Structural insight into sodium-dependent bile acid transport by members of the SLC10 family

This study resolves crystal structures of an ASBT homologue and a humanized version to reveal that, unlike NTCP, these SLC10 family transporters lack a transmembrane pore due to a flexible TM6 helix, and molecular dynamics simulations further demonstrate how membrane lipids facilitate the transport of bile acids with large hydrophobic groups.

The Gist

The Big Picture: The Body's Recycling Crew

Imagine your body is a massive city. In this city, bile acids are like specialized delivery trucks. Their job is to help your intestines pick up fats and vitamins from your food. Once they've done their job, they don't get thrown away; instead, they are recycled back to the liver to be used again. This is called the "enterohepatic circulation."

To get these trucks back to the liver, they need to cross a border wall (the cell membrane). They can't just walk through; they need a gatekeeper. That gatekeeper is a protein called ASBT (Apical Sodium-dependent Bile acid Transporter).

The Mystery: How Does the Gate Open?

Scientists have been trying to figure out exactly how ASBT works. They know it uses a "sodium battery" (sodium ions) to power the gate. But there was a confusing puzzle:

1. The Old Theory: Most transporters work like a revolving door. You enter one side, the door spins, and you exit the other. The door is never open to both sides at once.
2. The Recent Confusion: Recently, scientists looked at a similar gatekeeper called NTCP (found in liver cells) and found something weird. In its "open" state, it looked like it had a hole straight through the middle. If there's a hole, the gate isn't a revolving door; it's a tunnel. This broke the rules of how transporters were supposed to work.

The big question was: Is this "hole" a real feature of all these transporters, or was it just a fluke with NTCP?

The Experiment: Building a Better Model

The researchers in this paper decided to build a better model to solve the mystery.

• The Problem with Previous Models: The bacterial versions of this protein they studied before had an extra "helix" (a structural piece) that human proteins don't have. It was like trying to understand a human hand by studying a robot hand with an extra finger.
• The New Model: They found a bacterial protein from Leptospira (a type of bacteria) that looks almost exactly like the human ASBT gatekeeper. It has the right number of parts (9 helices instead of 10).
• The "Humanizing" Trick: To make it even more like the human version, they tweaked 10 specific spots on the protein to match human DNA. They call this the "humanized" version.

The Discovery: No Hole, Just a Flexible Flap

They took pictures (crystal structures) of this new protein in two states: Closed (facing the inside of the cell) and Open (facing the outside).

Here is what they found:

1. No Tunnel: Unlike the NTCP gatekeeper, this protein does not have a hole running through it when it opens.
2. The Secret Mechanism: The "door" is sealed shut by a flexible flap called TM6. Think of TM6 like a drawbridge or a curtain. When the gate opens to let the truck in, the curtain moves aside, but it doesn't leave a gaping hole; it just shifts to block the other side.
3. The Conclusion: The "hole" seen in NTCP might be a special quirk of that specific protein, or maybe NTCP is just very flexible. But for ASBT, the classic "revolving door" (alternating access) model still holds true. There is no leaky tunnel.

The Twist: The Lipid "Sidekick"

The researchers also looked at how the bile acid (the delivery truck) actually sits inside the gate.

• The Truck's Design: Bile acids have two parts: a "head" (polar, likes water) and a "body" (a greasy, hydrophobic steroid ring).
• The Surprise: The protein holds onto the "head" tightly, but the greasy "body" doesn't just sit inside the protein. Instead, it sticks out and hugs the surrounding fat molecules (lipids) of the cell membrane.
• The Analogy: Imagine trying to park a car with a very long, greasy trailer. You can't park it entirely inside a small garage. Instead, you park the driver in the garage, but the trailer sticks out and rests on the driveway.
• Why it matters: This "proteo-lipidic" interaction (protein + lipid) helps explain how these transporters can carry bulky, greasy molecules without getting stuck. The membrane itself becomes part of the parking spot.

Why Should You Care?

1. Drug Design: Many new drugs are designed to hitch a ride on these bile acid transporters to get into the body (prodrugs). Understanding that there is no "hole" and that the membrane lipids help hold the drug is crucial for designing better medicines.
2. Liver Health: Since NTCP is the entry point for the Hepatitis B virus, understanding the difference between NTCP and ASBT helps scientists figure out why the virus uses one but not the other, potentially leading to better cures.
3. Fixing the Theory: This paper corrects the scientific record. It shows that while some transporters might have holes, the standard "revolving door" mechanism is still the rule for this important family of proteins.

Summary in One Sentence

Scientists built a perfect model of a human bile acid transporter and discovered that, contrary to recent confusing reports, it works like a secure revolving door with a flexible curtain, and it uses the surrounding cell membrane as a helper to hold onto its greasy cargo.

Technical Summary

1. Problem Statement

The SLC10 family of transporters, specifically the Apical Sodium-dependent Bile acid Transporter (ASBT) and the Na+-dependent Co-transporting Polypeptide (NTCP), are critical for the enterohepatic recycling of bile acids. While the general mechanism of secondary transporters is the "alternating access" model (where the substrate binding site is never simultaneously accessible to both sides of the membrane), recent cryo-EM structures of mammalian NTCP in the outward-facing state revealed a paradox: an open pore running through the center of the protein. This violates the alternating access model, as it would allow uncontrolled diffusion.

Previous bacterial homologues (ASBTNM and ASBTYF) did not show this pore, but they possess an extra transmembrane helix (TM1) absent in human proteins. It remained unclear whether the open pore is a unique feature of NTCP, an artifact of specific crystallization conditions, or a general feature of the SLC10 family. Furthermore, the mechanism by which bulky hydrophobic bile acids are transported, particularly how they interact with the membrane environment during translocation, was not fully understood.

