Structural insights into cationic amino acid transport and viral receptor engagement by CAT1

Domain: physics
Published: 2025-12-18
Editorial outcome: major_revision

Abstract
Cationic amino acid transporter 1 (CAT1) transports cationic amino acids and plays pivotal roles in cancer proliferation, immune modulation, and nitric oxide metabolism. It also serves as the specific cellular receptor for certain murine leukemia viruses. Here, we report the cryo-electron microscopy (cryo-EM) structure of mammalian CAT1 in complex with its substrate ornithine and the receptor-binding domain (RBD) of Friend murine leukemia virus (FrMLV). CAT1 specifically recognizes the side-chain amino group of ornithine via residue S347 on transmembrane helix 8 (TM8), capturing the transporter in an inward-facing occluded conformation. Notably, the FrMLV RBD (frRBD) primarily engages the third extracellular loop (ECL3) of CAT1—a region marked by substantial species-specific variation that likely governs cross-species viral tropism. Together, our structural and biochemical results elucidate the molecular mechanism of substrate recognition and transport by mCAT1, and unveil the molecular basis for FrMLV receptor specificity. These findings provide a valuable framework for structure-based drug design targeting CAT1 in cancer and infectious diseases.

Introduction
CAT1, encoded by the SLC7A1 gene, is broadly expressed in various tissues such as muscle, liver, and immune cells, where it plays a critical role in maintaining cellular homeostasis by mediating the regulated transmembrane transport of essential cationic amino acids, including arginine, ornithine, and lysine1–5. Among these substrates, ornithine plays a pivotal role in nitrogen metabolism as an intermediate in the urea cycle and as a precursor for the biosynthesis of proline and polyamines, thus contributing to the regulation of normal cellular metabolism6. In contrast, dysregulation of arginine metabolism is closely associated with the development of several malignancies, including acute myeloid leukemia, acute lymphoblastic leukemia, and chronic lymphocytic leukemia. These tumors often exhibit a deficiency in argininosuccinate synthase 1 (ASS1) and are thus auxotrophic for extracellular arginine7–9. The primary arginine transporter, CAT1, is essential for sustaining this metabolic dependency, linking its activity to protein synthesis, metabolic regulation, and cellular signaling.

CAT1 belongs to the SLC7 subfamily of the amino acid–polyamine–organocation (APC) superfamily, a group critically involved in regulating metabolism, orchestrating immune responses, and promoting tumor progression10–12. Structural and functional studies of SLC7 family members, including the bacterial proton-dependent transporter GkApcT13 and AdiC14, the heteromeric amino acid transporter b[0,+]AT115–17, and the neutral amino acid transporters LAT118,19 and LAT220,21, have demonstrated that most SLC7 proteins require association with a heavy chain subunit from the SLC3 family for proper biogenesis and function. For example, LAT1 and LAT2 need to heterodimerize with 4F2hc, which stabilizes the light subunit and is essential for trafficking to the plasma membrane18,19. In contrast, b[0,+]AT1 associates with rBAT, where the light subunit stabilizes the heavy subunit, and recent studies have shown that formation of a higher-order “superdimer” of the b[0,+]AT1-rBAT complex is also required for correct biogenesis and membrane localization17,22. Such diversities in subunit organization, substrate specificity, and transport mechanisms highlight the remarkable functional versatility of the SLC7 family.

In contrast to these canonical features of the APC superfamily, CAT1 exhibits two distinct characteristics: a distinct 14-transmembrane domain architecture and an apparent ability to mediate substrate transport independently of auxiliary subunits3,4. Furthermore, accumulating evidence suggests that CAT1 mediates substrate transport through an ion-independent mechanism, reflecting a distinct mechanistic adaptation within the SLC7 family8,23. Nevertheless, due to the lack of high-resolution structural information, the molecular mechanism underlying CAT1 transport remains elusive.

Intriguingly, CAT1 also functions as a critical viral receptor in specific species. Mouse and bovine CAT1 (mCAT1 and bCAT1) have been identified as essential receptors for FrMLV24,25 and bovine leukemia virus (BoMLV)26,27, respectively, highlighting the intricate interplays between host transporters and viral invasion strategies. FrMLV utilizes mCAT1 as a high-affinity receptor, binding to the host cell surface to initiate infection. The envelope glycoprotein (Env) of FrMLV is synthesized as a precursor and subsequently cleaved into two subunits: the surface subunit (SU) and the transmembrane subunit (TM)28–30. The mature Env forms a trimeric SU-TM complex, in which the subunits are linked by labile interchain disulfide bonds. The SU subunit mediates viral attachment by interacting with specific extracellular domains of CAT1, while the TM subunit contains a hydrophobic fusion peptide and a transmembrane segment that likely facilitates membrane fusion upon receptor binding31. The RBD of SU specifically recognizes CAT1, anchoring the virus to the host cell and initiating the entry. This interaction underscores the functional versatility of CAT1 and suggests that viruses can exploit host transporters for infection32. Despite this detailed mechanistic understanding, the precise structural determinants on CAT1 that confer high-affinity and specific recognition of MLV envelope glycoproteins remain unclear.

In this work, we determine the 3.65 Å cryo-EM structure of full-length mCAT1 in complex with its substrate ornithine and the FrMLV receptor-binding domain (frRBD). The structure reveals mCAT1 in an inward-facing occluded conformation, with ornithine positioned in a negatively charged central cavity. We further show that the species-specific extracellular loop 3 (ECL3) adopts a conformation essential for high-affinity frRBD binding and for determining viral tropism. These results elucidate the structural basis of CAT1-mediated viral tropism and host specificity.

Results
The overall structure of the mCAT1/frRBD complex
In mammals, the CAT family transporters exhibit nearly identical substrate specificity for cationic amino acids, with their transport activity well-characterized through heterologous expression systems (such as Xenopus oocyte and FaO rat hepatoma cells)33–35. To investigate the molecular basis of substrate recognition and viral engagement, we generated recombinant expression constructs for full-length mCAT1 and frRBD. Biolayer interferometry (BLI) assays revealed a strong interaction between mCAT1 and frRBD, with a dissociation constant (KD) of approximately 5.91 nM (Fig. 1a). Then, we purified frRBD and incubated it with full-length mCAT1. The mCAT1/frRBD complex showed a single peak in size-exclusion chromatography (Fig. 1b). Cryo-EM grids were then prepared in the presence of ornithine, and single-particle analysis yielded a 3.65 Å reconstruction (Fig. 1c and Supplementary Fig. 1). The cryo-EM map enabled precise modeling of both proteins, revealing a 1:1 binding stoichiometry between mCAT1 and frRBD (Fig. 1c). While most of the structure was well-defined, several flexible regions remained unresolved, including the N-terminal residues (a.a. 1~31), the C-terminal residues (a.a. 601~622), and the intracellular loop ICL5 (linking TM10 and additional transmembrane helix ATM1). Despite previous studies suggesting oligomerization36, our data indicate that mCAT1 predominantly exists as a monomer, and only a minor fraction of dimeric particles was observed in 2D class averages (Supplementary Fig. 1).

The overall structure of the complex is approximately 100 Å in height and consists of a distinct transmembrane mCAT1 and a compact soluble RBD (Fig. 1c). Unlike prokaryotic CAT family members and most APC superfamily transporters, mCAT1 adopts a distinct 14 transmembrane-helix topology linked by seven extracellular loops (ECL1~7) and six intracellular loops (ICL1~6) (Fig. 1d). TM1–TM12 forms the transmembrane core enclosing a central substrate-binding cavity, which is the hallmark feature of the APC superfamily37, and excluding the auxiliary helices ATM1 and ATM2 outside (Supplementary Fig. 2a). At the periphery of this cavity, TM1 and TM6 are interrupted by short loop structures, segmenting them into TM1a/1b and TM6a/6b half-helices (Fig. 1d). The transmembrane core of mCAT can be well superimposed with its prokaryotic homolog, GkApcT13, with an RMSD of 1.64 Å for 338 Cα atoms on (Supplementary Fig. 2a), while ATM1 and ATM2 are only conserved within the CAT family, as well as in another two CAT-associated proteins, SLC7A4 and SLC7A1438. These two ATM helices are located at the periphery of the transport domain, distal from canonical substrate-binding sites and primarily interact with TM10 and TM11 through hydrophobic interactions and van der Waals contacts (Supplementary Fig. 2a, b), thus insufficient for direct substrate transport regulation.

