Lipid dependence of connexin-32 gap junction channel conformations

Domain: physics
Published: 2025-12-05
Editorial outcome: minor_revision

Abstract
Connexin-32(Cx32)gapjunctionchannels(GJCs)mediateintercellularcou-plinginvarioustissues,includingmyelinatingSchwanncells.MutationsinCx32,suchasW3S,areassociatedwithX-linkedCharcot-Marie-Tooth(CMT1X)disease.LipidsregulateCx32GJCpermeation,althoughtheregulatorymechanismisunclear.Here,wedeterminethecryo-EMstructuresofCx32GJCsreconstitutedinnanodiscs,revealingthatphospholipidsblocktheCx32GJCporebybindingtothesiteformedbyN-terminalgatinghelices.Thephospholipid-boundstateiscontingentonthepresenceofasterolmoleculeinahydrophobicpocketformedbytheN-terminus:theN-terminalhelixofCx32failstosustainaphospholipidbindingsiteintheabsenceofcholesterolhemisuccinate.TheCMT1X-linkedW3SmutantwhichhasanimpairedsterolbindingsiteadoptsaconformationoftheN-terminusincompatiblewithphospholipidbinding.OurresultsindicatethatdifferentlipidspeciescontrolconnexinchannelgatingdirectlybyinfluencingtheconformationoftheN-terminalgatinghelix.

Introduction
Cell communication is crucial for coordination of cells with their environment and generation of appropriate cellular responses.

Among different means of cellular communication, gap junction intercellular communication (GJIC) enables a fast and direct signal exchange between neighboring cells and allows electrical and metabolic coupling of the tissues1,2. In vertebrates GJIC is mediated by connexins, which assemble into gap junction channels (GJCs), that contain a central pore, connecting the cytoplasms of two neighboring cells and allowing solute permeation from one cell into the other1–3.

Each of the 21 connexin isoforms in humans has a distinct expression pattern, permeability, and gating properties, establishing the required GJIC between specific cell types4. The GJIC has to be tightly regulated to ensure the passage of appropriate solutes and to prevent the exchange of noxious signals between coupled cells2–4. Among the regulators, lipids (fatty acids, sterols, and phospholipids) have gained increased attention for their effects on GJC permeability. These hydrophobic small molecules have been shown to alter GJC permeability either directly by binding to the channel, or indirectly, via changes in the biophysical properties of the membrane or via activation of distinct signaling pathways5–8.

The potential role of direct lipid-protein interactions with the connexin channels has been illustrated by the recent structural studies of connexin hemichannels (HCs) and GJCs. Ordered lipid-like molecules have been identified at five different locations in recent cryo-electron microscopy (cryo-EM) structures of connexin HCs and GJCs: (i) as annular lipids, lining the extracellular lipid leaflet side of the channel (Cx46/50 GJC9, Cx43 GJC10,11, Cx36 GJC12, and Cx32 HC and GJC13), (ii) below the N-terminal helix (NTH) (Cx43 GJC10,11, Cx43 HC11, Cx32 HC13), (iii) between NTHs (Cx43 GJC10,11), (iv) lining the cytoplasmic entry of the pore (Cx36 GJC12), and (v) lining the inside of channel pore (Cx31.3 HC14, Cx36 12, Cx46/50 9, Cx32 GJC and HC 13 and Cx43 GJCs 10,11. The presence of these ordered lipid-like small molecules around or inside the channels suggest that lipids may have a direct effect on connexin channel structure and function.

Connexin-32 (Cx32) is a widely expressed isoform of the connexin channel family, found abundantly in the liver cells from which it was originally isolated15,16. Cx32 is also found in the myelinating Schwann cells, where it appears to be important for maintaining the cellular homeostasis, with roles for both HCs and GJCs17. Multiple mutations in Cx32 are associated with the X-linked Charcot-Marie-Tooth disease (CMT1X), a disorder accompanied by demyelination of the peripheral neurons and eventually leading to degeneration of the muscles, for which there is currently no cure17–20. Cx32 HCs play a role in secreting ATP at the Schwann cell surface21, and the GJCs are important to maintain lateral connections across the myelin layer22.

We have recently determined the structures of human Cx32 HCs and GJCs in detergent micelles, where we observed a conformation of the N-terminal gating helix of Cx32 supported by a lipid-like molecule in the lipid-1 site, which we interpreted as a molecule of cholesterol hemisuccinate (CHS)13. Our work showed that two CMT1X-linked mutants, W3S and R22G, have a structural and a functional defect in the HCs, with little or no observable structural effects on Cx32 GJCs, and no measurable effect on Cx32-mediated GJC activity in cell-based assays13.

In addition to our direct observation of sterol-like molecules bound to Cx32, the role of lipids in Cx32 regulation has been explored previously (Fig. 1A). Cx32 preferentially resides in cholesterol-rich caveolin-containing lipid rafts8. Phospholipids also directly associate with Cx32 and together with the sterols influence Cx32 HC permeability6. However, how phospholipids and sterols interact with Cx32 channels in the context of a lipid bilayer remains unclear.

In this work, to investigate the effect of phospholipids and sterols on Cx32 channels, we constituted the purified Cx32 channels into lipidic environment in nanodiscs, and determined their cryo-EM structures.

Results
Structures of Cx32 GJC in lipid environment

To address the effect of different lipids on Cx32 GJC, we purified Cx32 in digitonin, and reconstituted the protein into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and liver polar lipids (LPL) -containing nanodiscs (Fig. S1A, B). We chose two different lipid preparations for the following reasons: in the case of POPC we would obtain nanodiscs with clearly defined lipid content, and LPL would represent a close to physiological lipid composition, since Cx32 is known to be abundantly expressed in hepatocytes23. We prepared samples for cryo-EM by plunge-freezing the purified and reconstituted samples in liquid ethane, collected the cryo-EM data, and determined the structures of Cx32 GJC in nanodiscs, at 3.20 Å and 3.29 Å resolution for POPC and LPL, respectively (Figs. S2, S3 and S4A, B, Table 1). The structures were obtained by imposing the D6 symmetry. Refining the particles without imposing symmetry (C1) resulted in a similar reconstruction, albeit at a slightly lower resolution (Figs. S2–3, S5–6).

Overall, the structures of Cx32 GJC in POPC- and LPL-containing nanodiscs are very similar to each other, with an RMSD of 0.495 Å (Fig. 1B, C, Fig. S6), and each was overall similar to the structure of Cx32 GJC in detergent13. The four transmembrane helices (TM1-4) and the extracellular loops 1 and 2 (ECL1-2) of individual connexin subunits have the same conformation in the presence or absence of lipids, indicating that lipids do not affect the assembly of two HCs into a GJC, and do not affect the overall pore architecture. These similarities apply also to the orientation of the individual amino acid residues, with an RMSD of 0.259 Å and 0.636 Å between Cx32 GJC in detergent, and Cx32 GJC in POPC- and LPL-containing nanodiscs, respectively.

Nonetheless, the addition of lipids has a notable effect on the conformation of the Cx32 N-terminal helix (NTH), an α-helical region critical for Cx32 gating13,24,25 (Figs. 2–4). The NTH transitions from a flexible structural element unresolved in our cryo-EM reconstruction of Cx32 GJC in detergent13 to a well-ordered α-helical conformation, as well as moves from lining the channel pore to pointing towards the symmetry axis of the pore. This movement is independent from conformational changes in the TM1-4 or ECL1-2. This conformation resembles the NTH conformation of Cx32 HC that we previously determined using Cx32 samples in detergent13. Performing protomer focused classification (PFC) on the individual subunits of Cx32 GJC in nanodiscs did not expose any substantial variability in the NTH conformation (Fig. S7A, B), indicating that the NTH of all subunits in these conditions assume a similar conformation.

It is noteworthy that, unlike the Cx32 sample that previously led to cryo-EM reconstructions of both GJCs and HCs in detergent micelles13, upon nanodisc reconstitution we could observe predominantly the full GJCs. This may reflect a greater stability of the fully assembled GJCs, allowing these complexes to stay intact during detergent removal. In contrast, HCs may suffer in a number of ways during the lipid reconstitution procedure, which results in very few particles that can be used for cryo-EM image processing and structure determination of HCs in nanodiscs. Therefore our study was focused on the Cx32 GJCs.

Annular and pore-lining lipids

Several lipid-like densities have been identified in the Cx32 GJC structure in detergent. In particular, lipid-1, which lines the inside of the channel pore, and annular lipids on the extracellular membrane leaflet side of the channel. Due to the limited resolution of the 3D reconstructions, the lipid identities could not be unequivocally determined13. The densities at equivalent positions in Cx32 GJC structure in nanodisc are resolved better and could correspond to acyl chains (Fig. 2A). Acyl chains in similar positions were identified in the structures of Cx46/50 GJC9, Cx43 GJC10, and Cx36 GJC12. As we were not able to determine whether these acyl chains represent free fatty acids copurified with the protein, or whether they are parts of bound phospholipids, we left these regions of the EM maps unmodelled (the hexadecane in Fig. 2 is shown for illustration purposes).

Annular lipids are present at the interface between two neighboring connexin subunits (Fig. 2B). The lipid binding pocket is formed by TM1 and TM4 of one subunit and TM2 and TM3 of the neighboring subunit. The pocket is formed predominantly by hydrophobic amino acids. Similarly, lipid-1 binds to a hydrophobic pocket formed by neighboring connexin subunits, in this case formed by TM1 of one and TM1 and TM2 of the second (Fig. 2C). The lipid-1 density spans the majority of the transmembrane domain, from lipid-2 below the NTH to the interface between the transmembrane and extracellular domains.

Sterol-like and phospholipid-like molecules stabilize the NTH

Closer inspection of the density map around the NTH in both Cx32 GJC nanodisc structures, reveals two densities which could stabilize the observed NTH conformation (Fig. 2A). The first is positioned below the NTH (Fig. 2D) and is shaped like a sterol. This density is similar to the one we have observed at the lipid-2 site previously for Cx32 HCs in detergent13, as well as in the structures of Cx43 GJCs solved in both nanodiscs and in detergent10,11. However, the identity of the lipid-2 molecule could not be determined definitively. Although we modeled a cholesterol molecule into the corresponding part of the density map, this density could also accommodate other sterols, such as cholesteryl hemisuccinate (CHS) and digitonin, which were used during stages of protein purification, as described in “Methods” (Fig. S8A). This binding site is hydrophobic, with the W3 residue side chain in direct proximity, participating in the interactions with the bound lipid molecule. Residue W3 is conserved in all β-group connexins (Fig. 2F), suggesting its functional importance in interactions with the lipid-2 as a mediator of NTH conformational changes in Cx32 and other GJCs.