2. Methodology

The authors employed a multidisciplinary approach combining structural biology, protein engineering, and computational simulations:

• Protein Selection and Engineering:
  • Identified a bacterial homologue from Leptospira biflexa (ASBTLB) with 9 transmembrane helices (mimicking human ASBT/NTCP by lacking the extra TM1 found in other bacterial homologues) and 32% sequence identity to human ASBT.
  • Created a "humanized" mutant (ASBTLB10mut) by introducing 10 point mutations to increase sequence similarity to human ASBT in the substrate-binding region.
• Structural Biology (X-ray Crystallography):
  • Used Lipidic Cubic Phase (LCP) crystallization to solve structures in multiple states.
  • Inward-facing (IF): Solved the wild-type (WT) ASBTLB structure.
  • Outward-facing (OF): Solved the ASBTLB10mut structure, both with and without the bile acid deoxycholic acid (DCA) bound.
  • Resolutions ranged from 2.2 Å to 2.9 Å.
• Computational Modeling:
  • Generated AlphaFold2 predictions for human ASBT and NTCP to compare with experimental data.
  • Performed All-Atom Molecular Dynamics (MD) simulations (1 µs duration) on WT and mutant ASBTLB in both IF and OF states, embedded in a POPE:POPG lipid bilayer.
  • Simulations included bound DCA and two sodium ions (Na1, Na2) to analyze substrate anchoring and lipid interactions.
• Validation:
  • Used stability assays (CPM dye binding) to confirm bile acid binding.
  • Validated docking poses using Autodock Vina.

3. Key Results

A. Structural Conformations and the "Pore" Question

• Inward-Facing State: The WT ASBTLB structure revealed a clear cavity open to the intracellular side. Sodium ions (Na1 and Na2) were clearly resolved in the core domain, coordinated by conserved residues (e.g., S84, N85, E233 for Na1; Q47, Q237 for Na2).
• Outward-Facing State: The ASBTLB10mut structure (both apo and DCA-bound) adopted an outward-facing conformation. Crucially, no open pore was observed.
• Role of TM6: The absence of the pore is attributed to the position of Transmembrane Helix 6 (TM6). In ASBTLB, TM6 is flexible (hinged by Glycine residues) and packs against the core domain, effectively sealing the intracellular exit. In contrast, human NTCP structures show TM6 pulled away, creating the pore.
• Comparison to Human Proteins: The ASBTLB structures align closely with AlphaFold2 predictions for human ASBT (which also predict a closed pore) but differ significantly from experimental NTCP structures (which show an open pore). This suggests the open pore is not a universal feature of the SLC10 family but specific to NTCP or its specific conformational state.

B. Substrate Binding and Mechanism

• Substrate Location: DCA was resolved in the outward-facing structure, bound between TM1 and TM5. The carboxylate group forms hydrogen bonds with Thr82 and the backbone of Ala83, anchoring the polar head near the core domain crossover.
• Lipid-Mediated Transport: MD simulations revealed a "proteo-lipidic" mechanism. While the polar carboxylate is anchored to the protein, the hydrophobic sterol rings (A-D) of the bile acid interact extensively with the surrounding membrane lipids rather than being fully enclosed by the protein.
  • In the outward-facing state, the binding site opens sideways to the membrane, allowing lipids to stabilize the hydrophobic tail.
  • In the inward-facing state, the sterol moiety reorients toward the intracellular side, still maintaining contact with lipids.
• Conformational Change: The transition between states involves a rigid-body movement of the "panel" domain relative to the "core" domain (elevator mechanism), with a rotation of ~22° and a 5 Å translation. The substrate does not need to flip entirely within a closed protein shell; instead, the lipid environment facilitates the movement of the bulky hydrophobic group.

C. Sodium Coordination

• Sodium ions were stably bound in both IF and OF states in simulations. The coordination geometry remained consistent, suggesting sodium binding rigidifies the core domain and sets up the substrate binding site, consistent with the alternating access model.

4. Significance and Implications

• Resolution of the Transport Paradox: The study challenges the notion that the open pore is a mandatory feature of SLC10 transporters. It demonstrates that a canonical alternating access mechanism (without a continuous pore) is structurally feasible and likely the mechanism for ASBT. The open pore in NTCP may be a specific adaptation or a transient state unique to that protein.
• Novel Transport Mechanism: The paper proposes a proteo-lipidic transport mechanism. It suggests that for bulky, amphipathic substrates like bile acids, the membrane lipids are not just a passive barrier but an active component of the transport pathway. The protein anchors the polar head, while the lipid bilayer accommodates the hydrophobic tail, explaining how large conjugated molecules can be transported without requiring a massive internal cavity.
• Drug Discovery: Understanding the structural differences between ASBT and NTCP (specifically the TM6 flexibility and pore status) provides a structural basis for designing selective inhibitors. This is crucial for repurposing ASBT inhibitors for treating liver disorders (e.g., pruritis, cholestasis) without affecting NTCP-mediated hepatitis B virus entry or bile acid uptake in the liver.
• Validation of Models: The study validates that bacterial homologues with 9 TMs are excellent surrogates for studying human SLC10 transporters, provided specific mutations are introduced to mimic human binding pockets.

In summary, this work provides the first high-resolution structural evidence of a closed-pore, sodium-dependent bile acid transporter, reconciling the alternating access model with the transport of bulky hydrophobic substrates through a unique lipid-assisted mechanism.