Binding modes of substrate TCA in hNTCP
To understand the substrate transport mechanism, we first assess the potential substrate binding modes in the experimentally resolved open-pore and inward-facing conformations of hNTCP (PDB id: 7PQQ and 7PQG). The two conformations differ mainly in the interface between the core and the panel domains. The different TM arrangements lead to a difference of 4 Å, 5 Å, and 6 Å in the width of the extracellular gate, the middle pore, and the intracellular gate, respectively (Fig. 2a). Molecular docking of the substrate TCA results in two dominant docking poses in the open-pore hNTCP (poses 1 and 2, green and magenta in Fig. 2a, respectively) and one docking pose in the inward-facing hNTCP (pose 3, orange in Fig. 2a). The docking poses 1 and 2 share the same configuration of the sulfate head contacting the X-motif (98CSPGGNLSN106 on TM3 and 257ETGCQNVQ264 on TM8) while differ in the orientation of the sterol tail. In the docking pose 3 of inward-facing hNTCP, the sulfate head of TCA is also in contact with the X-motif, while the sterol tail points toward the cytoplasmic side. We note that the docking pose 3 in the intracellular pocket is different from substrate binding pose in the X-ray structure of TCA-bound ASBT (PDB id: 3ZUY)20, in which the sulfate head of TCA is oriented toward the solvent environment and the substrate is closer to the membrane-solvent interface.
We carried out three 5 μs MD simulations, each initialized with one of the three TCA-protein docking structures (Supplementary Table 1, Supplementary Fig. 1). The conformation of TCA in the complex was characterized by the relative position of its sulfate head along the membrane normal (Z-position) with the bilayer midplane at Z = 0 Å, and the angle between its tail-to-head vector and the normal vector of the membrane plane (Z-angle) (Fig. 2a). The time evolution of the Z-position and Z-angle showed that TCA reached to three different equilibrium states based on the initial docking pose (Fig. 2b-c). Starting with the docking pose 1, the Z-position of TCA slid up to approximately 7 Å and the Z-angle gradually rotates to ~130° to reach the equilibrium binding pose 1 in the extracellular pocket. Starting with the docking pose 2, the Z-position of TCA remains stable at ~2 Å and the Z-angle at ~85° throughout the simulation, maintaining the equilibrium binding pose 2 in the middle pore. Starting with the docking pose 3, the Z-position of TCA slid down to approximately Å and the Z-angle rotated to ~45° after 2 μs MD simulation to reach the equilibrium binding pose 3 in the intracellular pocket. Ultimately, three substrate binding sites located respectively in the top extracellular pocket, the middle pore, and the bottom cytoplasmic pocket, were captured with TCA stably bound from 5 μs MD simulations (Supplementary Fig. 2).
Interestingly, the hNTCP protein shows different dynamics in the three TCA-bound states. The TMD structure exhibits the most significant structural stability when initialized with the docking pose 2, with only ~2 Å RMSD change compared to the starting cryo-EM structure (Fig. 2d). Structural alignment confirms the high consistency between the experimentally determined open-pore hNTCP and the TCA-bound hNTCP with binding pose 2 (Fig. 2e). We note that in this state the coordinates of TCA overlap well with the partial cryo-EM density observed at the interface between the core and the panel domain (EMDB id: EMD-13596). While the limited experimental cryo-EM density alone was insufficient to directly model the full substrate-bound complex structure, here a clear binding mode of substrate in the open-pore hNTCP is obtained at the atomic level combining docking and long timescale MD simulations. A close examination on this binding mode shows that residues in the X-motif form multiple hydrogen bonds (H-bonds) with the head group of TCA to maintain its stability. In particular, the backbone NH groups of N103, L104 and V263 form three H-bonds with the sulfonate group, and the sidechain of N103 forms an additional H-bond with the substrate carbonyl group. Residues on the panel domain (S199 and T203 on TM6) contribute to holding the sterol tail of TCA.
Interestingly, although inserting TCA in the top extracellular pocket or the bottom cytoplasmic pocket of hNTCP induces large conformational changes, the complex structures reach equilibrium after approximately 2 μs (green and orange in Fig. 2d). While the TCA head group moves upward (for binding pose 1) or downward (for binding pose 3), it is still anchored by the X-motif through hydrogen bond interactions in either pocket (Supplementary Fig. 3). When TCA binds in the top extracellular pocket, its sterol tail is hold by the residues on the panel domain (S28 on TM1 and N209 on TM6), whereas the tail becomes free when it binds in the bottom cytoplasmic pocket (Supplementary Fig. 3). Derivation of these intermediate TCA-bound conformational states already reveals the essential process of TCA being transported from the extracellular to the intracellular matrix. Furthermore, the cooperation between substrate binding and synchronized protein conformational changes was captured in microsecond-timescale MD simulations.
