify key binding residues, which then the authors mutate to corroborate their importance on CDL binding. However, there are some aspects that absolutely need to be addressed concerning these simulations:\n- The setup for the simulations as well as the number of lipid species should be provided, along with the number of replicas.\n- The residence time for PE and CDL lipids is provided, but not that of PG lipids.\n- The mutations proposed in the manuscript, and tested using ssNMR, have not been tested using MD simulations. This is key to corroborate the accuracy of their findings.\n- A density analysis of the lipid bilayers should be provided to further confirm the enrichment of certain lipids in the vicinity of the protein dimer.\nNext, they use docking to incorporate bound lipids to the dimer interface. My main concern here is that MD simulations have already shown the binding of CDL lipids at the dimer interface, why is it needed to add extra lipids using docking? On top of that, the conformation of the docked lipids looks unrealistic for a structure embedded in a lipid bilayer. The authors should test different docking results to validate their results. In addition, the comparison between PG and CDL bound structures to the ssNMR one in Figure 4e is static. An analysis of the RMSD along the entire simulation should also be provided."}, "author_response": "Response to reviewers\nWe thank the reviewers for carefully reading our manuscript and providing helpful comments on its clarity and presentation. We have addressed each comment as indicated below:\nIn response to reviewer 1:\nThe authors have addressed most of my initial concerns. However, I remain concerned about the lack of a suitable transport assay to support their claims. While the author noted that conventional cell-based transporter assay failed for VsSemi-sweet, cell-based or proteoliposome reconstitution assay methods demonstrated sugar transport for SemiSweet from E. coli and L. biflexa. I wonder whether the conclusion in this manuscript is still valid for SemiSweet from E. coli and L. biflexa and if so, the authors could validate it using the established transporter assays to strengthen their study.\nThank you for your suggestions. In response, we have performed transport assays and MD simulation analyses to E. coli and L. biflexa SemiSWEET homologs.\n1. Validation of the Mechanism in Other Homologs:\nWe performed new analyses to assess the conservation of lipid binding. First, we calculated the residence times of lipids interacting with these homologs. Simulation results indicate that PE, PG, and CDL also co-purify with EcSemiSWEET and LbSemiSWEET, although the binding strength of CDL is weaker in these variants compared to VsSemiSWEET (Figure S5). Second, a detailed sequence and structural alignment of the lipid-binding interfaces revealed that the key residues for PG and CDL recognition are highly or fully conserved across all three homologs (Figure S36). This suggests that the fundamental regulatory mechanism mediated by these interfacial lipids is possibly universal among bacterial SemiSWEETs.\n2. New Experimental Evidence: Proteoliposome-Based Transport Assays:\nTo overcome the lack of a liquid scintillation counter at our institution, we partnered with Professor Jianping Li's laboratory (Nanjing Medical University) to conduct the crucial liposome 14C-sugar uptake assays. Since LbSemiSWEET exhibits stronger binding affinity with CDL, we established a proteoliposome-based transport assay for LbSemiSWEET following a previously published protocol (Feng et al., Cell, 2017, 169:96-107; Lee et al., Nat. Commun., 2015, 6: 6112). LbSemiSWEET with specific lipid binding was reconstituted into liposomes, and the uptake of 14C-glucose was measured. As shown in Figure R1, LbSemiSWEET bound to CDL exhibited robust transport activity (1.9 nmol/mg relative to protein-free liposomes). In contrast, transport activity was significantly impaired when the protein was bound to PG. These functional results provide direct experimental evidence that CDL and PG exert opposing functional effects—consistent with our proposed model in which CDL supports, whereas PG disrupts, the functional conformational cycle.\nIt is important to note that the transport activity we measured for LbSemiSWEET (1.9 nmol/mg) falls within the established range for known sugar transporters, which varies considerably due to structural and experimental differences. For context, reported activities range from ~0.06 nmol/mg for the fucose transporter FucP to ~2.4 nmol/mg for the xylose transporter XylE (Nieng Yan et al., Nature. 2012, 490:361-366; Nieng Yan et al., Nature. 2010, 467:734-738). Furthermore, while the reported value for EcSemiSWEET is higher (~8.5 nmol/mg/h relative to empty control liposomes; Lee et al., Nat. Commun., 2015, 6: 6112), the activity we observed for LbSemiSWEET is comparable and reasonable given the distinct substrates and key experimental variables, such as 14C-sugar concentration and incubation time. Therefore, we confidently conclude that CDL-bound LbSemiSWEET possesses authentic sugar transport activity.\nThe combination of our new functional transport data and MD simulation results for VsSemiSWEET and the bioinformatic analysis demonstrating conservation of the regulatory site across homologs provides a comprehensive and robust response to this point. These additions have decisively strengthened the manuscript and validated our central mechanistic claim.