An additional observed density, which we named lipid-3, is located between the neighboring NTH regions. The lipid-3 density has a similar shape in both POPC- and LPL-containing Cx32 GJC nanodisc structures (Fig. 2E, Fig. S8B, C). The density is consistent with the glycerophosphoric segment and for parts of the two aliphatic chains of POPC. The binding site for lipid-3 is formed by residues M1, N2, G5 and L9 of one Cx32 subunit and M1 of the neighboring subunit. The position equivalent to G5 in other β-group connexin isoforms is not conserved but occupied by hydrophobic residues (Fig. 2F), which could mediate the lipid-3 interaction in other β group connexins. Mutation of one of the conserved residues, L9W, is associated with CMT1X26, indicating the functional importance of this site. While our interpretation of the observed densities is that they correspond to the bound phospholipid molecules, we can not completely exclude a possibility that these densities may also correposnd to bent acyl chain of the lipids, or even some other small molecule species present in the sample (co-eluting with the purified protein and appearing as ordered density upon addition of the lipids and removal of the detergent).

Cx32 reconstituted in nanodiscs is effectively plugged by lipid molecules

The structure of Cx32 GJC reconstituted into nanodiscs shows a conformational change of the NTH that leads to a decrease in the pore diameter from approximately 15 Å in an open conformation to 12 Å (Fig. 3A, B, Fig. S8D). This NTH reorganization modifies the electrostatic potential of the cytoplasmic pore entry, which becomes more positively charged (Fig. 3C). We refer to this Cx32 GJC conformation as the ordered NTH state (NO-state). Including lipid-3 in pore diameter calculations shows an additional restriction of the pore diameter to only 4 Å (Fig. 3A, B), which effectively constricts and blocks the pore entry to ions or small molecules.

Lipid-2 binding is required for NTH stabilization

The positions of lipid-2 and lipid-3 in the vicinity of the Cx32 NTH regions, known to be the key gating regions of connexin channels, suggest that these two lipids may stabilize NTH in the NO-state. To assess the requirement for lipid-2 and lipid-3 in NTH stabilization, we determined the GJC structures of (i) Cx32 GJC purified without addition of the cholesterol analog CHS during protein purification, and (ii) Cx32 W3S mutant, which has an impaired lipid-2 binding site13 (Figs. S1C, D, S4, S9–S11). The structures were determined at 3.12 Å and 2.35 Å resolution, respectively, by imposing D6 symmetry, although the same reconstruction at a slightly lower resolution was obtained in C1 symmetry (Fig. S5).

The Cx32 GJC structure in nanodisc without addition of CHS during purification confirmed that the density corresponding to lipid-2 is indeed CHS, and this molecule is a prerequisite for stabilizing the NO-state (Fig. 4A, B). Removal of CHS from the purification procedure, followed by nanodisc reconstitution of Cx32, results in a 3D reconstruction where NTH tilts and aligns along the channel pore, displacing the lipid-2 density (Fig. 4B). This conformation is compatible with an open state of a GJC, similar to those observed for Cx2627 (Fig. 4D, Fig. S12).

W3S GJCs do not form an NO-state in lipidic environment

Mutation of the residue W3 to a serine produced a similar effect on Cx32 GJCs upon lipid reconstitution in the presence of CHS, as removing CHS did on the wild-type Cx32 (Fig. 4C, Fig. S13A). The NTH of W3S GJC in POPC is ordered and points towards the inside of the channel pore, in a conformation slightly different from that in the wild-type Cx32 maintaining a pore diameter of >10 Å consistent with an open channel (Figs. 4D–F, S12). The conformation of the NTH in the W3S GJC in lipidic environment is distinct from the constricted NTH conformation observed in the Cx32-W3S HC (Fig. S13B)13. It is important to note that while structurally the NTH conformations of the W3S GJC in POPC nanodiscs and W3S HC in detergent are distinct, the two states of the mutated protein are also dramatically distinct in their functional properties. Whereas the GJC function of the W3S mutant appears to be very similar to that of the wild-type Cx32, the HC activity of the W3S is dramatically reduced13. The current limitations of our protein preparations prohibit HC structure determination, and we are at loss to explain the reason why Cx32 HCs are refractory to structure determination upon lipid reconstitution. The optimal approach to correlate the structural and functional properties of W3S GJCs and HCs would be to compare the corresponding structures determined for the proteins reconstituted in the same lipidic environment. Stabilization of wild-type and W3S mutant Cx32 HCs in nanodiscs for cryo-EM structure determination will require careful future experimentation.

To determine whether there is a variability in the NTH conformation among the individual subunits of the W3S and the wild-type Cx32 (in the absence of CHS) GJCs, and whether any of the Cx32 subunits still feature lipid-2 or lipid-3 densities that may be averaged out due by imposing the D6 symmetry, we performed protomer-focused classification (Fig. S7C, D). In all classes for both analyzed datasets we could observe the NTH in only one corresponding major conformation. Neither lipid-2 nor lipid-3 could be identified in the classes, further confirming that the lipid-2 corresponds to the CHS molecules added during purification.

Altogether, our results with the wild-type Cx32 in the absence of CHS and the Cx32-W3S GJCs indicate the requirement of the sterol binding site (lipid-2 site) for stabilizing the NTH in a conformation that allows the phospholipids to bind to the lipid-3 sites.

Analysis of Cx32 NTH motion through molecular dynamics simulations

To assess the dynamic interplay between the NTH and the lipids present in the Cx32 pore, we performed molecular dynamics simulations (MD) using a minimal hexameric Cx32 HC system, embedded in a lipid bilayer (see “Methods” for details on the system setup).

For the purposes of our MD analysis, we used cholesterol (CHOL) instead of CHS, because CHOL is a natural sterol that is abundant in biological membranes and is likely to occupy the lipid-2 binding sites in the native Cx32 GJCs/HC. Secondary structure analysis was performed for residues located at the N- and C-termini of each NTH (residues 3–5 and 9–11) in wild-type Cx32 and Cx32 W3S HCs both in the presence and absence of bound lipids, POPC and CHOL. We monitored the average probability of the aforementioned residues adopting either an α-helical or any other type of conformation over the course of two replicate 1 μs MD simulations for each studied system (Fig. 5A, B, E, F). The results indicate a significantly lower probability of these residues forming an α-helix in Cx32 W3S compared to wild-type Cx32 (p≤0.05, Table S1) (Fig. 5A, B). Interestingly, the disruption of the α-helical secondary structure becomes more significant in Cx32 W3S as the simulation time increases (Fig. 5B). This underscores the instability of the NTHs carrying the W3S mutation, which were initially modeled by retaining the Cβ orientation of W3 in the wild-type protein. On the other hand, the presence of bound POPC and CHOL is likely to enhance the stability of the NTHs in both wild-type Cx32 (Fig. 5E) and Cx32 W3S (Fig. 5F) relative to the respective unliganded forms (Fig. 5A, B), although this trend did not reach statistical significance (p>0.05, Table S1).

Structural representations of the last frame (t=1 μs) in one of the two replicate trajectories for each system are provided to visualize the conformational changes undergone by the NTH residues during the MD simulations (Fig. 5C, D, G, H). The depicted structure of apo Cx32 W3S hexamer (Fig. 5D) clearly illustrates the tendency of some of its NTHs to unfold during the microsecond-long MD simulation. These results suggest that longer MD simulations may be required to observe the unfolding process of all the six NTHs. To a lesser extent, a similar phenomenon can be observed in the last frame of Cx32 W3S in the presence of bound lipids (Fig. 5H). In contrast, the NTHs of wild-type Cx32 remained largely intact at the end of the MD simulations in both conditions (Fig. 5A, G). The conclusions drawn from the visual inspection of the last frames are consistent with the secondary structure analysis presented above.

We extended the same analysis to four systems consisting of wild-type Cx32 and Cx32 W3S, each bound to either CHOL or POPC (Fig. S14). Our results show that the NTH stability in the wild-type protein bound to POPC is significantly higher than that of Cx32 W3S in the same condition (p≤0.05, Table S1). A similar trend was observed for the CHOL-bound systems (p=0.078, Table S1). Furthermore, the presence of either CHOL or POPC appears to stabilize the NTHs in both wild-type Cx32 and Cx32 W3S relative to their unliganded forms, although this effect was not statistically significant (p>0.05, Table S1).

The RMSF calculations for the NTHs (Fig. S15) underscore the increased flexibility in the N-terminal residues of Cx32 W3S mutant across all conditions, becoming more significant in the absence of lipids. In particular, the RMSF profile in Fig. S15A suggests that the destabilizing impact of the W3S mutation during the microsecond-long MD simulations is especially evident at both ends of the NTH, where residues exhibit greater flexibility compared to their counterparts in wild-type Cx32. This increased flexibility likely contributes to a progressive shortening of the NTH in the mutant protein, which motivated our prior selection of residues at both termini of the NTH for secondary structure probability analysis (Figs. 5 and S14, and Table S1). Complementary RMSD measurements (Fig. S16) also reveal a greater tendency of the N-terminal residues in the mutated protein to deviate from their initial α-helical conformation compared to those in wild-type Cx32.

To further evaluate the impact of the W3S mutation on the stability of the NTH, we carried out alchemical free energy calculations using the thermodynamic integration (TI) method. A thermodynamic cycle in which the mutation was performed in both in the native conformation of the NTH within the Cx32 HC and in a linear, capped tripeptide model (ACE-Gly-X-Gly-NME, where X is either W or S28; enabled the prediction of the folding free energy difference between the wild-type and the mutant proteins (ΔΔG, Eq. S1). Integration of the 〈∂V/∂λ〉 λ vs. λ profiles for each alchemical transformation, i.e., decharging, van der Waals, and recharging steps (Fig. S17), yielded a ΔΔG of −3.62±0.46 kcal/mol (Table S2). The negative value of the estimated ΔΔG indicates that the folding free energy of Cx32 wt is more favorable than that of Cx32 W3S, consistent with the destabilizing role of the analyzed mutation. The influence of lipids on NTH motion relative to TM1 was investigated by monitoring an angle during MD simulations, defined by the centers of mass of three atom groups at the NTH, the N-terminal end of TM1, and the center of TM1 (see Fig. S18). The angle distributions for wild-type Cx32 show that neighboring NTHs tend to alternate between conformations shifted upward and downward relative to the position observed in the cryo-EM structure of Cx32 in nanodiscs (Fig. S19). A similar trend was observed for Cx32 W3S, with broader angle distributions reflecting partial NTH unfolding (Fig. S20). In contrast, for wild-type Cx32 and W3S with POPC and CHOL at the lipid binding sites, the angle distributions are narrower, suggesting that the bound lipids restrict the NTH motion to positions closer to that observed in the experimental structure (Figs. S21 and S22).