The ornithine binding site of mCAT1
The additional cryo-EM density observed along the mCAT1 transport pathway closely aligns with the conserved substrate-binding sites identified in structurally characterized homologous amino acid transporters, including LAT220, xCT39, LAT119, GkApcT13, b[0,+]AT115 and AdiC14 (Fig. 2a and Supplementary Fig. 2c). This density could be fitted with an ornithine molecule (Fig. 2b), and three independent 100-ns molecular dynamics simulations showed that the modeled ligand remained stable (average RMSD~1.5 Å), supporting our assignment of ornithine (Fig. 2b, c). As expected, no unbinding events of ornithine were observed, consistent with the fact that substrate translocation in secondary transporters generally occurs on the millisecond-to-second timescale40–42, far beyond the reach of conventional MD simulations. Notably, although a 100-ns simulation cannot capture the complete translocation process, it is sufficient to evaluate the local stability of the substrate-binding pocket43,44.

Structural analysis indicated that the ornithine backbone amino group is positioned for a potential cation–π interaction with Y257, while its carboxyl group can form hydrophilic interactions with S44 and G47 (TM1) and S343 (TM8) (Fig. 2b and Supplementary Fig. 2d). These contacts are consistent with recognition modes observed in homologous transporters: residues between TM1a and TM1b (positions 44–50 in mCAT1) in LAT2, xCT, LAT1, GkApcT, BAT1 and AdiC typically coordinate the substrate carboxylate, and the serine on TM8 (S343 in mCAT1) parallels conserved serines in xCT and LAT1 (Supplementary Figs. 2d, 3). The ornithine side chain is further stabilized through a potential hydrogen bond with S347 on TM8 (Fig. 2b and Supplementary Figs. 2d, 3). These interactions were validated with functional assays. Electrophysiological recordings showed that S44A retained currents comparable to wild type, consistent with a backbone-mediated contact, whereas Y257A, S343A and S347A exhibited markedly reduced ornithine-induced currents, indicating their critical contributions to substrate coordination and transport (Fig. 2d, e). Fluorescence analysis confirmed that mutant and wild-type transfected cells expressed at comparable levels, supporting that these functional differences arose from altered transport activity rather than expression bias (Supplementary Fig. 4).

Comparison of mCAT1 with the prokaryotic transporter GkApcT shows conserved and divergent features. In GkApcT, E115 together with Y116 and S321 directly coordinates the arginine guanidinium group (Fig. 2f and Supplementary Fig. 2d), with E115 acting as a protonation site for H+-coupled transport, while D237 stabilizes the TM3–TM6 interface via a water-mediated bridge rather than direct substrate contact13. In mCAT1, the equivalent positions are S120, Y121, D263, and S347. Notably, the replacement of the glutamate (E115) with a serine at position 120 abolishes protonation, suggesting CAT1 functions as a membrane-potential-driven and proton-independent transporter38. Electrophysiological analysis supports this assignment: S120A and D263A mutations reduced transport, whereas Y121A retained near wild-type activity, suggesting that S120 and D263 contribute to substrate recognition and stabilization (Fig. 2d, e).

These findings are consistent with those for LAT1, in which the equivalent residue N258 (equivalent to D263 in mCAT1) does not directly coordinate neutral amino acid substrates but instead acts as a structural switch regulating access to the translocation pathway, a role essential for transport dynamics18. A related mechanistic theme is observed in AdiC, where the conserved residue E208 contributes to both substrate recognition and gating14,45. These comparisons suggest a recurring principle in APC transporters, whereby conserved polar residues may stabilize substrate binding or serve as conformational switches for gating.

The inward-facing occluded state and gating mechanism
In the determined mCAT1/frRBD complex, mCAT1 adopts an inward-facing occluded state (Fig. 2g, middle), resembling GkApcT in its substrate-bound state. Y257 (TM6) collaborates with G47-A48-G49 (TM1), V128 (TM3), and TM8 to seal the extracellular vestibule, thereby regulating substrate entry (Fig. 2g, left). This gating mechanism is reminiscent of the extracellular-side gating observed in previously characterized GkApcT and other members of the SLC7 family13,18,19. In contrast, the intracellular vestibule is more open, though a narrow conduit formed by TM1a, TM6b, and TM8 restricts access to the substrate-binding pocket (Fig. 2g, right). Moreover, the conserved aromatic Y257 in mCAT1, which corresponds to F252 in LAT1 (Supplementary Figs. 2, 3), is the critical gating residue regulating substrate translocation. Mutation of F252 to alanine (F252A) in LAT1 significantly reduces or abolishes transport activity18.

To examine conformational variability, we predicted multiple mCAT1 models using AlphaFold346, which has been shown to retain conformational memory of transporter dynamics47,48. Across the predicted states, the major conformation changes were located on TM1, TM6 and also two key residues, Y257 and F349 (Supplementary Fig. 5). Y257 reoriented with the extracellular vestibule, while F349 shifted at the cytoplasmic gate, together controlling pathway closure at both ends. In line with these observations, electrophysiological recordings showed that Y257A and F349A mutations significantly reduced transport activity, confirming their role as essential gating residues in mCAT1 (Fig. 2d, e).

Together, our structural and functional data reveal that substrate recognition in mCAT1 is mediated by a conserved set of polar interactions, while conformational transitions are governed by aromatic residues Y257 and F349, which act as dual gates at opposite ends of the transport pathway.

mCAT1 is a FrMLV receptor to specifically recognize its RBD domain
mCAT1 functions dually as a cationic amino acid transporter and as the entry receptor for FrMLV. To delineate the molecular basis of this viral recognition, we performed a structural analysis of the mCAT1/frRBD complex, thereby extending structural insights into viral receptor–ligand recognition, which have so far been obtained only for a limited number of well-characterized examples, including ASCT2–RD11449, NTCP–HBV50,51, NPC1–Ebola52, MFSD6–EV-D6853,54, and MFSD2A–SYNC255. The frRBD from mCAT1/frRBD complex exhibits a structural architecture similar to the unbound frRBD56 (RMSD = 0.871 Å), retaining its β-sandwich core with three variable regions (VRA, VRB, VRC) (Supplementary Fig. 6a, b). The longest segment, VRA (72 residues), folds into three α-helices (VRA-H1~H3) interconnected by loops (Supplementary Fig. 6a). The interaction between frRBD and mCAT1 is extensive, with the frRBD positioned above the extracellular domain of mCAT1. The interface, which buries a total surface area of approximately 728 Å², is characterized by a network of hydrogen bonds, electrostatic interactions, and hydrophobic contacts. The binding is primarily mediated by polar interactions between the VRA region of frRBD and mCAT1’s extracellular loops ECL3 and ECL6, as well as transmembrane helix TM1 (Fig. 3a).

The binding surface can be divided into three distinct patches. Patch 1 involves contacts between frRBD’s Loop H1H2, Loop H2H3, and VRA-H3 with mCAT1 ECL3 (Fig. 3b and Supplementary Fig. 6a). Y94′ is positioned near K222, while T129′ and N137′ approach E237, and N133′ lies adjacent to E221, forming polar interactions. R131′ is also located close to E221 and E237, suggesting electrostatic contributions that may further stabilize this region. Patch 2 is characterized by an extended hydrogen-bonding network between frRBD and mCAT1 (Fig. 3c). Here, S114′ and D120′ of frRBD interact with T321 and G236 of mCAT1. The carbonyl of C117′ is positioned near N232, D120′ is oriented toward the backbone amide of Y235, and W136′ lies close to the backbone amide of G236. These interdigitating contacts likely constitute a stabilizing core of the interface. Patch 3 comprises additional polar contacts, primarily involving potential salt bridges (Fig. 3d). R119′ of VRA-H2 is situated near E60 of TM1 and K234 of ECL3 of mCAT1, while T126′ oriented toward R511 (ECL6). Additionally, T126′ of Loop H2H3 is oriented toward R511 of ECL6, suggesting a hydrogen-bonding interaction. In addition to these patches, aromatic stacking may further stabilize the interface: P104′ and W136′ of frRBD are positioned for CH/π57 or π–π interactions with Y235 and F224 of mCAT1 (Fig. 3e).

Mutagenesis supported the critical role of these residues. Substitutions within Patch 1, particularly at E237, reduced the predicted binding free energy by ~1.2 kcal/mol (Fig. 3f), consistent with earlier studies that identified this residue as a determinant of molecular recognition31,32,58. Since mutagenesis primarily probes side-chain contributions, the structural data reveal additional backbone-mediated hydrogen bonds. Together, these findings indicate that both side-chain and backbone interactions cooperate to stabilize the complex.