Alternative conformations of hNTCP in TCA-bound and apo states
We further computed the sizes of the extracellular gate, the middle pore, and the intracellular gate of hNTCP along all MD simulations of TCA-bound hNTCP systems. While gauging the extracellular and the intracellular gates is sufficient to distinguish the global conformations for most SLCs such as secondary active major facilitator superfamily (MFS) transporters34, for hNTCP additional characterization on the middle pore is found to be indispensable. The width of the middle pore can be used to inspect the main mode of motion in the global conformational changes of hNTCP transport substrate.
The time evolution of the size of extracellular gate, middle pore, and intracellular gate indicates that the global conformation of hNTCP can exhibit the outward-facing, open-pore, and inward-facing states with TCA binding restriction (Fig. 3a-c, Supplementary Fig. 4). When TCA binds to the extracellular pocket, the width of the extracellular gate enlarges to ~22 Å and the middle pore shrinks to ~15 Å. Consequently, an additional outward-facing state of hNTCP is sampled with TCA binding to the extracellular pocket. When TCA binds to the middle pore, the extracellular gate adopts a smaller size of ~18 Å and the middle pore remains open for ~18 Å. When TCA binds to the cytoplasmic pocket, the extracellular gate adopts the smallest size of ~13 Å and the middle pore keeps closed at ~13 Å. Instead, the dimension of the intracellular gate makes no significant difference under the three TCA-bound states after 5 μs MD simulations, which shows significantly higher flexibility than the extracellular gate and middle pore (Fig. 4c).
Three additional 5 μs MD simulations were carried out for apo hNTCP starting from the outward-facing, open-pore, and inward-facing states (Supplementary Fig. 5) after removing both the substrate TCA and two Na+ from the complexes. Time evolution of the size of extracellular gate, middle pore, and intracellular gate showed that the extracellular gate was relatively stable, while the middle pore further shrunk to the narrower state, and the intracellular gate remained highly dynamics (Fig. 3d-f). The shrinkage of the middle pore suggests that the instantaneous removal of TCA drives hNTCP out of equilibrium. To better understand the movement of TMD in apo hNTCP, the projection of nice TM helices on the membrane plane was depicted from three intersections including the extracellular plane, the middle-membrane plane, and the intracellular plane. As shown in Fig. 3g, the projected positions overlapped between the three independent MD simulations of apo hNTCP, suggesting that each TM helix has the flexibility and mobility to access the conformational space sampled by another simulation. Additionally, the extracellular pocket (bounded by TM1, TM5, and TM8) and the intracellular pocket (bounded by TM3, TM6, and TM9) of apo hNTCP are illustrated in Fig. 3g. These two pockets not only have access to the water environment, but also are extensively exposed to the lipid bilayer environment, with phospholipid molecules entering both the extracellular and intracellular pockets, as observed in the MD simulations of apo hNTCP (Supplementary Fig. 6).
The observed multiple conformations of TCA-bound and apo hNTCP provide insights into the substrate transport mechanism of hNTCP. Structural characterization of three TCA-bound hNTCP reveals the high flexibility of the interface between the core and panel domains, which enables the conformational transitions of TMD to cooperate with substrate transport. Two helices, TM6 and TM9, dominantly contribute to the high dynamics of hNTCP through bending and turning respectively, with the fluctuations of ~15° in angles perpendicular to the membrane (Supplementary Fig. 7). By delineating the solvent-accessible space in both extracellular and intracellular sides, all representative alternative conformations of hNTCP sampled from MD simulations are ordered according to the states of substrate accesses from extracellular side to intracellular side (Fig. 4). First, hNTCP assumes a dynamic-apo state when no substrate access to the extracellular side, and the middle pore is closed. Second, hNTCP adopts an outward-bound state when the substrate enters the extracellular pocket. Next, hNTCP transitions to an open-pore state when the substrate binds to the middle pore. Subsequently, hNTCP manifests an inward-bound state when the substrate binds to the intracellular pocket. Finally, when the substrate completely detaches from the intracellular side, hNTCP reverts to the dynamic-apo state with a closed middle pore.
Coupling between Na+-binding and TCA-binding via X-motif conformational change
As hNTCP is a Na+-dependent secondary symporter, we investigated the role of Na+ in its substrate transport. In the three 5 μs MD simulations for TCA-bound hNTCP, two Na+ were built to coordinate with X-motif due to the reported importance of Na+ on the substrate transport28,35. Structural analyses shows that all the stable TCA-bound conformational states are coupled with two Na+ binding to the negatively charged X-motif (Supplementary Fig. 8). Except for the key residues (Q68, S105, N106, T123, E257, and Q261) hypothesized by evolutional analysis, residues G101, G102, and S119 at the Na1-site, and S99 and C260 at the Na2-site also contribute to the stable binding of two sodium ions (Supplementary Fig. 8c). Time evolution of distances between Na+ and coordinated oxygen atoms reveals that these residues in the X-motif subtly cradle the two Na+ ions. We then carried out two 5 μs MD simulations each initialized with two Na+ but no TCA binding to the two experimentally determined conformations. Two Na+ ions were found to be stable during the 5 μs open-pore hNTCP simulation. However, the Na+-binding sites in inward-facing hNTCP became unfavorable without TCA as both Na+ escaped from their binding sites and the one initialized in the Na1-site entered the cytoplasm solvent after 500 ns (Supplementary Fig. 9).