Overall, the results presented here highlight the destabilizing effect of the W3S mutation on the Cx32 NTH. Moreover, we observed that the binding of CHOL and/or POPC is likely to exert a stabilizing influence on these helices in both the mutant and wild-type proteins. Disruption of the CHOL binding site due to the unfolding of the NTH regions in the W3S mutant make the presence of ordered lipids within the pore less likely based on our cryo-EM data.

Dynamic behavior of lipids within the Cx32 pore region

To investigate the conformational dynamics of lipids bound to wild-type Cx32 and Cx32 W3S during the 1-μs replicate MD simulations, we tracked the RMSD values for their heavy atoms relative to the corresponding starting conformations in the initial structure (Fig. 6A–H). The distribution of RMSD values for CHOL indicates that these molecules remained largely in native-like conformations when bound to the wild-type protein, as evidenced by a high and narrow peak around ~2.5 Å (Fig. 6B). In contrast, a wider RMSD distribution was observed for CHOL bound to Cx32 W3S (Fig. 6B), likely due to partial disruption of the NTH residues in the mutated system (Fig. 5F, H). This structural change may compromise the integrity of the CHOL binding site.

In both systems, POPC molecules exhibit broad RMSD distributions throughout the simulations, reflecting their high intrinsic flexibility and binding to a relatively shallow surface on the protein (Fig. 6A). Notably, the main peak of RMSD values for the POPC molecules bound to Cx32 W3S is shifted toward higher values compared to the wild-type system’s distribution. This suggests that the mutation can also compromise the binding of POPC to the NTH region of Cx32. Figure 6B shows the central structures of wild-type Cx32 and Cx32 W3S bound to POPC and CHOL, determined through clustering analysis. Transparent surfaces were generated to enclose the conformational space explored by the ligands’ central structures after superposition of all chains within each system. These structures highlight the loss of the native binding mode of CHOL to Cx32 W3S and reveal a tendency for POPC molecules to adopt conformations in the mutant that are more detached from the NTH compared to the wild-type system. Thus, the MD simulations suggest that CHOL and POPC exhibit stronger binding to wild-type Cx32 than to Cx32 W3S, consistent with experimental findings.

Dominant motions of wild-type Cx32 monomers potentially enabling lipid entry into the pore

Despite mounting evidence supporting the presence of lipids into the Cx32 pore, the entry path of the external lipids remains unknown. To shed some light into this issue, we assessed the dominant motion of the Cα atoms of every Cx32 monomer across all the simulated wild-type HCs using principal component analysis (PCA). Our results indicate that the two largest motions, corresponding to the principal components 1 and 2 (PC1 and PC2), account for nearly 50% of the total protein motion (Fig. 7A). By projecting the sampled phase space on these two PCs, we observed different regions (Fig. 7B), characterized by structural properties that could be relevant to understand the lipid entry, as we will be described hereinafter.

To characterize the dominant motions of wild-type Cx32 monomers, we extracted conformations along PC1 and PC2 corresponding to the minimum, mean, and maximum values of each PC. Structural representations of these frames show that PC1 describes the upward and downward displacement of the NTH along the pore axis, whereas PC2 reflects the lateral displacement of TM2 relative to TM3 (Fig. 7C). Notably, the motion associated with PC1 encompasses the previously described open (downward conformation) and NO states, as well as the identified upward conformation (Fig. 7B, C). These findings are consistent with earlier results derived from NTH–TM1 angle distributions of wild-type Cx32 (Fig. S19). Given that the displacements of the NTH and TM2 represent the most prominent dynamic modes of wild-type Cx32, they are hypothesized to contribute to lipid translocation from the bilayer into the pore.

Interestingly, a wild-type Cx32 monomer frame sampled from a phase space region characterized by negative PC1 values and large positive PC2 values (Fig. 7B) exhibits a partially open tunnel connecting the bilayer with the pore interior (Fig. 7C). However, to enable the passage of lipids such as POPC and cholesterol, this tunnel would require a wider opening, especially at the TM2–TM3 interface. Another monomer, extracted from a region with positive PC1 and a PC2 value higher than that of the previous structure (Fig. 7C), possesses a substantially widened TM2–TM3 opening that could, in principle, allow lipid passage (Fig. 7E). However, in this conformation, the NTH occludes the exit into the pore. These structural analyses suggest that a combination of a large displacement of TM2 away from TM3, coupled with an upward movement of the NTH, would be necessary to enable lipid entry into the pore. Such a concerted motion was not fully captured in the MD simulations conducted here, likely reflecting the inability of unbiased MD to efficiently sample phase space regions separated by high energy barriers. It is also possible that the insertion of lipids into the TM2–TM3 opening, a phenomenon not observed in the analyzed trajectories, could facilitate the upward displacement of the NTH thereby contributing to the formation of the TM1–TM2 exit pathway.

To further evaluate the feasibility of lipid translocation into the pore via the TM2–TM3 and TM1–TM2 openings, we performed steered molecular dynamics (SMD) simulations in which a cholesterol and a POPC molecule, positioned near the TM2–TM3 path entrance, were independently pulled toward the pore. These simulations were initiated from a trajectory frame displaying a fully open TM2–TM3 pathway, as shown in Fig. 7E. Prior to lipid pulling, the NTH–TM1 angle was increased to ~100° to fully open the initially closed TM1–TM2 pathway. Frames extracted from lipid-pulling simulations suggest that concerted movements of the NTH and TM2 can create a tunnel sufficiently wide to allow lipid translocation into the pore (Fig. 7F). However, the conclusions drawn from this approach remain preliminary and constitute an extrapolation of the dominant motions observed during the MD simulations of wild-type Cx32 HCs. A further limitation arises from the absence of the TM2–TM3 connecting segment in the simulated system, as this region is unresolved in the cryo-EM structure. Its presence could restrict TM2–TM3 motion, potentially rendering lipid translocation through this pathway less likely.

Methods
Lipid preparation

The lipids (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) or bovine liver polar lipid extract (LPL)) (Avanti Polar Lipids) dissolved in chloroform at a concentration of 25 mg/ml were transferred to a glass tube and dried under the nitrogen stream until completely dried. A buffer consisting of 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 2% n-Dodecyl β-D-maltoside (DDM) was added to reach lipid concentration of 8.33 mg/ml. The lipids were placed in a sonication bath at 45°C until the lipids were completely dissolved; the clarified lipid stocks were aliquoted and stored at −20°C until use.

MSP2N2 expression and purification

A single colony of E. coli BL21 (DE3) cells transformed with MSP2N2 plasmid was used to inoculate 100 ml of LB medium supplemented with 50 µg/ml kanamycin and grown overnight at 37°C and 180 rpm. Terrific broth (TB) medium (1 L), supplemented with 50 µg/ml kanamycin, was inoculated with 30 ml of the overnight culture, and grown at 37°C and 180 rpm to the OD of ~3.0. Protein expression was induced by addition of IPTG at a final concentration of 1 mM for 3 h at 37°C and 180 rpm. Cells were harvested by centrifugation for 30 min at 3700 × g and 4°C. The cell pellets were resuspended in 50 ml of 40 mM Tris-HCl pH 8.0, 0.3 M NaCl and 1% Triton X-100 per 1 L of cell culture, supplemented with 1 mM PMSF and 10 µg/ml DNase I and lysed by sonication for 15 min on ice at 50% amplitude. The lysate was clarified by centrifugation for 30 min at 20,000 × g. The cleared lysate was added to Ni-NTA resin (2 ml per 1 L of cell culture) and incubated for 30 min at 3°C with constant rotation. The resin was collected in a gravity column, washed with 20 column volumes (CV) of 40 mM Tris-HCl pH 8.0, 0.3 M NaCl, 50 mM Na-cholate and 20 mM imidazole, then 20 CV of the same buffer with 50 mM imidazole and eluted with 5 CV of the same buffer with 400 mM imidazole. The protein was desalted into 20 mM Tris-HCl pH 8.0, 0.1 M NaCl, 0.5 mM EDTA using G-25 PD-10 desalting column, concentrated using an Amicon Ultra 10 kDa cut-off concentrator, aliquoted and stored at −80°C until use.

Cell culture

HEK293F cells were maintained in 15 cm plates in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 1% Penicillin-Streptomycin (PenStrep) at 37°C and 5% CO2. For transfection using branched polyethyleneimine (PEI), the medium was replaced with DMEM supplemented with 2% FSC PenStrep. The DNA and PEI dilutions were prepared separately in un-supplemented DMEM, using 40 µg DNA per plate and PEI in 1:2 ratio of DNA to PEI (w/w). The dilutions were mixed and incubated at room temperature for 5 min and added to the cells in the drop-wise manner. The cells were cultured at 37°C and 5 % CO2 for 48 h, after which they were harvested by scraping and stored at −80°C.