Functional validation was obtained from pseudovirus infection assays. Alanine substitutions in frRBD residues T129′, R131′, N133′, R119′, E123′ and T126′, as well as the double mutant P104′/W136′, all reduced viral entry efficiency, although none of the single mutations completely abolished infection (Fig. 3g and Supplementary Fig. 7). Consistently, single-dose BLI sensorgrams showed that purified frRBD variants (Y94′, R119′, R131′ or N133′) exhibited slightly reduced binding responses to mCAT1 compared with wild type (Supplementary Fig. 8a). Together, these results confirmed that the mCAT1/frRBD interface is mediated by multiple polar and aromatic contacts, with side-chain contributions from Patch 1 and Patch 3 providing additional support, while the backbone-mediated hydrogen-bonding network in Patch 2 likely constitutes the predominant stabilizing force.

ECL3 rigidity encodes species-specific susceptibility to FrMLV infection
To elucidate the structural determinants of FrMLV-mCAT1 species specificity, sequence alignment of CAT1 across different species was performed and reveals substantial divergence in ECL3 region, particularly among primates and other non-rodent species. Notably, hCAT1 contains multiple insertions within this region, and key interacting residues—K222, F224, N232, Y235, and E237—are poorly conserved in humans, Ailuropoda melanoleuca, and other non-rodents (Fig. 4a), likely accounting for the inability of FrMLV to infect these hosts. A disulfide bond linking ECL3 and ECL4 (C226–C309) is conserved in both mouse and human CAT1 (Fig. 4a, b), suggesting that while the overall structural scaffold is maintained, the specific binding determinants have diverged.

To functionally assess this hypothesis, we established stable cell lines expressing either mCAT1 or hCAT1 (Supplementary Fig. 9). Cells expressing mCAT1 supported efficient FrMLV entry, while those expressing hCAT1 did not (Fig. 4c). Domain-swapping experiments pinpointed ECL3 as the critical determinant: replacing the ECL3 loop of mCAT1 with the corresponding segment from hCAT1 (mCAT1-hLoop) markedly reduced pseudovirus infection, while the reciprocal replacement rendered hCAT1 (hCAT1-mLoop) permissive to FrMLV entry (Fig. 4c). This finding was corroborated by binding assays, which showed that introducing human ECL3 into mCAT1 substantially weakened frRBD interaction (Supplementary Fig. 8b). Collectively, these results demonstrate that ECL3 is the central mediator of viral recognition and a key determinant of host range.

We next investigated the role of the conserved disulfide bond. Mutation of C309 to serine in mCAT1 led to a marked reduction in viral entry efficiency in pseudovirus assays (Fig. 4d). BLI measurements confirmed a corresponding decrease in binding affinity compared to wild-type protein (Fig. 4e). Notably, the interaction remained within the nanomolar range, indicating that the disulfide bond contributes positively to high-affinity binding but is not absolutely required. In addition to the disulfide bond, extensive π–π stacking interactions among residues in the ECL1, ECL3, and ECL4 were observed, which may contribute to the structural rigidity of ECL3 (Figs. 3g, 4b). This rigidity appears to be essential for maintaining the precise spatial conformation required for high-affinity MLV binding at the host cell surface.

Additionally, we extended our analysis to multiple MLV lineages, including several FrMLV subtypes, other exogenous MLVs, and endogenous murine retroviruses. The Env proteins of FrMLV subtypes were highly conserved, with PVC-211 differing from the prototypical strain by only a few substitutions at non-interface positions (Supplementary Fig. 10). Pseudovirus assays confirmed that MoMLV is able to infect Lenti-X 293T cells stably expressing mCAT1 (Fig. 4f). Accordingly, sequence alignment revealed strong conservation of the receptor-binding interface residues across diverse MLV lineages, with MoMLV preserving these key sites (Fig. 4g and Supplemental Fig. 10). These observations indicate that mCAT1 recognition is conserved among multiple MLV strains and related retroviruses, facilitated by the preservation of critical interfacial residues.

Taken together, our findings demonstrate that the rigid spatial configuration of ECL3, stabilized by disulfide bonding and interdomain π–π interactions, forms a high-fidelity binding platform for frRBD. The divergence of key ECL3 residues in non-rodent species structurally encodes the barriers to FrMLV tropism, thereby establishing ECL3 architecture and rigidity as critical determinants of species-specific viral entry.

Methods
Ethics statement
All experimental procedures complied with institutional and field-specific ethical guidelines. As this study did not involve human subjects, animal experiments, or materials requiring biosafety approval, no ethics committee approval was necessary.

Statistics & Reproducibility
No statistical method was used to predetermine sample size. No data were excluded from the analyses.
The experiments were not randomized.
The investigators were not blinded to allocation during data collection or analysis.

Molecular cloning
The full-length mouse CAT1 (mCAT1, UniprotKB: P30823), human (hCAT1, UniprotKB: P30825), and the receptor-binding domain (frRBD) of Friend murine leukemia virus (MLV, isolate 57) envelope glycoprotein (UniprotKB: P03390, residues 36~270) were synthesized by Anhui General Biology and human codon-optimized. The genes were then subcloned into the mammalian expression vector pCAG. The mCAT1 was fused with an N-terminal 3xFlag tag, a 2xStrepII tag, and a C-terminal EGFP, and the chimeric protein mCAT1-hLoop was generated by replacing the ECL3 sequence (QLTEEDFGNTSGRLCLNNDTKEGKPGVGGF) of mCAT1 with the human variant (QLTENKSSPLCGNNDTNVKYGEGGF). The expression plasmid for frRBD (or mutants) was fused with a CD5 N-terminal secretory signal peptide at the N-terminus and a 10xHis tag at the C-terminus. All plasmids used for transfections were prepared using the GoldHi EndoFree Plasmid Maxi Kit (CWBIO).

Protein expression and purification
HEK293F mammalian cells (Thermo Fisher Scientific) were cultured in SMM 293-TII medium (Sino Biological) at 37°C and 5% CO2 in a Multitron-Pro shaker (Infors, 130 rpm). When the cell density reached 2.0 × 10^6 cells/mL, plasmids were transiently transfected into the cells. For transfection of one liter of cell culture, approximately 1.5 mg of plasmid was premixed with 3 mg of polyethyleneimine (PEI) (Yeasen Biotechnology) in 50 mL of fresh medium and incubated for 30 minutes before being added to the cells. After 48 hours of overexpression, the cells were harvested by centrifugation at 4000 × g for 15 minutes. The cell pellet was resuspended in a buffer containing 25 mM Tris (pH 8.0), 150 mM NaCl, and a protease inhibitor mixture (Protease Inhibitor Cocktail, TargetMol). Next, 1% (w/v) lauryl maltose neopentyl glycol (LMNG) (Anatrace) and 0.1% (w/v) cholesteryl hemisuccinate Tris salt (Anatrace) were added to the suspension, and it was incubated at 4°C for 2 hours. After ultracentrifugation at 100,000 × g for 1 hour, the supernatant was collected and loaded onto anti-Flag M2 affinity resin (GenScript Biotech). The resin was washed with buffer containing 25 mM Tris (pH 8.0), 150 mM NaCl, and 0.06% LMNG (w/v). The protein was eluted with the wash buffer supplemented with 0.2 mg/mL Flag peptide. The eluted protein was then concentrated and subjected to size-exclusion chromatography (SEC) on a Superose 6 Increase 10/300 GL column (Cytiva) in a buffer containing 25 mM Tris (pH 8.0), 150 mM NaCl, and 0.06% LMNG.

The expression and purification of frRBD (WT and mutans) followed a protocol similar to that used for purifying the RBDs of coronaviruses60,61. The cells were harvested by centrifugation at 4000 × g for 15 minutes, and the secreted proteins were purified using Ni-NTA Agarose (Cytiva). The Ni-NTA Agarose was washed twice with wash buffer containing 25 mM Tris (pH 8.0), 150 mM NaCl, and 50 mM imidazole. The protein was eluted with elution buffer containing 25 mM Tris (pH 8.0), 150 mM NaCl, and 500 mM imidazole. The eluted protein was concentrated and further purified using size-exclusion chromatography (SEC) on a Superdex 200 Increase 10/300 GL column (Cytiva) in a buffer containing 25 mM Tris (pH 8.0), 150 mM NaCl.