In addition, we performed three 3 μs MD simulations each initialized with previously sampled stable TCA-binding conformations but the two Na+ removed. Without Na+-binding, the binding stability of TCA significantly decreased in all three simulations (Supplementary Fig. 10). In particular, TCA bound to the middle and bottom positions (binding poses 2 and 3) quickly underwent rotation after a few hundred nanoseconds, allowing its hydrophilic sulfate head group to access the water environment (Fig. 5a-c), which suggests a synergistic relationship between Na+-binding and substrate binding. In the absence of Na+ occupancy in hNTCP, the TCA substrate displayed common amphiphilic properties and oriented in the same direction as lipid molecules.
It is interesting to note that there is little direct interaction between the two Na+ ions and TCA. Energy calculations along MD trajectories with both Na+ and TCA stably bound showed that the total interaction energy is less than -0.1 kcal/mol in all three binding poses (Supplementary Fig. 11). Structure clustering and comparisons revealed that the major effect of Na+-binding is the tightening of the X-motif (Fig. 5d), which is well-coordinated with the sulfate head of TCA. The X-motif assumes a tightly folded state with Na+-binding, whereas it is more unwound and flexible without Na+-binding, reducing its ability to form stable contacts with TCA. Further interaction energy calculations illustrated that Na+-binding indirectly enhances the interaction between hNTCP and TCA with energies changed by -40±6 kcal/mol upon Na+-binding and confirmed that the changes are dominated by the interaction energies between the X-motif and TCA (Supplementary Fig. 12 and 13). Our results suggest that Na+-binding induces the conformational change of X-motif and thus modulates the local environment for substrate binding in hNTCP. The delicate cooperation among Na+-binding, X-motif conformational change, and TCA-binding in hNTCP is also implied in the alignment of the Na+-bound X-motif in the open-pore and the inward-facing hNTCP (Fig. 5e). Transition from the open-pore to the inward-bound conformation accompanies the movement of two Na+ ions further towards cytoplasm and minor conformational shifts of the folded X-motif.
Na+-binding drives substrate translocation in hNTCP
After establishing that Na+-binding impacts substrate binding in hNTCP, we further investigate the effect of Na+-binding on the thermodynamic properties of substrate translocation with enhanced sampling MetaD simulations. Four sets of well-tempered 2D-MetaD simulations were initialized using the stable TCA-bound outward-facing and the inward-facing state, each with or without Na+-binding (Supplementary Table 1). The Z-position and Z-angle of TCA were used as collective variables (CVs) to sample its translation and rotation in hNTCP. The resulting free energy profiles of TCA translocation in the extracellular and the intracellular pockets were constructed under different Na+ binding states (Fig. 6), whose convergence was checked with three parallel runs (Supplementary Fig. 14). An evident alteration in the shapes of the free energy profiles reveals the determining role of Na+-binding on consecutive TCA translocation.
When TCA is located in the extracellular pocket, the shape of the free energy surface is completely reversed by Na+-binding, with the global minimum shifting from the hydrophilic sulfate head group being close to the extracellular water environment (Z-position=19.8 Å) to it translocating into the middle pore and coordinating with the X-motif (Z-position=3.6 Å) (Fig. 6a). Without Na+-binding, TCA is most favorable with a head-up, tail-down configuration (Z-angle around 0°), which is presumably how the TCA diffuses into the pocket. After Na+ ions enter their binding sites at the X-motif, a new free energy minimum emerges that is 10.2±1.1 kcal/mol more favorable, providing the thermodynamic incentive for TCA to orientate into a head-down, tail-up configuration with Z-angle around 145° (binding pose 1). Our simulations reveal that Na+-binding to X-motif activates TCA transport.