Connexin purification

The protein purification was performed as described previously13. The adherent HEK293F cells were transfected with a pACMV plasmid encoding Cx32 or Cx32-W3S mutant, in frame with a C-terminal 3C-YFP-twinStrep tag. Following a 48 h period after transfection, cells harvested from 100 15-cm culture plates were resuspended in buffer A (25 mM Tris-HCl pH 8.0, 150 mM NaCl) supplemented with protease inhibitors (1 mM benzamidine, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml pepstatin, 1 μg/ml trypsin inhibitor and 1 mM PMSF) and 10 µg/ml DNase I. The cells were lysed by 300 sonication pulses at 35% amplitude (Sonics Vibra-Cell, cycle: 0.5 s pulse on, 0.5 s pulse off). The lysate was clarified by ultracentrifugation at 4°C, 142,000 × g (Ti45 rotor, Beckmann Coulter) for 50 min. The pellet was transferred to fresh buffer A supplemented with protease inhibitors and homogenized. The membranes were solubilized by addition of 1% DDM and 0.2% cholesteryl hemisuccinate (CHS) for 1 h at 4°C, with constant rotation. In the case of purifying Cx32 in the absence of CHS, CHS was omitted in this membrane solubilization step. The membranes were clarified by ultracentrifugation at 4°C, 142,000 × g (Ti45 rotor) for 50 min. The solubilized and clarified membranes were incubated with 2 ml of anti-GFP nanobody coupled CNBr-sepharose resin for 30 min at 4°C, with constant rotation. The resin was applied to a gravity column, washed with 40 column volumes of buffer C (25 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1 % digitonin). The protein was eluted by cleavage using 3C protease (0.4 mg) for 2 h with rotation at 4°C. The eluted protein was concentrated to a volume <800 µl using Amicon Ultra 100 kDa cut-off concentrator.

Nanodisc reconstitution

For nanodisc reconstitution, the purified Cx32 protein was mixed with MSP2N2 and lipids, either 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) or liver polar lipids (LPL), diluted in buffer C to 200 µl, in a molar ratio of 6:2:200 (Cx32:MSP2N2:lipid) and incubated at room temperature at constant rotation for 1 h. The Bio-Beads SM2 Resin (BioRad, 50 mg) was added to the mixture, placed at 4°C with constant rotation overnight. The protein was separated from the BioBeads into a fresh Eppendorf tube, centrifuged for 5 min at 24,000 × g and 4°C using a cooling benchtop centrifuge (Eppendorf). The reconstituted protein was further purified by HPLC using a Superose 6 Increase 10/300 GL column pre-equilibrated with buffer A. The size exclusion chromatography (SEC) fractions corresponding to Cx32 in nanodisc were pooled together and concentrated to ~2 mg/ml (samples: Cx32 POPC nanodisc, Cx32 LPL nanodisc, W3S POPC nanodisc) or ~1.3 mg/ml (sample: Cx32 no CHS POPC nanodisc) for cryo-EM sample preparation.

Cryo-EM sample preparation and data collection

For cryo-EM sample preparation, 3.5 µl aliquots of concentrated protein reconstituted in nanodiscs were applied to glow discharged Quantifoil Cu R1.2/1.3 200-mesh grid. The grids were blotted for 3 s and plunge-frozen in liquid ethane using Vitrobot Mark IV (Thermo Fisher).

The cryo-EM datasets were collected using a Titan Krios electron microscope (Thermo Fisher), equipped with a K3 direct electron detector camera (Gatan) and a GIF-Quantum energy filter, with a slit width of 20 eV. The defocus range was set from −0.5 µm to −2.5 µm. The data was collected as movies dose-fractionated into 40 frames using EPU 2.0. The exposure time for each micrograph was 0.9 s, with a total dose of 61.6 e-/Å2 for Cx32 POPC nanodisc dataset, 61.2 e-/Å2 and 50 e-/Å2 for Cx32 LPL nanodisc datasets, 55 e-/Å2 for Cx32 without CHS POPC nanodisc dataset, and 55 e-/Å2 for W3S POPC nanodisc dataset.

Cryo-EM data processing

Optics groups were assigned to individual movies using a script provided by Dr. Pavel Afanasyev33. The movies were motion-corrected using MotionCor234. The Cx32 POPC nanodisc and Cx32 LPL datasets were CTF-corrected using Gctf35, and Cx32 no CHS POPC nanodisc, and W3S POPC nanodisc datasets using CTFFind436. The two Cx32 LPL nanodisc datasets were merged immediately after CTF refinement. Approximately 1000 particles were picked per dataset, extracted, and subjected to one round of 2D classification in Relion 4.0.137. The best classes, showing features of GJC and HC, were selected for reference-based autopicking in Relion 4.0.1. The autopicked particles were subjected for several rounds of 2D classification, until clear GJC features were observed. Best particles were selected for 3D classification, using Cx32 GJC in detergent as a reference (low pass filtered to 40 Å)13, in D6 symmetry. The best class, showing clear GJC features, was then subjected to 3D auto-refinement with imposed symmetry followed by CTF refinement and particle polishing and final refinement. Autorefinement without imposing any symmetry (C1) was used to assess the influence of symmetry on map quality and features. The local resolution was determined using ResMap38. The detailed steps of the processing pipeline for each sample are described in detail in Figs. S2–S3, S7, S9–10 and Table 1.

Protomer-focused classification was performed by symmetry expanding the particles of the final autorefinement job, using ‘relion_particle_symmetry_expand’ command, using D6 symmetry. The reference map for a single protomer was generated in ChimeraX39, based on the corresponding model, using the ‘Color Zone’ and ‘Split Map’ commands. The reference was used for mask generation in Relion 4.0.1, with 10 Å low pass filtering, extending the binary map with 5 pixels and adding a 20-pixel soft edge. The particles were then subtracted based on the generated mask, recentred on the mask, and rescaled to box size of 200 × 200 pixels. The particles were used for 3D classification without image alignment and symmetry imposition, using appropriately recentred and rescaled mask and reference, to 4, 6 or 8 classes. The best class number was selected by evaluating if more classes reveal more information in the regions of interest without concomitant drastic decrease in resolution (Fig. S7).

Model building and refinement

The models of Cx32 GJC in POPC nanodisc, Cx32 without CHS GJC in POPC GJC nanodisc, and Cx32 GJC in LPL nanodisc were built based on the single subunit of Cx32 WT HC detergent model (PDB ID: 7ZXN). The models of W3S GJC in POPC nanodisc and R22G GJC in POPC nanodisc were built based on the single subunit of W3S and R22G HC detergent models, respectively (PDB ID: 7ZXT and 7ZXO). The models were built in COOT40 by aligning the subunit template into the density and refining amino acid positions. The cytoplasmic regions (CL: L106-H126 (Cx32 POPC, Cx32 LPL, W3S POPC); CT: A218-C283 (Cx32 POPC, Cx32 LPL, W3S POPC) were not built due to not being resolved in the density map. The cholesterol (lipid 2) (chemical ID: CLR) was built into Cx32 POPC nanodisc maps, based on Cx32 HC detergent model (PDB ID: 7ZXN). POPC molecule (chemical ID: POV) was built into Cx32 POPC and LPL nanodisc maps, using first rigid body fit and later real space refine functions in COOT. The side-chain clashes and overlaps were minimized using Chiron41. All models were refined using ‘phenix.real_space_refine’ function and validated using MolProbity42 in PHENIX43. The HOLE analysis of the pore radius was performed in COOT.

Electrostatic surface potential analysis

The electrostatic potential was calculated in PyMOL using APBS Tools 2.1, using AMBER99 force field.

System setup for molecular dynamics simulations

The coordinates for a Cx32 wt HC bound to six POPC and six CHOL (Cx32 wt:POPC:CHOL) molecules were extracted from the cryo-EM structure. The protonation states of the protein’s ionizable residues and the transmembrane region were predicted using the H++ and PPM web servers, respectively44,45. ACE and NME caps were added to residues M105 and I127, respectively, in each Cx32 chain, corresponding to gaps found in the cryo-EM structures.

CHARMM-GUI46 was employed to embed the protein into a lipid bilayer containing major components of plasma membranes from mammalian cells, i.e., 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), palmitoyl sphingomyelin (PSM), 1-palmitoyl-2-oleoylphosphatidylserine (POPS) and cholesterol (CHOL), that are parametrized in the lipid21 force field of Amber 22 (https://opm.phar.umich.edu/biological_membranes/lipid_composition). The resulting bilayer comprised a total of 146 CHOL molecules, 126 POPC molecules, 68 PSM molecules, 68 POPE molecules and 22 POPS molecules. Water molecules were added to form a cuboid box measuring 134.37 Å × 135.51 Å × 102.23 Å, with edges positioned at least 10 Å away from the protein surface and the bilayer stretching along the xy plane. Table 2 summarizes the composition and box dimensions of all simulated systems.

The system’s PDB file, assembled using CHARMM-GUI, was processed by the program charmmlipid2amber.py from AmberTools22 in order to convert the CHARMM naming convention into AMBER47. The PDB files for other systems prepared for MD simulations, which included Cx32 wt with no lipids in the pore (apo Cx32 wt), apo Cx32 W3S, Cx32 W3S with POPC and CHOL in the pore (Cx32 W3S:POPC:CHOL), Cx32 wt with only POPC in the pore (Cx32 wt:POPC), Cx32 wt with only CHOL in the pore (Cx32 wt:CHOL), Cx32 W3S with only POPC in the pore (Cx32 W3S:POPC) and Cx32 W3S with only CHOL in the pore (Cx32 wt:CHOL), were generated from the processed PDB of Cx32 wt:POPC:CHOL, either the initial structure or an equilibrated frame (see below). In all cases, “CONECT” records for S-S bonds in the proteins were obtained using pdb4amber of Amber2247, which ensured the correct formation of such bonds before further processing for all-atom MD simulations.

The program tleap was employed to obtain the topologies and the coordinate files for the studied systems and to add the counterions (4 Na+, Table 2) necessary to neutralize the simulation boxes. The force fields ff19SB and lipid21 were chosen to parametrize the protein and the lipids, including those in the bilayer or within the protein pore. The water molecules were treated with the OPC model47. Hydrogen mass repartitioning was carried out to increase the timestep from 2 to 4 fs during the production runs48. It is worth noting that the initial structures to generate the topology and coordinate files of systems involving the W3S mutation were derived from the equilibrated structures (see below) of the wild-type systems, by manually renaming the W3 residue to SER in the PDB files and deleting the side-chain atoms beyond the CB. The missing hydroxyl group of the Ser residue was added automatically by tleap.

Molecular dynamics simulations

Prior to the production runs, all systems were subjected to energy minimization (EM) and equilibration steps. A total of 3000 EM cycles were performed for each system using the program pmemd.MPI47. The minimized systems were subjected to heating in two subsequent simulations. During the first phase, the temperature was linearly increased from 10 to 100 K during 500 ps. Random initial velocities drawn from a Maxwell-Boltzman distribution at 10 K were assigned to every atom at the beginning of this heating process. Then, during the second phase, the temperature was linearly increased from 100 to 303 K during 600 ps. In both cases, the heavy atoms of the protein and all lipids were restrained to their initial positions using a harmonic potential defined by a spring constant of 10 kcal·Å−2·mol−1. The cell volume was kept constant and the temperature was controlled using the Langevin thermostat, with a collision frequency of 1.0 ps−1.