Cryo-EM sample preparation and data acquisition
The purified mCAT1 and frRBD proteins were combined in a molar ratio of 1:3 and incubated for one hour (with 0.06% LMNG). The mixtures were subjected to SEC (Superose 6 Increase 10/300 GL, Cytiva) in a buffer containing 25 mM Tris (pH 8.0), 150 mM NaCl. The peak fractions from SEC were collected and concentrated. The protein samples were concentrated to approximately 8 mg/mL, and 1 mM ornithine was added before applying the sample to grids. Aliquots (3.5 μL) of the protein were placed onto glow-discharged holey carbon grids (Quantifoil Au R1.2/1.3). The grids were blotted for 4 seconds and flash-frozen in liquid ethane cooled by liquid nitrogen using a Vitrobot (Mark IV, Thermo Scientific). The prepared grids were transferred to a Krios microscope (Thermo Scientific) operating at 300 kV, equipped with a Gatan K3 detector and GIF Quantum energy filter. Movie stacks were automatically collected using EPU software (Thermo Scientific) with a slit width of 20 eV on the energy filter and a defocus range from −1.2 to −2.2 μm in super-resolution mode at 81,000 nominal magnification. Each stack was exposed for 2.56 seconds with an exposure time of 0.08 seconds per frame, resulting in a total of 32 frames per stack. The total dose rate was 50 e-/Å² per stack.

Data processing
The movie stacks were motion-corrected using MotionCor262,63. After patch CTF estimation, particles were automatically picked using templates generated from the initial 2D classification of manually picked particles in cryoSPARC64. After 2D classification, particles with clear features were selected and subjected to ab initio reconstruction to obtain the initial models. Multi-hetero refinement without symmetry was performed using cryoSPARC64, followed by non-uniform refinement, global CTF refinement, local refinement, and DeepEMhancer65, yielding a 3D reconstruction of the full structure. Resolution was estimated using the gold-standard Fourier shell correlation (FSC) 0.143 criterion66 with high-resolution noise substitution67.

Model building and structure refinement
For model building, atomic models predicted by AlphaFold346 were used as templates and were molecular dynamics flexibly fitted68 into the cryo-EM maps. These models were manually adjusted using Coot69 to refine the atomic details. Each residue was checked considering its chemical properties during model building. Several segments with no corresponding density were excluded from the model. Structural refinement was performed in Phenix70, with secondary structure and geometry restraints applied to prevent overfitting.

Bio-layer interferometry
Bio-layer interferometry (BLI) experiments were carried out on an Octet system (ForteBio) to assess protein–protein interactions. StrepII-tagged mCAT1, the C309S mutant, or the mCAT1-hLoop chimera were immobilized onto pre-equilibrated streptavidin (SA) biosensors. The loaded sensors were then exposed to serial dilutions of purified frRBD or its mutants, and real-time binding kinetics were recorded. After the association phase, the biosensors were transferred into the regeneration buffer to monitor dissociation. Sensorgrams were processed using the Octet Data Analysis software to derive kinetic parameters and binding affinities for the interactions between mCAT1 variants and frRBD proteins. All measurements were performed in two or three independent replicates to ensure reproducibility.

Prediction of mutation effects on mCAT1/frRBD binding affinity
The effects of single-point and multi-site missense mutations on protein-protein binding affinity were predicted using mCSM-PPI271 and mmCSM-PPI72, respectively. These AI-based algorithms employ graph-based structural signatures to evaluate how mutations perturb residue interaction networks, evolutionary profiles, complex network metrics, and energy terms. The predictions integrate these features with substitution scores derived from normal mode analysis and dynamic terms, providing more accurate assessments than traditional force field-based approaches. For our analysis, the 3D structure of the mCAT1/frRBD complex was uploaded to the mCSM web server (https://biosig.lab.uq.edu.au/mcsm_ppi2/, https://biosig.lab.uq.edu.au/mmcsm_ppi/). All single-point mutations and combinatorial mutations at the interface residues were systematically input for evaluation. The server’s user-friendly interface facilitated efficient processing and output of optimized binding affinity predictions.

Generation of stable cell lines
Full-length CAT1 from Mus musculus and Homo sapiens was cloned into the lentiCRISPRv2 vector73 under the control of the SFFV promoter. A Flag tag was fused at the C terminus, followed by a self-cleaving P2A sequence (ATNFSLLKQAGDVEENPGP), an mCherry reporter, and a ribosome-binding site driving the expression of a puromycin resistance gene. Two additional chimeric constructs were generated using the same strategy. Lentiviral particles were produced by co-transfecting Lenti-X 293T cells (Clontech, 632180) with the transfer plasmids, psPAX2, and pMD2.G. Supernatants were collected 72h post-transfection, clarified, filtered (0.45 μm), and concentrated using protamine sulfate (80 μg/mL, Macklin) and chondroitin sulfate C (80 μg/mL, Macklin) as described previously74. Target Lenti-X 293T cells were transduced in the presence of 5 μg/mL polybrene, and stable cell lines were obtained through puromycin selection and subsequent passaging to eliminate untransduced cells.

Pseudovirus production and entry assays
Full-length MLV Env proteins (FrMLV isolate 57 [P03390], and MoMLV isolate Shinnick [P03385]), with or without point mutations, were cloned into the pCAG vector with a C-terminal 10×His tag. Pseudoviruses (PSVs) were generated using the pNL4-3.Luc.R⁻E⁻ backbone, which carries a luciferase reporter but lacks functional Env. Lenti-X 293T cells were co-transfected with pNL4-3.Luc.R⁻E⁻ (20 μg) and Env-expressing plasmids (10 μg) using PEI (90 μg). Supernatants were harvested 48h post-transfection, clarified by centrifugation (12,000 rpm, 10 min, 4°C), aliquoted, and stored at −80°C.

For entry assays, Lenti-X 293T cells stably or transiently expressing the indicated mCAT1 orthologs or mutants were seeded into 96-well plates (5×10⁴ cells/well) and infected with PSV in the presence of 8 μg/mL polybrene. After 18h, the medium was replaced with fresh D10, and cells were cultured for another 48h. Luciferase activity was measured using the Luciferase Assay System (Promega, E1501) on a Varioskan LUX plate reader (Thermo Fisher Scientific). All assays were performed in triplicate.

Immunofluorescence staining of stable cell lines
Stable Lenti-X 293T cells expressing CAT1 orthologs or chimeras with C-terminal Flag tags were fixed and permeabilized with 100% methanol for 10 min at room temperature. Cells were incubated with anti-Flag (DDDDK) antibody (ABclonal, AE005) in PBS/1% BSA for 1h at 37°C, washed, and stained with ABflo® 647–conjugated goat anti-mouse IgG (ABclonal, AS059) for 1h at 37°C in the dark. Nuclei were counterstained with Hoechst 33342 (1:100 in HBSS) for 30 min at 37°C. Images were acquired with a CSU-W1 SoRa spinning disk confocal system (Nikon) and processed using Fiji (ImageJ v1.54f).

Expression of mCAT1 mutants
Lenti-X 293T cells were transiently transfected with mCAT1–eGFP mutants (30 μg plasmid DNA per 10 cm dish) using PEI (90 μg). After 4h, the medium was replaced with D10, and cells were incubated for 48h. Cells were then replated for Western blot, Electrophysiology assays or Pseudovirus entry assays, and fluorescence imaging. For imaging, nuclei were stained with Hoechst 33342 (1:100 in HBSS) and syncytia formation was visualized under the CSU-W1 SoRa confocal system (Nikon) and analyzed with Fiji.