When TCA is in the intracellular pocket, the global minima in the free energy profiles are similar in both Na+-binding states, with the sulfate head of TCA accessing the intracellular water environment. This suggests that the inward-bound state leads to spontaneous release of the substrate. The difference in the free energy landscapes again highlights the importance of Na+-binding in this process (Fig. 6b). When the Na+-binding sites were occupied, the free energy landscape is relatively flat with multiple minimum and barriers in between, such that the transition would be a relatively slow process. When there is no Na+-binding, the configuration with Z-position=-5.6 Å, Z-angle=43° (binding pose 3) becomes significantly more positive in free energy than the global minima, leading to a fast downhill process for TCA dissociation. The results from MetaD simulations are consistent with the observations from microsecond conventional MD (cMD) simulations of systems 3 and 9 (Supplementary Table 1), both suggesting that the dissociation of Na+ ions facilitates the reorientation of TCA for its translocation in hNTCP.
Substrate translocation relies on alternative-state transition to access the intracellular side
The transition between the Na+-binding, outward-bound state and the Na+-binding, inward-bound state were further studied using MetaD simulations. The relationship between local gating motions and global structural transitions is a crucial aspect of the transport mechanism, and two sets of well-tempered 2D-MetaD simulations were performed over 900 ns to shed light on this topic (Supplementary Table 1). The Z-angle of TCA were used as one CV to describe the substrate movement, while the conformations of hNTCP were characterized by the native contact differences ( ) in TMD as another CV. In particular, the native contact difference was used to characterize the conformations along the transition path from outward-facing to open-pore, and was used to characterize the conformational transition from open-pore to inward-facing (Fig 7a, see Methods). The construction of free energy surface profiles reveals the relationship between TCA translocation and the conformational changes of hNTCP. We note that and are two distinct CVs for driving different conformational sampling, and the corresponding 2D projection of free energy surfaces are not comparable.
The free energy profile of the outward-bound to open-pore transition shows that the outward-bound and open-pore states of hNTCP are both thermodynamically favorable (Fig. 7b). The transition from the outward-facing to the open-pore conformation does not need to involve the reorientation of TCA and is thermodynamically favored. When the hNTCP assumes the open-pore conformation, the TCA can switch between two orientations which has Z-angles of 135° and 85°, respectively. Based on the free energy profile of open-pore to inward-bound transition, a slight conformational shift towards the inward-facing conformation allows TCA to rotate further down with a Z-angle smaller than 90° (Fig. 7c). The open-pore state is thermodynamically more favorable than inward-bound state when the hydrophilic sulfate head group of TCA interact with X-motif, and an intermediate minima was observed at ~ 0. The transition entails more subtle coupling between the substrate orientation and protein dynamics and involves overcoming multiple energy barriers. Our free energy calculation results are consistent with the fact that the outward-facing conformation was not captured in structural biology experiments, while the open-pore and the inward-facing conformations were. Taking together, the global conformational transitions of hNTCP that enable TCA transport are examined from a thermodynamic perspective, facilitating to the construction of transport cycle.
Overall scheme of the hNTCP transport cycle
Earlier, the elevator-type alternating-access model was presumed for several transporter families including SLC1027,36. In this mechanism, substrate translocation accessibility is obtained by overcoming a fixed barrier of one domain movement against another relatively rigid domain, with substrate binding and release in each alternative state facilitated by local gating transitions in the moving domain. This elevator-type alternating-access mechanism is only partially relevant to hNTCP; the extensive MD simulations, starting with experimentally resolved open-pore and inward-facing structures, provide a more vivid picture on its transport cycle. Combining the dynamic and thermodynamic evidence in apo and TCA-bound hNTCP, a complete molecular mechanism of substrate transport is proposed (Supplementary Movie 1) including the following key steps:
- Under Apo condition, hNTCP is intrinsically dynamic and populates a range of conformations including outward-facing and inward-facing ones, as evidenced in three 5 μs cMD simulations (Supplementary Table 1, MD systems 4-6). When apo-hNTCP adopts outward-facing configurations, amphiphilic molecules such as TCA can access into the extracellular pocket but cannot directly enter the middle pore of TMD for further transport.
- Upon Na+-binding, substrate translocation is thermodynamically activated as TCA can flip from the extracellular side into the middle pore where it stably stays (MD systems 1-2, 7-8, 12, 14). Na+-binding enhances the coordination of the X-motif with the sulfate head group of TCA by tightening it up.
- The Na+-bound and TCA-bound hNTCP undergoes conformational transition from the outward-facing to the open-pore conformation. This open-pore state is stable and provides space for the rotation of the TCA tail group to occur (MD systems 12, 14, 16-17).
- The hNTCP transitions into an inward-facing conformational state as the sterol tail of TCA flips down. With Na+ bound, TCA maintains stable in the inward-facing hNTCP on the microsecond timescale (MD system 3).
- Finally, Na+-unbinding facilitates the detachment of TCA from the intracellular pocket of hNTCP. With the release of the two Na+ ions into the cytoplasm, TCA flips again, exposing its sulfate head group to the cytoplasmic solvent (MD systems 13-15). The release of TCA allows hNTCP to return to the dynamic-apo state in step (1) and restarts the transport cycle.