Following the heating phase, the systems underwent four NPT equilibration steps, during which position restraints were applied exclusively to the heavy atoms of the protein and the lipids within the pore (if present). The restraint constant was reduced from 8 to 2 kcal·Å⁻²·mol⁻¹ in 2 kcal·Å⁻²·mol⁻¹ strides over the four 8 ns MD simulations. During these equilibration steps, the systems were simulated at constant temperature and pressure of 303 K and 1 bar, which was achieved by using the Langevin thermostat and the Monte Carlo barostat, respectively. Finally, all systems were subjected to 1 μs production runs in the NPT ensemble, in identical conditions to those used during the NPT equilibration except for the position restraints, which were eliminated. All the steps, from heating to production runs, were carried out in duplicate with pmemd.cuda of Amber2249. Short-range electrostatic and van der Waals interactions were explicitly calculated using a 10 Å cutoff, while long-range electrostatic interactions were treated with the Particle Mesh Ewald (PME) method.

It is worth noting that, as each Cx32 HC contains six monomers, the two replicate MD trajectories per system correspond to twelve independent monomer simulations, providing statistical robustness for the monomer-based analyses.

Thermodynamic integration free energy calculations

Thermodynamic integration (TI) free energy calculations were performed to assess the impact of the W3S mutation on the stability of the Cx32 NTH. The folding free energy difference between the wild-type and mutant proteins (ΔΔG) was determined based on a thermodynamic cycle described in detail elsewhere, in which the reference unfolded state is modeled as a capped tripeptide (ACE-GXG-NME), where X represents the residue subjected to mutation28. According to this cycle, ΔΔG is calculated as (Eq. 1):

ΔΔG = ΔGwt Fold - ΔGmut Fold = ΔGW3→S3 Unfolded - ΔGW3→S3 Folded (1)

where ΔGwt Fold and ΔGmut Fold correspond to the folding free energies of the wild-type and the mutant proteins, respectively. On the right-hand side, ΔGW3→S3 Unfolded and ΔGW3→S3 Folded stand for the free energy changes associated with the alchemical W3S mutation in the unfolded and folded states, respectively. The calculation of these free energy changes was performed in three distinct stages for both the folded and unfolded systems: (i) decharging of the W3 residue, (ii) van der Waals transformation of W3 into S3 with atomic charges set to zero, and (iii) recharging of the S3 residue. All alchemical transformations proceeded through a series of intermediate hybrid states controlled by the coupling parameter λ, which was varied from 0 to 1 in increments of 0.05. In total, 126 MD simulations were performed to complete the three alchemical transformations for both the folded and unfolded states. System preparation and MD simulations followed a previously established protocol with minor modifications50. Specifically, no dummy particles were introduced, and the alchemical transformations for the folded and unfolded states were conducted in separate simulation boxes, as the mutation does not involve a net-charge variation. To improve simulation efficiency, hydrogen mass repartitioning was applied, allowing the use of a 4 fs integration timestep. Cuboid boxes with edges placed at least 9 or 13 Å away from the solute (HCs or capped tripeptides) were generated for every system (HCs or capped tripeptides). The HC was simulated in an aqueous environment without the lipid bilayer to reduce computational cost, a simplification justified by the absence of direct interactions between the NTH and membrane lipids. The parameters for the protein and peptide atoms were derived from the ff19SB force field. Table S3 presents a detailed description of the simulation boxes and their composition for each system employed for TI free energy calculations.

For each λ value, 20 ns of productive MD simulation was performed in both folded and unfolded states, with all simulations run in duplicate using distinct initial velocities, assigned during the heating step. To enhance convergence in the folded state during the van der Waals transformation, the simulations at λ values of 0.75, 0.80, and 0.85 were extended to 100 ns, with the λ = 0.80 state further subjected to quadruplicate runs (Fig. S17).

The free energy changes corresponding to the decharging, van der Waals, and recharging steps for both the folded and unfolded states were determined by integrating the respective 〈∂V/∂λ〉 λ versus λ profiles obtained from the simulations. Integration was performed using the trapezoidal rule, and the standard errors of the mean (SEM) calculated for each λ point across replicate simulations were propagated to estimate the SEM associated with the resulting free energy values. To ensure equilibration, the initial 25% of frames from each production run were excluded from the analysis.

Steered molecular dynamics simulations

Steered molecular dynamics (SMD) simulations were performed to introduce one POPC and one CHOL molecule, each positioned within the lipid bilayer, into the open tunnel conformation formed between the TM2–TM3 and TM1–TM2 openings of a wild-type Cx32 monomer in the hemichannel (HC). The simulations were initiated from a trajectory frame exhibiting a fully open TM2–TM3 pathway (Fig. 7E), which had undergone heating and NPT equilibration steps as described previously for conventional MD simulations. To open the tunnel on the TM1–TM2 side, the N-terminal helix (NTH) of this monomer was displaced upward using an angular collective variable (CV) involving residues 3–10, 19–21, and 26–35 (Fig. S18). This CV was gradually increased from 1.09 rad to 1.7 rad over a 50 ns SMD simulation using a harmonic potential with a spring constant of 20 kcal·mol⁻¹·rad⁻², carried out with pmemd.cuda.

The final frame of this SMD simulation displayed a fully open tunnel connecting the pore to the bilayer. In separate 50 ns SMD simulations, a CHOL or POPC molecule located near the TM2–TM3 tunnel entrance was pulled into the tunnel. The collective variables were defined as the Euclidean distance between the center carbon atom of each lipid and a dummy particle fixed at the tunnel center. To prevent translational or rotational motions of the entire HC with respect to this dummy particle, a previously described approach requiring the presence of two additional fixed dummy particles was employed50. A spring constant of 20 kcal·mol⁻¹·Å⁻² was applied during the pulling process.

Trajectory analysis

Structural analyses along the trajectories were carried out with different commands of the program cpptraj of Amber2247. The secstruct and rmsf commands were employed to monitor the secondary structure and atomic fluctuations of residues within the N-terminal region of Cx32 along the MD trajectories, respectively. Moreover, the rms and angle commands were used to calculate root-mean-square deviations for the bound lipids and to monitor the angle formed between the NTH and TM1 during the MD simulations, respectively47.