Western blot
PSV-containing supernatants were concentrated using protamine sulfate (80 μg/mL) and chondroitin sulfate C (80 μg/mL). Viral pellets were lysed with Cell Culture Lysis Reagent (Promega, diluted 1:5), mixed with 5× SDS loading buffer, and incubated at room temperature for 10 min. Samples were resolved by SDS–PAGE, transferred, and probed with anti-His monoclonal antibody (CWBIO, CW0286M), followed by HRP-conjugated goat anti-mouse IgG (CWBIO, CW0102). Detection was performed with BeyoECL Plus (Beyotime, P0018S) using an Amersham Imager 680 (Cytiva). Plasmids encoding the wild-type, mutants, and negative-control constructs were transfected into 10-cm culture dishes. After 36h of transfection, cells were harvested by centrifugation at 400 × g for 5 min and lysed with 1% DDM in buffer containing 25 mM Tris-HCl and 150 mM NaCl at 4°C for 2h with gentle rotation. The lysates were clarified by centrifugation at 12,000 rpm for 30 min, and the resulting supernatant was diluted tenfold with the buffer containing 25 mM Tris-HCl and 150 mM NaCl to minimize interference of DDM during electrophoresis. Samples were then mixed with 5× SDS loading buffer and incubated at room temperature for 10 min. Proteins were separated by SDS–PAGE, transferred to PVDF membranes, and probed with either an anti-Flag monoclonal antibody (HUABIO, M1403-2) or an anti-β-actin monoclonal antibody (Proteintech, 66009-1-Ig), followed by HRP-conjugated goat anti-mouse IgG secondary antibody (Proteintech, SA00001-1). Protein bands were visualized using the BeyoECL Plus chemiluminescence detection kit (Beyotime, P0018S) and imaged with an Amersham Imager 680 system (Cytiva).

Electrophysiology
Lenti-X 293T cells were maintained in DMEM (Gibco) medium supplemented with 10% fetal bovine serum (FBS; Biological Industries) at 37°C in a 5% CO2 atmosphere. Transfection when the cells reached 80% confluence. After 24h of transfection, the cells were cultured overnight on poly-L-lysine-treated coverslips. Whole-cell recordings were performed using borosilicate glass pipettes pulled with a P-1000 puller (Sutter Instrument). The whole-cell currents were recorded using a system consisting of an Axopatch 700B amplifier and a Digidata 1550B digitizer (Molecular Devices). A step protocol (from −80 to +80 mV in 10-mV increments, 2000-ms duration) was used to record the ionic currents. The junction potential was compensated for prior to recording. The compositions of the pipette and bath solutions were based on those used for mCAT1 whole-cell recording in oocytes75. The pipette solution contained 130 mM CsCl, 2 mM MgCl2, 1 mM CaCl2, and 20 mM HEPES (pH 7.2). The bath solution contained 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 20 mM HEPES, and 20 mM L-ornithine (pH adjusted to 7.4 with NaOH). All data were acquired using pCLAMP software (Molecular Devices) and analyzed with pCLAMP 10.6 (Molecular Devices) and OriginPro 2019 (OriginLab).

Molecular dynamics simulations
The initial protein structure was obtained from cryo-electron microscopy (cryo-EM) data (PDB ID: 9UAT). The frRBD domain was not included in the simulated system. Membrane orientation was assigned using the Orientations of Proteins in Membranes (OPM) database web server to ensure correct insertion and positioning within the lipid bilayer. The system was built using the Maestro interface (Schrödinger Release 2023-2), followed by solvation with explicit TIP3P water molecules and neutralization with NaCl at 0.15 M physiological salt concentration. The final simulation box was orthorhombic with dimensions 92.7 × 83.8 × 98.8 Å3, containing 68,270 atoms in total (including protein, ligand, lipid, water, and ions).