\nFigure S5. Residence times of lipids interacting with SemiSWEET proteins from different species. a, E. coli SemiSWEET. b, L. biflexa SemiSWEET. c, Vibrio sp. SemiSWEET. Residues with lipid residence times of 100–500 ns are colored light cyan, those with 500–1000 ns are shown in green, and residues with residence times exceeding 1000 ns are depicted in red.\nFigure R1. Glucose uptake activities (nmol 14C-glucose/mg protein) for LbSemiSWEET with different lipids binding, compared to protein-free control liposomes (mean±s.e.m., n = 3). Asterisk indicate a significant difference from the control (*P < 0.01, Student's t-test). Sample preparation involved a lipid-exchange procedure, native co-purified lipids were washed with LDAO detergent, followed by incorporation of the specified lipids and reconstitution into proteoliposomes at an 80:1 lipid-to-protein ratio. Proteoliposomes were extruded through a 0.4 μm membrane, collected by ultracentrifugation at 100,000 g for one hour, and washed twice with 50 mM potassium phosphate, 2 mM MgSO , pH 4.5 (KPM buffer). Liposomes were resuspended in KPM buffer at 50 mg/mL. Uptake was initiated by the addition of 8 μL liposome to 200 μL of KPM buffer containing 2 mM glucose and 8 μM of 14C-glucose (100 μCi mL-1). After a five-minute incubation, the liposomes were collected on 0.22 μm filters (Millipore) and washed with 1 mL cold KPM buffer twice. Filters were then incubated with 4 mL scintillation fluid for quantification.\nIn response to reviewer 2:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nThank you for your constructive suggestions.\nIn response to reviewer 3:\nThe authors provided a very solid response letter to all reviewer's questions.\nWe are grateful to the reviewer for acknowledging the thoroughness of our responses. We believe that addressing the reviewers' comments has substantially improved the quality and impact of our manuscript.\nIn response to reviewer 4:\nRegrettably, my assessment of the manuscript remains consistent with the concerns outlined in my previous review. In particular, the NMR data continues to present significant challenges in terms of interpretability. The spectra are highly complex, and without clearer annotation or supporting evidence, it is difficult to independently verify the authors' structural assignments. As such, my evaluation is ultimately dependent on the authors' own interpretation and description of the data, which limits the robustness of the review process. I maintain that this article is better suited to a more specialized journal.\nThank you for your feedback. We agree with the reviewer that the interpretation of solid-state NMR data is not straightforward. However, this does not mean that we can interpretate NMR data according to our demands individually. NMR community has established adjective, definite rules for analysis of NMR data. For example, to assign the signals corresponding to distance restraints, we used the following criteria, as described in Page 38 in the main text.\n“The signals corresponding to 13C-13C distance restrains were assigned based on the following criteria:\n1) The peaks that could be assigned as 13C-13C distance constraints exhibited symmetry, were well-resolved, and had a sufficient signal-to-noise ratio (SNR ≥ 6) in the 2D CORD spectra. 2) All possible assignments of the cross peaks were collected. A tolerance of approximately 0.2 ppm was set for both direct and indirect dimensional 13C chemical shifts, considering the linewidth of the peaks (0.2 to 0.6 ppm) in the CORD spectra.”\nAll methods for processing and analysis of NMR data have been detailedly provided in the Method section of the manuscript.\nAlthough analysis of solid-state NMR data is not straightforward, all main solid-state NMR results have been strictly cross-validated by different solid-state NMR experiments and by various techniques, such as solution NMR, mutation analysis, and MD simulations. Specifically, the binding of CDL to the protein was verified by the following experimental and simulation data.\n1. Different solid-state NMR spectra. i) 2D CORD 13C-13C spectra (mixing time of 50 ms ) before and after delipidation to identify the binding of lipids to VsSemiSWEET(Fig.1). ii) 2D CORD 13C-13C spectra (mixing time of 500 ms ) of VsSemiSWEET to identify the binding sites of CDL to the protein(Fig. 3a). iii) 2D NCA spectra of VsSemiSWEET to characterize the oligomeric stability of the mutation, verifying the binding sites.(Fig. 4c and 4d). iv) 2D NCA and 2D 13C-13C spectra of VsSemiSWEET with different lipid bindings to identify the binding sites of different lipids( Fig. 5d,5e,5f and 5g, Fig. S32). v ) 1D 1H-13C INEPT spectrum of VsSemiSWEET to identify bound lipid.\n2. Different solution NMR spectra. i ) Solution 1D 31P spectra of lipid bound VsSemiSWEET before and after delipidation. ii ) 2D 1H-13C HSQC spectra to verify bound lipids. (Fig. S1). iii) 1D 31P spectra of lipid bound VsSemiSWEET mutants to identify the binding sites (Fig. 3c and 3d) .\n3. SDS-PAGE. i）SDS-PAGE results for wild-type VsSemiSWEET, the K32R and lipid-binding-deficient mutants (K32A, H40A and H37A/H40A) mutants（Fig. 4a）. ii) SDS-PAGE of VsSemiSWEET with bound lipids to examine the oligomeric state of VsSemiSWEET with the binding of different lipids(Fig. s22).\n4. MD simulations have been extensively employed to characterize different aspects of the lipid binding to the protein (Fig. 3b and 3e, Fig. 4e and 4f, Fig. 5a and 5b, Fig.6, Fig. S19, S23, S24, S25,S27, S29, S31 and S33)\nCollectively, in this study, the interpretation of solid-state NMR data is highly confidential, with the highly crossed verifications from different techniques and different experiments.