Clustering analysis was performed with the cluster command of cpptraj to determine the representative structure of Cx32 and the bound lipids during the MD simulations. The hierarchical agglomerative method was chosen for clustering and the RMSD the heavy atoms of the bound lipids and of residues belonging to the lipid binding site were used as the metric for clustering47.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Cryo-EM structures of large-pore forming channels have revealed lipidic molecules within the pore, suggesting a novel mechanism in which lipids contribute to channel gating. This manuscript presents high-resolution cryo-electron microscopy (cryo-EM) structures of connexin-32 (Cx32) gap junction channels (GJCs) reconstituted in lipid nanodiscs. The study builds on previous structural work by the authors, which identified lipid densities in Cx32 channels prepared in detergents. The current work identifies three lipid-binding sites (lipids 1–3) and provides evidence suggesting that lipid 2 is likely cholesterol, based on electron density and the structures obtained without cholesterol-hemisuccinate (CHS) during purification. Lipid 3 is located within the pore, potentially obstructing the passage of large molecules. A disease-associated mutation, W3S, appears to exclude both lipid 2 and 3, resulting in a constitutively open channel. These findings suggest a novel lipid-mediated gating mechanism in Cx32.\n\nHowever, the study reveals major discrepancies between the cryo-EM data and molecular dynamics (MD) simulations. The cryo-EM structures suggest that the W3S mutation excludes lipid 2 while stabilizing the N-terminal helices (NTHs) in an ordered conformation. In contrast, MD simulations predict that NTHs are largely disordered in the W3S mutant and that cholesterol stabilizes NTH. Furthermore, protomer-focused classification of the cryo-EM data shows minimal conformational heterogeneity, whereas MD simulations indicate significant subunit variability. These inconsistencies raise concerns regarding the interpretation and physiological relevance of the lipid densities. The authors acknowledge these limitations but further clarification is warranted.\n\nMajor Comments\n1. The mechanism by which lipids, particularly lipid 3, exit the pore to allow channel opening is not well explained. Additionally, it is unclear how cholesterol at lipid 2 site exits or redistributes within the membrane environment. If the functional behavior of Cx32 is sensitive to cholesterol levels, could lipid 2 relocate to a non-cholesterol-rich leaflet? Further simulations or experimental data would help support the proposed lipid-gating model.\n2.The mechanism by which membrane lipids enter the pore remains speculative. This is a crucial gap, as it directly impacts the plausibility of the lipid-gating hypothesis. Targeted MD simulations could help elucidate whether such lipid infiltration is physically and biologically reasonable.\n3. Although previous cryo-EM maps in detergents showed lipid densities in Cx32 hemichannels, this study focuses exclusively on gap junction channels. The absence of hemichannel data from nanodisc preparations makes the interpretation incomplete. Including these structures, or at least discussing them in greater depth, would provide a more holistic understanding of lipid effects across Cx32 assemblies.\n4. It is unclear whether lipid 2 is observed in Cx32 structures purified without CHS and reconstituted into LPL nanodiscs. Clarifying this point is essential to assess the relevance of lipid 2 and to rule out CHS-related artifacts.\n\nMinor Comments\n1. The manuscript currently relies on qualitative comparisons of MD simulation results. Incorporating statistical analysis would provide a more robust framework for evaluating these results.\n2. Line 46: \"The potential role of direct of lipid-protein interactions...\" should be \"The potential role of direct lipid-protein interactions...\"\n3. Line 99~: The sentence “The NTH transitions from a disordered to an ordered α-helical conformation…” is introduced without sufficient context. A brief description or reference to the disordered state would help the reader understand the significance of this transition.\n4. Line 170: \"The NTH of Cx32 GJC in POPC...\" Please specify that the NTH structure described in POPC refers to the W3S mutant, to avoid confusion.\n5. Line 172~: The difference between NTH conformations in the W3S GJC versus the Cx32-W3S hemichannel is noted, but its structural or functional significance is not elaborated. Further explanation would strengthen the discussion.", "reviewer_2": "The manuscript by Lavriha and colleagues describes a structural biology study of connexin 32 gap junction channel (Cx32GJC) with particular emphasis on the role lipids play in the regulation of this interesting protein. In vivo, Cx32CJG forms oligomeric complex to connect cytoplasm of two neighbouring cells allowing passage of solutes. It is known that lipids play important role in regulating the activity of Cx32CJG and other connexins. In this manuscript authors performed a very careful structural biology study whereby Cx32CJG was reconstituted in lipid nanodiscs. This allowed visualization of another molecule of lipidic origin that, through complex interplay with cholesterol molecule, affected the structural properties of the complex resulting in significantly changed opening of the channel. The structural biology study is supplemented by molecular dynamics characterization of the complex with bound lipids. The study is well written and presented and provides additional insights into the regulation of connexin gap junction proteins by small molecules.\n\nA lot of care was invested to show the presence of the additional lipid (in the manuscript it is referred to as lipid-3) at different experimental conditions. However, I think the main shortcoming of the work is that the density of this molecule is poorly defined in terms of size. Authors assign phospholipid molecule to this density, as it could correspond to the part around glycerol moiety and parts of the acyl chains. The text describing this is carefully used, but I think extra care should be taken not to overinterpret this result at its current stage (i.e. discussion in the introduction section, lines 289-296 on sterols and phospholipids- but these later may not be phospholipids). Could it be that this density could be assigned to some other molecule deriving from the purification procedure and assembly of Cx32CJG into nanodiscs? Could this represent a bent acyl chain? Could the same result be obtained by using their previous method (ref 13) obtaining Cx32CJG oligomers in detergent, but in the presence of some phospholipid(s)?\n\nThe interplay between different lipids and structural changes in connexin proteins was previously demonstrated. The results should be, therefore, carefully presented in corresponding parts of the manuscript (abstract, introduction, discussion) and discussed in the view of this previous work, in particular in relation to work from reference 10.\n\nThe last paragraph of the discussion describes the effects of gating induced by lipids in other proteins and it is claimed that clear parallels could be drawn by these and their system. But what are these parallels, this is not apparent to me? Is it the same binding to the interior of the channel, affecting the Nterminal region? Please be more specific what these clear parallels are.\n\nFigure 2: what are yellow rectangles in Figure 2A? It should be explained in the legend to figure. If they represent lipid bilayer, are they not too thick (i.e. in comparison to the ones shown in Figure 1B and C)?\nFigure 3: What does abbreviation ONS means in panel A, define please.\nFigure 3: Panel A shows a comparison between protein in detergent (previous work) and nanodiscs (current work). The PDB Id of the structure used for this panel should be stated in the legend (and the reference).\nFigure 7: Panel B of Figure 7 does not contribute anything conceptually and this state of the protein could be described only in the text.\n\nMaterials and methods section: what was the source of POPC and LPL (lines 19,20)? Please define.\nThe purification of connexin (lines 51-66) is not clear to me. Why do you need anti-GFP nanobody coupled CNBr Sepharose for purification? Does the construct contains GFP with appropriate cleavage site (as in the next step something is cleaved off)? Please be more specific here about the construct that was used for the study.\nFigure S1: There are two bands for Cx32 on gels shown in Figure S1 in A,B and no protein in panel C.. Please comment.\nLegend to Figure S8 does not correspond to the figure.", "reviewer_3": "The manuscript “Lipid dependence of connexin-32 gap junction channel conformations” by Lavriha et al. describes a series of cryoEM structures of Connexin-32 wildtype and the disease-associated mutation W3S. The structures provide insights into the gating mechanism of connexin channels, supporting a role of sterols and phospholipids to directly bind to the inner channel pore, thereby stabilizing the N-terminal helix in a closed channel conformation. Accompanying molecular dynamics simulations strengthen the idea that phospholipids and sterols dock to different sites in the inner pore of Connexin-32 and stabilize the N-terminal helix. The manuscript is overall very well written and easy to follow. The authors present a thorough study, which focuses on a topic of wide interest in this area, and which highlights the importance of lipids in regulating gap junction channels. I only have minor points that should be addressed by the authors:\n1. The authors describe that the Cx32 wildtype structure was used as the starting structure for all MD simulations. However, the authors should also include how they introduced the mutation into the system prior to the MD simulations, and how they decided, which rotamer of serine to choose, so that the observed destabilization of the N-terminal helix is not a result of the choice of serine rotamer in that local area.\n2. A key effect of the binding of sterols and phospholipids to the channel pore appears to be the support of the N-terminal helix to rearrange from the open to the NO state. Such conformational changes occur over long timescales, and are not captured by the performed 1 µs simulations. Other studies have shown that e.g. Hamiltonian Replica Exchange MD are required to compute the free energy of the open-to-closed transition (e.g. Mhashal et al., ACS Catalysis, 2020)."}, "author_response": "RESPONSE TO REVIEWER’S COMMENTS\nReviewer #1 (Remarks to the Author):\nCryo-EM structures of large-pore forming channels have revealed lipidic molecules within the pore,\nsuggesting a novel mechanism in which lipids contribute to channel gating. This manuscript presents\nhigh-resolution cryo-electron microscopy (cryo-EM) structures of connexin-32 (Cx32) gap junction\nchannels (GJCs) reconstituted in lipid nanodiscs. The study builds on previous structural work by\nthe authors, which identified lipid densities in Cx32 channels prepared in detergents. The current work\nidentifies three lipid-binding sites (lipids 1–3) and provides evidence suggesting that lipid 2 is likely\ncholesterol, based on electron density and the structures obtained without cholesterol-hemisuccinate\n(CHS) during purification. Lipid 3 is located within the pore, potentially obstructing the passage of large\nmolecules. A disease-associated mutation, W3S, appears to exclude both lipid 2 and 3, resulting in a\nconstitutively open channel. These findings suggest a novel lipid-mediated gating mechanism in Cx32.\nHowever, the study reveals major discrepancies between the cryo-EM data and molecular dynamics\n(MD) simulations. The cryo-EM structures suggest that the W3S mutation excludes lipid 2 while\nstabilizing the N-terminal helices (NTHs) in an ordered conformation. In contrast, MD simulations predict\nthat NTHs are largely disordered in the W3S mutant and that cholesterol stabilizes NTH. Furthermore,\nprotomer-focused classification of the cryo-EM data shows minimal conformational heterogeneity,\nwhereas MD simulations indicate significant subunit variability. These inconsistencies raise concerns\nregarding the interpretation and physiological relevance of the lipid densities. The authors acknowledge\nthese limitations but further clarification is warranted.\nMajor Comments\n1. The mechanism by which lipids, particularly lipid 3, exit the pore to allow channel opening is not well\nexplained. Additionally, it is unclear how cholesterol at lipid 2 site exits or redistributes within the\nmembrane environment. If the functional behavior of Cx32 is sensitive to cholesterol levels, could lipid\n2 relocate to a non-cholesterol-rich leaflet? Further simulations or experimental data would help support\nthe proposed lipid-gating model.\n2.The mechanism by which membrane lipids enter the pore remains speculative. This is a crucial gap,\nas it directly impacts the plausibility of the lipid-gating hypothesis. Targeted MD simulations could help\nelucidate whether such lipid infiltration is physically and biologically reasonable.\nRESPONSE: We are grateful to the reviewer for these questions. The points 1 and 2 are related, so we\nconsolidated our answers to both questions here. Following these comments of the reviewer we have\ninitiated MD-based analysis of the putative lateral access movement of the lipid molecules into the pore.