All molecular dynamics (MD) simulations were carried out with the Desmond simulation engine (Schrödinger Release 2023-2) using the OPLS4 all-atom force field. Default relaxation protocols were applied prior to production runs, consisting of restrained minimization and short equilibration simulations in the NVT and NPT ensembles. After equilibration, three independent production simulations were initiated with different random seeds to ensure independence of trajectories and allow statistical assessment of convergence. Each production run was performed for 100 ns under NPT ensemble, with temperature maintained at 310 K and pressure at 1 atm using default thermostat and barostat as implemented in Desmond. A 9 Å cutoff was used for short-range nonbonded interactions, while long-range electrostatics were treated with the particle mesh Ewald (PME) method. Time step was set to 2 fs. Protein Cα atoms were used as the reference for structural alignment, while the RMSD of ornithine was calculated using its heavy atoms. All trajectory analyses were performed with the built-in tools of the Schrödinger suite.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_2": "The manuscript “Structural insights into cationic amino acid transport and viral receptor engagement by CAT1” authored by\nLingyun Xia et al. presents the CryoEM structure of mouse CAT1 (mCAT1) in inward-facing occluded conformation with the\nsubstrate L-ornithine bound, and in complex with the RBD domain of Friend murine leukemia virus (FrMLV). This structure is\nthe first one for a mammalian Cationic Amino acid Transporter (CAT), only after the structure of the bacterial CAT\nhomologue GkApcT. mCAT1 structure confirms the GkApcT structure in inward-facing occluded state (Jungnickel, Parker\nand Newstead, Nat Commun 2018). Two issues within mCAT1 manuscript merit publication in Nat Commun: i) description\nof residues that the structure propose to be key for the substrate specificity of CAT1, and ii) structural bases for FrMLV\nspecificity. Indeed, these two points are highlighted in the abstract by the authors. Interestingly, the occlusion of the\ntransporter confines the substrate L-ornithine facilitating an electrostatic volume that clearly define the substrate L-ornithine.\nIn many other structures of Slc7 family transporters in non-occluded conformation, the side-chain of the amino acid\nsubstrates is not well defined by the electrostatic volume. Beside these very positive considerations about this manuscript, I\nhave several points that the author might consider to improve their manuscript.\nRelevant points\n1. The functional validation of the interacting residues between virus RBD and mCAT1 by Bio-layer interferometry is\nexcellent. In contrast, as a “functional validation” of the L-ornithine interacting residues, the authors rely in the superposition\nof a mutated version of mCAT1 structure bound to L-arginine in the binding site(PDB ID: 6F34; also in Jungnickel, Parker\nand Newstead, Nat Commun 2018). In this validation (Fig. 2c), the side-chain amino group of L-ornithine has a H-bond with\nthe side-chain hydroxyl of Ser 347. In contrast, the side-chain L-arginine (guanidinium) presents H bonds with carboxylate of\nAsp 237 in GkApcT; a residue too far away to permit the side-chain of L-ornithine to bind the homologous D263 in mCAT1.\nMoreover, Glu 115, responsible for proton coupling in GkApcT, is Ser 120 in mCAT1, a transporter that is not proton\ndependent. In summary, all fits, but the authors claim in their abstract that “our structural and biochemical results\nelucidate the molecular mechanism of substrate recognition … (of mCAT1)”. The authors might consider to validate the impact of\nmutations of residues Ser 347 (e.g., to alanine) and Asp 263 (e.g., to alanine) on L-ornithine and L-arginine transport\nrespectively. It will be interesting to see whether Vmax or Km of transport would be affected in these mutants. Alternatively,\neven though not so clarifying, the authors might delete the “biochemical” results supporting substrate recognition in the\nabstract.\n2. It is very interesting that mCAT1 presents a disulphide bridge between ECL3 and ECL4. The two cysteine residues\ninvolved are conserved in mammalian CAT1 transporters (Fig. 4a) suggesting a transport function relevance. As the authors\nshow clearly, this disulphide bridge is key for the RBD virus binding, and specificity relies in sequence changes in different\nspecies within the relevant interactors. Thus, the conservation of the disulphide bridge between ECL3 and ECL4 might have\nan impact on transport activity. In the LAT subfamily of SLC7 transporters, ECL3 and ECL4 show several non-covalent\ninteractors, and the extracellular domain of the ancillary subunit (4F2, also named SLC3A2 or CD98hc) may stabilize this\ninteraction via connection with ECL3. Indeed, this could be at the bases of the role of this ancillary protein on LAT stability\nand transport. Indeed, the authors indicate in the second paragraph of the introduction that the ancillary protein (e.g., 4F2)\nmodifies the substrate specificity of the light subunit. Again, I propose the authors to consider testing whether elimination of\nthe ECL3-4 disulphide bridge impact on the transport activity of mCAT1 (e.g., changes in Vmax or Km).\n3. Is it known if the RBD blocks mCAT1 transport? The binding of FrMLV-RBD binds to ECL3 and ECL4, extracellular loops\nthat would block conformational transitions to outward-facing states.\nMinor comment\nIn the second paragraph of the introduction there are several issues related to the role of the ancillary proteins in the\nHeteromeric Amino acid transporters (lines 49-51). 4F2 stabilizes its light subunits, but b0,+AT stabilizes rBAT.\nHeterodimerización (4F2 associated transporters) is needed for the biogenesis of the transporter to the plasma membrane,\nand superdimer (rBAT/b0,+AT is also needed for biogenesis of the transporter to the plasma membrane (look at rBAT\npapers by Shushi Nagamori and Manuel Palacín).", "reviewer_3": "The manuscript by Xia et al. presents the cryo-EM structure of mammalian CAT1 (mouse) in complex with its substrate\nornithine and the FrMLV receptor-binding domain, aiming to elucidate mechanisms of substrate recognition and viral\nreceptor interaction. While the study touches on relevant biological themes, the overall novelty and depth of analysis are\nlimited, and key mechanistic insights remain underdeveloped. I have some general and specific comments on the\nmanuscript.\nGeneral comments:\nA) The overall statistics for the described structure are rather poor (high clash score, high percentage of CaBLAM outliers,\n…). For numerous side chains that have been mentioned as ‘critical’ in the manuscript, the position cannot be\nunambiguously assigned due to missing density. Therefore, I would be cautious in interpreting these data. The same is true\nfor the potentially bound substrate ornithine.\nB) The authors claim to present mechanistic insights – to me, it is rather the description of a single structure with very limited\nexperimental work. This becomes obvious throughout the manuscript because the authors repeatedly use the word\n‘suggest’, indicating that the conclusions are mainly speculative.\nAs such, the manuscript requires substantial improvement to meet the standards of a high-impact journal.\nSpecific comments:\n- Figure 1a: Please add the global fit of a 1:1 model to calculate the Kd to the graph. Do the curves return to the baseline in\nthe dissociation phase? It seems that there is some unspecific interaction with the surface. The x-axis should start with 0\nseconds. Please state the number of replicates (also in the MM section)\n- Please show the Cryo-EM quality assessment (density for all TMs, ligands, etc), state the map contour level, and map type\nshown. Also, add a local resolution map to supplementary figure 1\n- Line 107: Please introduce ATM1\n- Supplementary figure 1: I cannot see a blue box with dimers – the box is red\n- Please add labels for all ICLs and ECLs in figure 1d; it is also worth highlighting the conserved disulfide bridge\n- Line 114: ‘with TM1~TM12 forming a pseudosymmetric transmembrane core’: statement unclear-> TM1 and TM12 are not\nforming the binding cavity: please clarify\n- Line 119: ‘1.644 Å’ – two digits after the comma are enough\n-Include a full sequence alignment of various homologues, also highlighting the conservation of the two extra TM’s. Do the\nauthors have any idea about the functional role of these TM’s? Can they be removed?\n- Figure 2A: Please show the density for ornithine as a panel figure; state the map type and threshold. After checking the\nprovided map, I’m not fully convinced whether the ligand can be unambiguously assigned to ornithine. Without prior\nknowledge from the bacterial/LAT1 and 2 transporter work, it is very unlikely to assign this density to the ligand density. A\ncomparison to an apo structure would be very helpful. In addition, functional data on binding relevant mutants and/or MD\nsimulations could further strengthen the hypothesis that this density could be assigned to ornithine. The section on the\nornithine binding mode needs to be rephrased, and statements like ‘..exhibited a strong fit, …’ should be avoided.\n- In supplementary figure 2, a comparison of the binding sites of different homologues bound to different AS is stated.\nHowever, the residues forming the binding sites and interactions with the AS are neither shown nor stated. This makes a\ncomparison very difficult.\n- Line 149: remove one ‘its’\n- Line 155: What is Extended Data Fig. 3?\n- Line 178: To my knowledge, LAT1 is a transporter and not a channel\n- Line 179: Why is the protein AdiC mentioned here? It has not been introduced before\n- Line 185-187: Transition to receptor-bound chapter missing: 4 well-characterized examples of what?? I guess you mean\nreceptor interaction. In this context, I would also mention the MFSD2A–SYNC2 complex.\n- Lines 194-197: I recommend not discussing a 1Å shift in a 3.7 Å structure. Furthermore, the authors discuss reorientations\nof sidechains (e.g. E123’), but this side chain is poorly defined in the EM map\n- Lines 205 – 222: The authors describe several interactions between mCAT1 and frRBD. However, the positions of some of\nthe mentioned side chains are only poorly defined in the EM map (e.g., E237, R511, …), so it is very speculative whether\nthese residues, for example, form a hydrogen or not. I advise checking the validity of the position of the mentioned residues,\nwhich are supposed to be involved in various interactions.\n- Lines 222 – 232: The authors used a computational tool to dissect the strength of the binding interface between mCAT1\nand frRBD. Although I agree that it is not necessary to mutate all residues potentially involved in the interaction, but I\nrecommend testing at least some of the computational predictions experimentally. This only requires the generation of a few\nmutants and testing them in binding assays.\n- Figure 4c, d: Please indicate the time frame for the on-reaction and the off-reaction; add corresponding fit; start at 0 s. The\nauthors do not state what conditions under reducing conditions mean. This information is missing, and how can they be sure\nthat the disulfide bond is reduced? One could also mutate one of the Cys residues to a Ser and check for binding.