\nWe should emphasize the broad interest of this work again. While solid-state NMR is a specialized technique, the findings presented in this study address a universal theme in membrane protein biology: how bound lipids with distinct physicochemical properties regulate the oligomerization and functionality of membrane proteins differentially. The results presented here are thus of broad interest to readers in structural biology, membrane biophysics, and functional studies of membrane proteins. Therefore, we believe that Nat. Commun. provides an ideal platform for disseminating our work to a broader scientific audience.\nIn response to reviewer 5:\nIt is well established that the composition of membrane bilayers plays a pivotal role in the regulation of multiple processes. However, how distinct lipid species can affect the activity of membrane proteins is still poorly understood in most cases. In this paper, Yang and co-workers use solid-state NMR and molecular dynamics simulations to study the role of membrane lipids, PG and CDL, in the dimerization and functional activity of a bacterial sugar transporter, VsSemiSWEET.\nThis manuscript makes use of multiple solid-state NMR techniques to resolve the protein structure and identify bound lipids. While the use of these methods is explained with high technical detail, which can make the manuscript difficult to navigate for non-expert readers, the use of MD simulations to investigate the role of the bound lipids is confusing and requires further analysis and clarification, as explained below.\nFirst, they have carried out MD simulations of the VsSemiSWEET dimer in lipid bilayers with no initially bound lipids. The results from these simulations show a preference for CDL over PE and identify key binding residues, which then the authors mutate to corroborate their importance on CDL binding. However, there are some aspects that absolutely need to be addressed concerning these simulations:\n- The setup for the simulations as well as the number of lipid species should be provided, along with the number of replicas.\n- The residence time for PE and CDL lipids is provided, but not that of PG lipids.\n- The mutations proposed in the manuscript, and tested using ssNMR, have not been tested using MD simulations. This is key to corroborate the accuracy of their findings.\n- A density analysis of the lipid bilayers should be provided to further confirm the enrichment of certain lipids in the vicinity of the protein dimer.\nNext, they use docking to incorporate bound lipids to the dimer interface. My main concern here is that MD simulations have already shown the binding of CDL lipids at the dimer interface, why is it needed to add extra lipids using docking? On top of that, the conformation of the docked lipids looks unrealistic for a structure embedded in a lipid bilayer. The authors should test different docking results to validate their results. In addition, the comparison between PG and CDL bound structures to the ssNMR one in Figure 4e is static. An analysis of the RMSD along the entire simulation should also be provided."}, {"round": 3, "reviewer_comments": {"reviewer_1": "the authors have addressed all concerns raised. I support publication.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "In the revision, the authors have addressed my methodological concerns, and the technical quality of the MD simulations is suitable for publication.\nOn the other hand, the manuscript remains very technical, hard to read, and of limited interest to a broad audience. My opinion is that it is better suited for a more specialised journal."}, "author_response": "Response to reviewers\nWe thank the reviewers for carefully reading our manuscript and providing helpful comments on its clarity and presentation. We have addressed each comment as indicated below:\nIn response to reviewer 1:\nThe authors have addressed all concerns raised. I support publication.\nThank you for your constructive feedback throughout the review process.\nIn response to reviewer 2:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nThank you for your valuable feedback.\nIn response to reviewer 5:\nIn the revision, the authors have addressed my methodological concerns, and the technical quality of the MD simulations is suitable for publication.\nWe are pleased that the revised methodology and MD simulations now meet the required standard for publication, and thank you for your valuable suggestions.\nOn the other hand, the manuscript remains very technical, hard to read, and of limited interest to a broad audience. My opinion is that it is better suited for a more specialised journal.\nThank you for your candid feedback on the manuscript's technical depth. While solid-state NMR is a specialized technique, the findings presented in this study address a universal theme in membrane protein biology: how bound lipids with distinct physicochemical properties regulate the oligomerization and functionality of membrane proteins differentially. The results presented here are thus of broad interest to readers in structural biology, membrane biophysics, and functional studies of membrane proteins. Therefore, we believe that Nat. Commun. provides an ideal platform for disseminating our work to a broader scientific audience."}]

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