\nWe provide here a possible explanation of how the lipids may be able to access their cognate binding\nsites. We should stress that these MD simulations are at the moment not directly supported by\nexperiments, as this presents a distinct challenge in itself. However, the observations now included in\nthe revised manuscript (please see below) are an important first step towards a better understanding\nof the lateral lipid movement in connexin channels.\nTo elucidate potential pathways for lipid entrance, we conducted principal component analysis (PCA)\nto capture the most significant motions of the Cα atoms of the Cx32 monomer, using data from all\nsimulations of wild-type Cx32 HCs presented in this study (see Figures 7A-C in the revised manuscript).\nIn total, the analysis encompassed 6 chains per system × 2 replicate simulations × 4 systems containing\nCx32 wild-type × 1 μs per simulation, amounting to 48 μs of simulation time. As described in the updated\nmanuscript, the dominant motions associated with the first two principal components correspond to: (1)\n1\nvertical displacement of the N-terminal helix (NTH) relative to the membrane plane, and (2) the lateral\ndisplacement of TM2 relative to TM3. These two movements could facilitate the formation of a transient\nlateral access pathway through which cholesterol and POPC might, in principle, enter (see Figures 7D-\nE in the resubmitted manuscript). Pulling simulations indicate that lipid entrance would require a greater\nvertical displacement of the NTH than what was observed in conventional MD simulations of the Cx32\nwt HCs (Figure 7F). Nevertheless, it can be hypothesized that the initial insertion of a lipid between\nTM2 and TM3 may trigger a wider vertical movement of the NTH. It is important to note, however, that\nthis hypothesis needs to be experimentally validated. Moreover, our simulations are limited by the\nabsence of the flexible TM2–TM3 linker, which is unresolved in available structural data and was\ntherefore not modeled. The presence of this linker could potentially constrain the separation between\nTM2 and TM3 observed in our simulations. These aspects are detailed in the revised version of the\nmanuscript.\n3. Although previous cryo-EM maps in detergents showed lipid densities in Cx32 hemichannels, this\nstudy focuses exclusively on gap junction channels. The absence of hemichannel data from nanodisc\npreparations makes the interpretation incomplete. Including these structures, or at least discussing\nthem in greater depth, would provide a more holistic understanding of lipid effects across Cx32\nassemblies.\nRESPONSE: We are grateful to the reviewer for highlighting this discrepancy. This prompted us to\nimprove the description of the outcome of nanodisc reconstitutions. We were essentially unable to\nobtain the hemichannel reconstructions for the lipid-reconstituted Cx32 channels. While this was\npossible for the detergent samples, upon nanodisc reconstitution the remaining particles that could lead\nto a 3D reconstruction were predominantly those of gap junction channels. We clarified this in the\nrevised manuscript (page 4, line 108):\n“It is noteworthy that, unlike the Cx32 sample that previously led to cryo-EM reconstructions of both\nGJCs and HCs in detergent micelles 13, upon nanodisc reconstitution we could observe predominantly\nthe full GJCs. This may reflect a greater stability of the fully assembled GJCs, allowing these complexes\nto stay intact during detergent removal. In contrast, HCs may suffer in a number of ways during the lipid\nreconstitution procedure, which results in very few particles that can be used for cryo-EM image\nprocessing and structure determination of HCs in nanodiscs. Therefore our study was focused on the\nCx32 GJCs.”\n4. It is unclear whether lipid 2 is observed in Cx32 structures purified without CHS and reconstituted\ninto LPL nanodiscs. Clarifying this point is essential to assess the relevance of lipid 2 and to rule out\nCHS-related artifacts.\nRESPONSE: As we observed virtually identical results with the POPC- and LPL-reconstituted Cx32\nsamples, we opted to focus on the POPC-reconstituted nanodiscs only when it came to the samples\nwhere CHS was omitted during the purification: LPL reconstitutions were not performed in the absence\nof CHS. This was in part motivated by the very poor basal yields of purified protein upon omission of\nCHS from the purification procedure, which is not uncommon for membrane proteins. Performing the\nexperiments in the presence of a chemically defined lipid species, POPC, appeared to be the most\nstraightforward approach producing interpretable results.\nMinor Comments\n1. The manuscript currently relies on qualitative comparisons of MD simulation results. Incorporating\nstatistical analysis would provide a more robust framework for evaluating these results.\nRESPONSE: We have now included the statistical analysis of the key parameters resulting from the\nMD simulation analysis (Table S1). The probability of adopting an α helix or an alternative conformation\n(labeled as Other in the new figures 5 and S14) was assessed for every NTH in the HCs throughout\nthe two replicate simulations conducted for every system. Averaging of these probabilities over the\ncourse of the simulations (discarding the first 600 ns) produced 12 probability values for the segment\noccurring as an alpha helix and 12 values for that occurring as a random coil. We assessed the\n2\nnormality of the data and then applied appropriate statistical tests to compare the probabilities in a pair-\nwise fashion for the systems of interest in different conditions (Table S1). As shown in the updated\nversion of the manuscript, the probability differences between Cx32 wt and Cx32 W3S are statistically\nsignificant (p<0.05).\nWe would like to stress that the secondary structure probabilities shown in the graphs correspond\nspecifically to residues 3-5 and 9-11, at both ends of the NTH, rather than to the full 3-10, as wrongly\nindicated in the previous version. Residues 6, 7, and 8 were deliberately excluded from the analysis\nbecause the center of the alpha helix exhibited less conformational variability, as observed especially\nin Figure S15A. Their inclusion would have biased the average values toward higher helicity and thus\nmasked the destabilizing effect of the mutation on both NTH ends. The figure captions have been\nrevised to reflect these clarifications. Moreover, differently from the previous versions of Figures 5 and\nS14, the updated figures use a binary classification of the secondary structure as (1) α helix or (2) other,\nwhich groups all other categories. By doing so, the bar heights are the same in all of the analyzed time\nintervals, which makes it easier to detect differences in the probability of the selected residues adopting\nan α-helical conformation in the studied systems.\nIn addition, to further evaluate the impact of the W3S mutation on NTH stability, we performed\nthermodynamic integration free energy calculations. These calculations were carried out for a\nthermodynamic cycle involving the W3S mutation both in the context of the hemichannel NTH and in a\ncapped peptide (ACE-Gly-X-Gly-NME, where X is either Trp or Ser). This linear peptide models the\nunfolded state. The resulting ΔΔG of approximately -3.62 kcal/mol supports a clear stabilization of the\nfolded (α-helical) state over the unfolded state by Trp relative to Ser. The new results and the\nmethodology are included in the revised manuscript (Table S2 and Figure S17).\n2. Line 46: \"The potential role of direct of lipid-protein interactions...\" should be \"The potential role of\ndirect lipid-protein interactions...\"\nRESPONSE: We have corrected this line accordingly (page 2, line 46).\n3. Line 99~: The sentence “The NTH transitions from a disordered to an ordered α-helical\nconformation…” is introduced without sufficient context. A brief description or reference to the\ndisordered state would help the reader understand the significance of this transition.\nRESPONSE: We thank the reviewer for spotting this ambiguous statement. This may be a semantic\ndistinction, but indeed we can not claim that NTH is “disordered” – it may be better to state “flexible” or\n“unresolved”. It is indeed possible that NTH remains alpha-helical while being flexible. To more\naccurately reflect our observations, this sentence now reads (page 3, line 99):\n“The NTH transitions from a flexible structural element unresolved in our cryo-EM reconstruction of\nCx32 GJC in detergent 13 to a well ordered..”.\n4. Line 170: \"The NTH of Cx32 GJC in POPC...\" Please specify that the NTH structure described in\nPOPC refers to the W3S mutant, to avoid confusion.\nRESPONSE: We have updated the text accordingly (page 6, line 181):\n“The NTH of W3S GJC in POPC is ordered and points towards the inside of the channel pore, in a\nconformation slightly different from that in the wild-type Cx32 maintaining a pore diameter of >10 Å\nconsistent with an open channel (Figure 4D-F, S12).”\n5. Line 172~: The difference between NTH conformations in the W3S GJC versus the Cx32-W3S\nhemichannel is noted, but its structural or functional significance is not elaborated. Further explanation\nwould strengthen the discussion.\nRESPONSE: We have added the following text into the corresponding part of the discussion (page 6,\nline 185):\n3\n“It is important to note that while structurally the NTH conformations of the W3S GJC in POPC\nnanodiscs and W3S HC in detergent are distinct, the two states of the mutated protein are also\ndramatically distinct in their functional properties. Whereas the GJC function of the W3S mutant appears\nto be very similar to that of the wild-type Cx32, the HC activity of the W3S is dramatically reduced 13.\nThe current limitations of our protein preparations prohibit HC structure determination, and we are at\nloss to explain the reason why Cx32HCs are refractory to structure determination upon lipid\nreconstitution. The optimal approach to correlate the structural and functional properties of W3S GJCs\nand HCs would be to compare the corresponding structures determined for the proteins reconstituted\nin the same lipidic environment. Stabilization of wild-type and W3S mutant Cx32 HCs in nanodiscs for\ncryo-EM structure determination will require careful future experimentation.”\nReviewer #2 (Remarks to the Author):\nThe manuscript by Lavriha and colleagues describes a structural biology study of connexin 32 gap\njunction channel (Cx32GJC) with particular emphasis on the role lipids play in the regulation of this\ninteresting protein. In vivo, Cx32CJG forms oligomeric complex to connect cytoplasm of two\nneighbouring cells allowing passage of solutes. It is known that lipids play important role in regulating\nthe activity of Cx32CJG and other connexins. In this manuscript authors performed a very careful\nstructural biology study whereby Cx32CJG was reconstituted in lipid nanodiscs. This allowed\nvisualization of another molecule of lipidic origin that, through complex interplay with cholesterol\nmolecule, affected the structural properties of the complex resulting in significantly changed opening of\nthe channel. The structural biology study is supplemented by molecular dynamics characterization of\nthe complex with bound lipids. The study is well written and presented and provides additional insights\ninto the regulation of connexin gap junction proteins by small molecules.\nA lot of care was invested to show the presence of the additional lipid (in the manuscript it is referred to\nas lipid-3) at different experimental conditions. However, I think the main shortcoming of the work is\nthat the density of this molecule is poorly defined in terms of size. Authors assign phospholipid molecule\nto this density, as it could correspond to the part around glycerol moiety and parts of the acyl chains.\nThe text describing this is carefully used, but I think extra care should be taken not to overinterpret this\nresult at its current stage (i.e. discussion in the introduction section, lines 289-296 on sterols and\nphospholipids- but these later may not be phospholipids). Could it be that this density could be assigned\nto some other molecule deriving from the purification procedure and assembly of Cx32CJG into\nnanodiscs? Could this represent a bent acyl chain? Could the same result be obtained by using their\nprevious method (ref 13) obtaining Cx32CJG oligomers in detergent, but in the presence of some\nphospholipid(s)?\nRESPONSE: This is a great point by the reviewer. We thought along the same lines as the reviewer,\npondering the different possibilities. In the end we decided that the most likely explanation for what we\nhave seen in our structures were six partially ordered phospholipid molecules that roughly match the\nbifurcated densities in the vicinity of the NTH regions. This seemed to be the most obvious explanation\nat the time – and it still is. However, the reviewer is right – it is entirely possible that this density feature\ncould potentially match a bent acyl chain, or yet another molecule that co-elutes along with the protein.