\n- The authors show a very simplified Table 1 (B-factors, Validation scores, … missing)"}, "author_response": "Response: We truly appreciate the time you invested in reviewing our work and the careful, constructive feedback you provided. Your comments were very helpful in identifying areas where our manuscript could be made stronger, both in terms of experimental support and clarity of interpretation.\nYou noted in particular the need for more robust functional evidence and a more cautious approach to interpreting structural features. In the revised manuscript, we have made a number of changes in direct response to these points. First, we reprocessed the cryo-EM data, which improved the final map quality to 3.65 Å and yielded better validation statistics; this updated map is presented together with local resolution analysis and expanded density panels. We also expanded Table 1 to include B-factors, validation scores, and other standard statistics. Where the density was less well resolved, we have rephrased our descriptions and avoided definitive statements. Second, we added several new experimental datasets. These include molecular dynamics simulations (three independent 100-ns runs), electrophysiological measurements of substrate-binding mutants (S44A, S120A, Y121A, Y257A, D263A, S343A, S347A, and F349A), pseudovirus infection assays with targeted interface mutations in frRBD, and BLI binding experiments with selected RBD variants. Together these data provide complementary evidence for the roles of key residues in substrate recognition and viral receptor binding. Finally, we carefully revised the wording throughout the manuscript so that the distinction between direct experimental evidence and structural inference is clear.\nWe are grateful for your insightful critiques, which guided us to reinforce the mechanistic depth of our study and to present our findings in a more rigorous and balanced manner.\nRelevant points\nQ1: The functional validation of the interacting residues between virus RBD and mCAT1 by Bio-layer interferometry is excellent. In contrast, as a “functional validation” of the L-ornithine interacting residues, the authors rely in the superposition of a mutated version of mCAT1 structure bound to L-arginine in the binding site(PDB ID: 6F34; also in Jungnickel, Parker and Newstead, Nat Commun 2018). In this validation (Fig. 2c), the side-chain amino group of L-ornithine has a H-bond with the side-chain hydroxyl of Ser 347. In contrast, the side-chain L-arginine (guanidinium) presents H bonds with carboxylate of Asp 237 in GkApcT; a residue too far away to permit the side-chain of L-ornithine to bind the homologous D263 in mCAT1. Moreover, Glu115, responsible for proton coupling in GkApcT, is Ser 120 in mCAT1, a transporter that is not proton dependent. In summary, all fits, but the authors claim in their abstract that “our structural and biochemical results elucidate the molecular mechanism of substrate recognition … (of mCAT1)”. The authors might consider to validate the impact of mutations of residues Ser 347 (e.g., to alanine) and Asp 263 (e.g., to alanine) on L-ornithine and L-arginine transport respectively. It will be interesting to see whether Vmax or Km of transport would be affected in these mutants. Alternatively, even though not so clarifying, the authors might delete the “biochemical” results supporting substrate recognition in the abstract.\nA1: Thanks for this thoughtful and constructive comment. In the revised manuscript, we have substantially strengthened our analysis of the substrate-binding pocket in mCAT1.\nFirst, we systematically classified the conserved substrate-binding residues across the SLC7 family based on available structural data, which confirmed the plausibility of our ornithine placement within the pocket (Fig. L1). This structural assignment was further supported by three independent 100-ns molecular dynamics simulations, which consistently showed that ornithine remained stably bound with an average RMSD of ~1.5 Å, reinforcing the validity of the modeled ligand conformation (Fig. L2).\nFig. L1 (revised Supplementary Figs. 2c, d)\nSecond, as suggested, we performed mutagenesis of the predicted binding residues (S44, Y257, S343, and S347) and assessed their function by whole-cell electrophysiology (Figs. 2d, e). Consistent with the structural model, mutations at Y257, S343, and S347 significantly reduced ornithine-induced currents, whereas S44A retained near wild-type activity, in agreement with a backbone-mediated contact. These results provide direct evidence for the involvement of these residues in ornithine coordination. In addition, as suggested by the reviewer, we examined D263A and S347A mutants (Fig. L2). Both showed impaired transport activity: S347A due to loss of a direct polar contact, and D263A likely through disruption of conformational transitions required for gating, even though this residue does not directly contact ornithine in our structure.\nThese new data provide convergent evidence that our assignment of the ornithine binding model is accurate, while also acknowledging that additional transport intermediates may exist. We have included these new data in the revised manuscript (Figs. L1, L2). Accordingly, the text in the Results section has been updated to describe these findings.\nFig. L2 (revised Figs. 2c-e)\nQ2. It is very interesting that mCAT1 presents a disulphide bridge between ECL3 and ECL4. The two cysteine residues involved are conserved in mammalian CAT1 transporters (Fig. 4a) suggesting a transport function relevance. As the authors show clearly, this disulphide bridge is key for the RBD virus binding, and specificity relies in sequence changes in different species within the relevant interactors. Thus, the conservation of the disulphide bridge between ECL3 and ECL4 might have an impact on transport activity. In the LAT subfamily of SLC7 transporters, ECL3 and ECL4 show several non-covalent interactors, and the extracellular domain of the ancillary subunit (4F2, also named SLC3A2 or CD98hc) may stabilize this interaction via connection with ECL3. Indeed, this could be at the bases of the role of this ancillary protein on LAT stability and transport. Indeed, the authors indicate in the second paragraph of the introduction that the ancillary protein (e.g., 4F2) modifies the substrate specificity of the light subunit. Again, I propose the authors to consider testing whether elimination of the ECL3-4 disulphide bridge impact on the transport activity of mCAT1 (e.g., changes in Vmax or Km).\nA2: We greatly appreciate your thoughtful suggestion to examine the functional role of the conserved disulphide bond between ECL3 and ECL4. To address this, we generated the C309S mutant to disrupt the linkage. Consistent with your expectation, our BLI assays showed that the mutation reduced frRBD binding affinity, although the interaction remained within the nanomolar range (Fig. L3). Likewise, pseudovirus infection assays demonstrated that C309S decreased viral entry efficiency, though the effect was moderate compared with wild type (Fig. L3).\nIn addition, as you proposed, we assessed whether this disulphide bond contributes to transport activity. Electrophysiological recordings of the C309S mutant showed somewhat larger currents relative to wild type, suggesting a possible impact on transport kinetics; however, statistical analysis (two-tailed t-test) did not reveal significant differences (Fig. L2). This indicates that while the ECL3–ECL4 disulphide bond is clearly important for viral recognition, its influence on substrate transport appears, if anything, to be subtle.\nFig. L3 (revised Figs. 4d, e)\nQ3. Is it known if the RBD blocks mCAT1 transport? The binding of FrMLV-RBD binds to ECL3 and ECL4, extracellular loops that would block conformational transitions to outward-facing states.\nA3: Thank you for raising this important point. As you suggested, binding of the FrMLV RBD to ECL3 and ECL4 might in principle interfere with conformational transitions of mCAT1. To examine this possibility, we directly tested the effect of purified frRBD protein on transport activity. Whole-cell patch-clamp recordings in Lenti-X 293T cells stably expressing mCAT1 showed that addition of 1 μg frRBD protein to the recording solution did not alter ornithine-induced currents, and the current amplitudes were comparable to vehicle controls (Fig. L4).\nThese results indicate that engagement of ECL3 by frRBD does not measurably interfere with the transport of ornithine under our experimental conditions. Thus, the receptor-binding function of ECL3 and the substrate transport function of the conserved binding pocket appear to operate largely independently. That said, we cannot exclude the possibility that more subtle effects on transport may occur under physiological conditions not fully captured by our assay.\nFig. L4 (revised Supplementary Fig. 11)\nMinor comment\nQ: In the second paragraph of the introduction there are several issues related to the role of the ancillary proteins in the Heteromeric Amino acid transporters (lines 49-51). 4F2 stabilizes its light subunits, but b[0,+]AT stabilizes rBAT. Heterodimerización (4F2 associated transporters) is needed for the biogenesis of the transporter to the plasma membrane, and superdimer (rBAT/b[0,+]AT is also needed for biogenesis of the transporter to the plasma membrane (look at rBAT papers by Shushi Nagamori and Manuel Palacín).\nA: We sincerely thank you for pointing out this important clarification regarding the roles of ancillary proteins in heteromeric amino acid transporters. Following your suggestion, we have revised the second paragraph of the Introduction (highlighted in yellow in the revised manuscript) to provide a more accurate description and have included the relevant references (Nagamori et al.; Palacín et al.).\nThe revised text now reads as follows (lines 45–57 in the revised manuscript, Introduction, highlighted in yellow):\n“Structural and functional studies of SLC7 family members, including the bacterial proton-dependent transporter GkApcT13 and AdiC14, the heteromeric amino acid transporter b[0,+]AT115-17, and the neutral amino acid transporters LAT118,19 and LAT220,21, have demonstrated that most SLC7 proteins require association with a heavy chain subunit from the SLC3 family for proper biogenesis and function. For example, LAT1 and LAT2 need to heterodimerize with 4F2hc, which stabilizes the light subunit and is essential for trafficking to the plasma membrane18,19. In contrast, b[0,+]AT1 associates with rBAT, where the light subunit stabilizes the heavy subunit, and recent studies have shown that formation of a higher-order “superdimer” of the b[0,+]AT1-rBAT complex is also required for correct biogenesis and membrane localization17,22. Such diversities in subunit organization, substrate specificity, and transport mechanisms highlights the remarkable functional versatility of the SLC7 family.”\nReviewer #3\nThe manuscript by Xia et al. presents the cryo-EM structure of mammalian CAT1 (mouse) in complex with its substrate ornithine and the FrMLV receptor-binding domain, aiming to elucidate mechanisms of substrate recognition and viral receptor interaction. While the study touches on relevant biological themes, the overall novelty and depth of analysis are limited, and key mechanistic insights remain underdeveloped. I have some general and specific comments on the manuscript.