\nThe simple logic of interpreting this density as the one lipid species that we have supplemented during\nreconstitution makes sense, however we agree that this needs to be worded even more carefully, as in\nthe revised text (page 5, line 152):\n“While our interpretation of the observed densities is that they correspond to the bound phospholipid\nmolecules, we can not completely exclude a possibility that these densities may also correposnd to\nbent acyl chain of the lipids, or even some other small molecule species present in the sample (co-\neluting with the purified protein and appearing as ordered density upon addition of the lipids and removal\nof the detergent).”\nRegarding the idea of mixing the lipid into the detergent sample, while it does seem like a potentially\nattractive approach, we opted to stay clear of it. The whole point of our study was to exclude the mixed\npopulation of lipids and detergents, and we reasoned at the design stage that we risk obscuring the\ninterpretation even more by mixing lipids in without removing the detergents completely from the\nsample.\nThe interplay between different lipids and structural changes in connexin proteins was previously\ndemonstrated. The results should be, therefore, carefully presented in corresponding parts of the\nmanuscript (abstract, introduction, discussion) and discussed in the view of this previous work, in\nparticular in relation to work from reference 10.\nRESPONSE: We are grateful to the reviewer for pointing this out. The excellent study described\nreference 10 was on Cx43. This is a different homologue of the connexin family, with potentially different\nregulatory mechanisms. Following the suggestion of the reviewer we have added new text in the\ndiscussion, to reflect the previous work. The added text reads as follows (page 11, line 419):\n“For example, a comprehensive structural analysis of the conformational changes in Cx43 revealed the\nexistence of distinct conformation states of the NTH: the gate-covering, pore lining, and flexible\nintermediate NTH states 10. Moreover, Lee et al. found that the presence of CHS, varied pH and use of\na C-terminally truncated Cx43 construct can influence the NTH conformation 10. It remains to be\ndetermined ..”\nThe last paragraph of the discussion describes the effects of gating induced by lipids in other proteins\nand it is claimed that clear parallels could be drawn by these and their system. But what are these\nparallels, this is not apparent to me? Is it the same binding to the interior of the channel, affecting the\nNterminal region? Please be more specific what these clear parallels are.\nRESPONSE: This is a good point, and there is a certain risk in going deep in this argument. Any detailed\ncomparisons at this stage would be too speculative. The updated manuscript now features a minimally\nspeculative final statement (page 12, line 392):\n“Lipid-mediated regulation has emerged as a common denominator in those studies. We anticipate that\nfuture investigations probing the molecular mechanisms of the large pore ion channel family members\nwill reveal the common mechanistic features of the lipid-mediated modulation of these proteins’\nstructure and function, which may reflect our own observations on lipid-reconstituted Cx32 channels.”\nFigure 2: what are yellow rectangles in Figure 2A? It should be explained in the legend to figure. If they\nrepresent lipid bilayer, are they not too thick (i.e. in comparison to the ones shown in Figure 1B and C)?\nRESPONSE: The pale yellow rectangles indeed roughly correspond to the two lipid bilayers. We\nadjusted this slightly, to make the rectangle boundaries more symmetrical. These rectangles are largely\nmeant a visual aid to help the reader orient themselves with respect to the view of the protein displayed\nin the figure. We hope that in the current adjusted form this visual aid is acceptable.\nFigure 3: What does abbreviation ONS means in panel A, define please.\nRESPONSE: We have updated the figure – this should be “NO-state” (“ONS” was an older working\nabbreviation in this figure that we overlooked). We have adjusted the figure accordingly.\nFigure 3: Panel A shows a comparison between protein in detergent (previous work) and nanodiscs\n(current work). The PDB Id of the structure used for this panel should be stated in the legend (and the\nreference).\nRESPONSE: We have updated the Figure 3 legend accordingly:\n“Pore representations of Cx32 GJC solved in detergent (PDB ID: 7ZXM 13) ..”\nFigure 7: Panel B of Figure 7 does not contribute anything conceptually and this state of the protein\ncould be described only in the text.\nopted now to keep it, for the reasons outlined below (we hope the reviewer will agree with our choice).\nThis figure was not meant both as the mechanistic insight into the lipid-lipid or lipid-protein interactions\nof Cx32, as well as a visual recapitulation of our experimental observations. In principle we could\nexclude this panel from the manuscript without a major loss to the overall message. However, we found\nthat this figure works very well to explain and summarise our story. For example, we found the complete\nfigure, including panels A and B to be very instrumental in explaining the results during a recent Gordon\nResearch Conference on lipid biology (Waterville Valley, July 2025), where these results were\npresented as a poster (VMK). Based on this, we think including this pannel may be very helpful for a\ngeneral reader to get an overview of the results in a simple and accessible format.\nMaterials and methods section: what was the source of POPC and LPL (lines 19,20)? Please define.\nRESPONSE: The lipids were purchased from Avanti Polar Lipids, and we have updated the methods\nwith this information (Page 2, line 20).\nThe purification of connexin (lines 51-66) is not clear to me. Why do you need anti-GFP nanobody\ncoupled CNBr Sepharose for purification? Does the construct contains GFP with appropriate cleavage\nsite (as in the next step something is cleaved off)? Please be more specific here about the construct\nthat was used for the study.\nRESPONSE: We have clarified this in the methods. Indeed anti-GFP nanobody is a powerful tool we\nuse for purificaiton of almost all membrane porteins in our lab – this has in many cases produced\noutstanding results, as the nanobody is highly specific and often superior to other established affinity\npurification reagents. The method relies on capturing the fluorescent protein-tagged protein on the\nnanobody resin, followed by elution by 3C protease cleavage (a 3C site is located between Cx32 and\nthe C-terminal YFP in our construct). Page 3, line 52:\n“The adherent HEK293F cells were transfected with a pACMV plasmid encoding Cx32 or Cx32-W3S\nmutant, in frame with a C-terminal 3C-YFP-twinStrep tag. Following a 48 h period after transfection,\ncells harvested from 100 15-cm culture plates were resuspended in buffer A ..”\nFigure S1: There are two bands for Cx32 on gels shown in Figure S1 in A,B and no protein in panel C..\nPlease comment.\nRESPONSE: The presence of more than one bands Cx32 is not unusual, as we do not heat our\nmebrane proteins and thus some of the protein may retain partial structure in the presence of SDS\nmicelles during SDS PAGE. Non-uniform migration of membrane proteins on SDS PAGE is not\nuncommon, as they do not behave the same as globular soluble proteins (for which one would typically\nheat the samples before loading on the SDS PAGE gel).\nThe panel C does contain Cx32 protein, but it appears that the sample also contains an excess of\nMSP2N2, which manifests in a much stronger staining of the MSP2N2 band by coomassie blue in\ncomparison. This exact sample was used for structure determination, and thus we have included this\ngel image. There can be two possible explanations: (1) An excess of MSP2N2 was introduced in this\ncase during reconstitution due to the operator error; (2) Identical MSP2N2 ratios were used for this\nprotein as for the wild-type Cx32 proteins in panels A, B and D, but W3S protein may have been much\nless stable under the reconstitution conditions, resulting in some of the protein precipitating, attaching\nto the Biobeads, etc. Either of the two options may apply here.\nLegend to Figure S8 does not correspond to the figure.\nRESPONSE: We are very grateful to the reviewer for spotting this, and we have corrected this in the\nrevised version. The new legend reads as follows (page 17, line 299):\n“Figure S8. Effect of lipid-2 on Cx32 GJC NTH. (A) Lipid-2 density can accommodate cholesterol, or\ncholesteryl hemisuccinate (CHS), and digitonin, used during protein purification. (B) Cx32 GJC\nreconstituted in LPL-containing nanodisc has the similar densities as Cx32 GJC, reconstituted in POPC\n6\ncontaining nanodisc. (C) Densities of lipid-2 and lipid-3 in the cryo-EM map of the Cx32 GJC in LPL-\ncontaining nanodiscs. (D) HOLE analysis of the pore conduction pathway of Cx32 GJC in LPL-\ncontaining nanodiscs. The pathway colored in yellow includes POPC in the calculation, whereas the\ngrey excludes it. The arrows represent the points of pore constriction due to NTH rearrangement.“\nReviewer #3 (Remarks to the Author):\nThe manuscript “Lipid dependence of connexin-32 gap junction channel conformations” by Lavriha et\nal. describes a series of cryoEM structures of Connexin-32 wildtype and the disease-associated\nmutation W3S. The structures provide insights into the gating mechanism of connexin channels,\nsupporting a role of sterols and phospholipids to directly bind to the inner channel pore,\nthereby stabilizing the N-terminal helix in a closed channel conformation. Accompanying molecular dynamics\nsimulations strengthen the idea that phospholipids and sterols dock to different sites in the inner pore\nof Connexin-32 and stabilize the N-terminal helix. The manuscript is overall very well written and easy\nto follow. The authors present a thorough study, which focuses on a topic of wide interest in this area,\nand which highlights the importance of lipids in regulating gap junction channels. I only have minor\npoints that should be addressed by the authors:\n1. The authors describe that the Cx32 wildtype structure was used as the starting structure for all MD\nsimulations. However, the authors should also include how they introduced the mutation into the system\nprior to the MD simulations, and how they decided, which rotamer of serine to choose, so that the\nobserved destabilization of the N-terminal helix is not a result of the choice of serine rotamer in that\nlocal area.\nRESPONSE: We thank the reviewer for spotting this, and we have updated the methods section\naccordingly.\nThe section: “System setup for molecular dynamics simulations” in the resubmitted version of SI\nincludes the following sentence at the end: “It is worth noting that the initial structures to generate the\ntopology and coordinate files of systems involving the W3S mutation were derived from the equilibrated\nstructures (see below) of the wild-type systems, by manually renaming the W3 residue to SER in the\nPDB files and deleting the side-chain atoms beyond the CB. The missing hydroxyl group of the Ser\nresidue was added automatically by tleap”.\nMoreover, we have included the following information in the main text: “This underscores the instability\nof the NTHs carrying the W3S mutation, which were initially modeled by retaining the Cβ orientation of\nW3 in the wild-type protein” (page 7, line 220).\nBy following this approach, we guaranteed that the deviations from the α-helical conformation observed\nover the course of the MD simulations stemmed from the nature of the new residue (Ser) and not as a\nconsequence of having chosen a rotameric form that destabilizes the NTH.\n2. A key effect of the binding of sterols and phospholipids to the channel pore appears to be the support\nof the N-terminal helix to rearrange from the open to the NO state. Such conformational changes occur\nover long timescales, and are not captured by the performed 1 µs simulations. Other studies have\nshown that e.g. Hamiltonian Replica Exchange MD are required to compute the free energy of the open-\nto-closed transition (e.g. Mhashal et al., ACS Catalysis, 2020).\nRESPONSE: This is an intriguing point, which we considered carefully. Our new PCA results show that\nthe NTH vertical motion that can lead to open and NO states are captured during the microsecond-long\nMD simulations (Figure 7C). Therefore, there is in principle no need to use enhanced sampling\ntechniques for that purpose in this particular system. Moreover, the angle histograms observed in Fig.\nS18 to S22 (Figures S17 to 21 in the previous SI version) already suggested the occurrence of\nsignificant vertical motions of the NTH relative to TM1. However, the inclusion of PCA in the new\nversion, further reinforces this point. Based on this, in the current revised version we have opted to omit\nthe proposed reference, although we could probably find a way to integrate the reference to Mhashal\net al., 2020, in case the reviewer deems this essential."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have adequately addressed the concerns raised by the reviewers. The revised manuscript includes additional principal component analysis and provides clearer explanations for previously ambiguous points, enhancing the transparency and comprehensiveness of the study. This is an interesting and well-executed study, and I have no further comments.", "reviewer_2": "I have no further comments, the authors have addressed all my comments.", "reviewer_3": "The authors have addressed all my comments raised in the previous revision. The revised paper has improved and I have no further concerns."}, "author_response": ""}]

Decision letter