\nResponse\nThank you very much for your detailed review and the thoughtful comments you shared. Your feedback was essential in helping us improve both the presentation of our data and the overall rigor of our analysis.\nIn response to your concerns regarding structural quality, we reprocessed our cryo-EM dataset, improving the map to 3.65 Å resolution with stronger validation metrics. We also added density views for all transmembrane helices and the ligand, clearly indicated contour levels and map types, and provided a local resolution map. Table 1 has been expanded to include B-factors and validation scores. Regarding substrate recognition, we combined comparative analysis of homologous transporters with molecular dynamics simulations and electrophysiological assays of binding-site mutants. These new data strengthen the case for ornithine binding while also ensuring that our descriptions avoid overstated claims. For viral receptor recognition, we rewrote the interaction descriptions to better reflect the limitations of the density. We also added experimental validation in the form of pseudovirus entry assays and BLI binding tests with selected mutants, which support the proposed interface interactions. In addition, we corrected and clarified several points you highlighted, including terminology (e.g. “gating” instead of “channel”), the introduction of AdiC, and the transitions between structural and receptor-binding sections.\nWe greatly value your careful and constructive feedback, which has helped us refine the manuscript substantially. We believe these changes address your concerns and strengthen the manuscript both in precision and in clarity.\nGeneral comments\n1) The overall statistics for the described structure are rather poor (high clash score, high percentage of CaBLAM outliers, …). For numerous side chains that have been mentioned as ‘critical’ in the manuscript, the position cannot be unambiguously assigned due to missing density. Therefore, I would be cautious in interpreting these data. The same is true for the potentially bound substrate ornithine.\nQ1: We thank you for this valuable observation. In the revised work, the cryo-EM data were reprocessed with enhanced particle polishing and CTF refinement, yielding a 3.65 Å map with improved validation metrics; the clash score (6.08) and CaBLAM outliers (0.7%) are now within acceptable ranges (Table L1).\nFor interface residues in the mCAT1/frRBD complex where side-chain density remains less well defined, we complemented the structural analysis with mutagenesis, pseudovirus infection assays (Fig. L1, left), and binding tests (Fig. L1, right), which consistently support their contribution to viral recognition. For the ornithine-binding pocket, molecular dynamics simulations (Fig. L2, left) and electrophysiological assays of site-directed mutants confirmed the importance of key residues for substrate transport (Fig. L2, middle and right).\nWe have also revised the manuscript text to present these findings with appropriate caution in cases where density is ambiguous.\nTable 1 Statistics of cryo-EM data collection, processing, and model refinement.\n(EMDB: EMD-63995)\n(PDB: 9UAT )\nData collection and processing\nEM equipment Krios (Thermo Scientific)\nVoltage (kV) 300\nDetector Gatan K3 Summit\nEnergy filter Gatan GIF Quantum, 20 eV slit\nElectron dose (e-/Å2) 50\nPixel size (Å) 1.087\nDefocus range (μm) -1.2 ~ -2.2\nNumber of collected micrographs 2,402\nSample mCAT1/frRBD\n3D Reconstruction\nSoftware cryoSPARC\nNumber of used particles 106,963\nResolution (Å) 3.65\nSymmetry C1\nRefinement\nModel resolution (Å)\n3.9\n(FSC threshold = 0.5)\nSoftware Phenix\nCell dimensions\na=b=c (Å) 278.272\nα=β=γ (˚) 90\nModel composition\nAtoms 11613 (Hydrogen 5788)\nProtein residues 767\nLigands (Orn) 1\nB factors (Å2)\nProtein 78.73\nLigand (Orn) 66.20\nR.m.s. deviations\nBond lengths (Å) 0.002\nBond angles (°) 0.498\nValidation\nMolProbity score 1.44\nCaBLAM outliers 0.7%\nCC (Mask) 0.71\nClash score 6.08\nRotamers outliers (%) 2.74\nRamachandran plot\nFavored (%) 97.45\nAllowed (%) 2.55\nDisallowed (%) 0\nTable L1 (revised Table 1)\nFig. L1 (revised Fig. 3g (left) and Supplementary Fig. 8a (right))\nFig. L2 (revised Figs. 2c-e)\n2) The authors claim to present mechanistic insights – to me, it is rather the description of a single structure with very limited experimental work. This becomes obvious throughout the manuscript because the authors repeatedly use the word ‘suggest’, indicating that the conclusions are mainly speculative.\nQ2: We fully appreciate your concern that in the original submission, several of our mechanistic statements were based largely on structural descriptions. In the revised manuscript, we have substantially strengthened the experimental foundation in several ways:\n1. Substrate recognition.\n1) Molecular dynamics simulations (three independent 100-ns runs) showed that ornithine remained stably bound with an average RMSD of ~1.5 Å (Fig. L2, left).\n2) Electrophysiology assays demonstrated that mutations of predicted binding residues (Y257A， S343A, S347A) significantly reduced ornithine-induced currents, whereas S44A retained near wild-type activity, consistent with its backbone-mediated role (Fig. L2, middle and right).\n3) Additional mutants (S120A, D263A) also showed decreased transport, highlighting their contribution to substrate stabilization (Fig. L2, middle and right).\n2. Viral recognition.\n1) Pseudovirus infection assays showed that mutations at the RBD interface (R119′, E123′, T126′, R131′, N133′, W136′, W136′/P104′) led to a noticeable reduction in viral entry efficiency, although none abolished infection entirely, underscoring the cooperative role of multiple contacts (Fig. L1, left).\n2) Binding assays (BLI) with purified RBD variants (R119′, R131′, N133′) revealed moderately reduced affinities, consistent with their involvement in receptor engagement (Fig. L1, right)..\n3) Domain-swapping experiments demonstrated that replacing mCAT1 ECL3 with the human counterpart markedly reduced viral entry, while the reciprocal replacement conferred susceptibility to FrMLV (Fig. L3, left).\n3. C309S disulphide mutant.\n1) This mutant reduced frRBD binding (still within nM range) and moderately impaired viral entry (Fig. L3, middle and right).\n2) Electrophysiology showed somewhat elevated currents, though without statistical significance, suggesting limited impact on transport (Fig. L2, middle and right).\n4. In addition, we have undertaken a careful revision of the manuscript's language throughout: where conclusions are directly supported by new data, we present them explicitly; where interpretation is based on structural inference, we have tempered the language to avoid overstatement.\nWe believe these new experiments and textual revisions significantly enhance the mechanistic depth of the manuscript and provide a balanced link between structure and function.\nFig. L3 (revised Figs. 4c-e)\nSpecific comments\n- Figure 1a: Please add the global fit of a 1:1 model to calculate the Kd to the graph. Do the curves return to the baseline in the dissociation phase? It seems that there is some unspecific interaction with the surface. The x-axis should start with 0 seconds. Please state the number of replicates (also in the MM section)\nA: We sincerely thank you for these constructive suggestions. Following your advice, we have made several improvements:\n1. Model fitting: A global 1:1 binding model fit has now been included in revised Fig. 1a (Fig. L4), and the corresponding K values are reported.\nD\n2. Dissociation phase and specificity: We agree that the dissociation curves do not fully return to baseline. To further assess the possibility of non-specific binding, we constructed an mCAT1-hLoop chimera in which the murine ECL3 was replaced by the human counterpart. As expected from the pseudovirus assays—where this chimera was sufficient to alter susceptibility to FrMLV (Fig. L3, left). BLI experiments showed that frRBD exhibited almost no detectable binding to the mCAT1-hLoop probe, and we did not observe evidence for non-specific surface interactions (Fig. L4, left). We therefore consider the incomplete return to baseline most likely to reflect the slow dissociation kinetics of the mCAT1/frRBD complex rather than unspecific binding.\n3. Curve presentation: The x-axis has been adjusted to start at 0 seconds, in accordance with your suggestion (Fig. L3, L4).\n4. Replicates: All BLI experiments were performed in three independent replicates, and this information has been added to the Methods section.\nWe hope that these revisions improve the clarity and rigor of the binding analysis.\nFig. L4 (revised Fig. 1a (left) and Supplementary Fig. 8a (right))\n- Please show the Cryo-EM quality assessment (density for all TMs, ligands, etc), state the map contour level, and map type shown. Also, add a local resolution map to supplementary figure 1\nA: We are very grateful for this helpful suggestion. In the revised manuscript, we have made the following improvements to better illustrate the quality of the Cryo-EM data:\n1. Ligand density: The density corresponding to the ligand is now included in revised Fig. 2b (Fig. L5), shown as a mesh at a contour level of 2 σ, with the contour levels explicitly indicated in the figure legend.\nFig. L5 (revised Fig. 2b)\n2. Transmembrane helices: Density maps for all TMs are now displayed in Supplementary Fig. 1 (Fig. L6), shown as a mesh at a contour level of 3 σ.\n3. Local resolution: A local resolution map has been added to Supplementary Fig. 1 (Fig. L6), highlighting the difference between global and local resolution across the structure.\nWe believe these additions will help readers more clearly assess the overall quality and reliability of the Cryo-EM reconstruction.\nFig. L6 (revised Supplementary Fig. 1)\n- Line 107: Please introduce ATM1\nA: We thank you for this helpful comment. ATM1 is now properly introduced with its full name, Additional Transmembrane helix 1. The revised text now reads as follows (lines 105–108 in the revised manuscript, highlighted in yellow):\n“While most of the structure was well-defined, several flexible regions remained unresolved, including the N-terminal residues (a.a. 1~31), the C-terminal residues (a.a. 601~622), and the intracellular loop ICL5 (linking TM10 and additional transmembrane helix ATM1).”\n- Supplementary figure 1: I cannot see a blue box with dimers – the box is red\nA: We sincerely thank you for catching this oversight. The box indicating the dimers in Supplementary Fig. 1 has now been corrected, and the figure has been updated accordingly.\n- Please add labels for all ICLs and ECLs in figure 1d; it is also worth highlighting the conserved disulfide bridge\nA: We thank you for this helpful suggestion. In the revised manuscript, Figure 1d has been updated to include labels for all seven extracellular loops (ECLs) and six intracellular loops (ICLs), and we have also highlighted the conserved disulfide bridge between ECL3 and ECL4. We believe these additions improve the clarity of the structural annotation.\n- Line 114: ‘with TM1~TM12 forming a pseudosymmetric transmembrane core’: statement unclear-> TM1 and TM12 are not forming the binding cavity: please clarify\nA: We apologize for the ambiguity in our original wording. Our intention was not to suggest that TM1 and TM12 directly form the binding pocket, but rather to indicate that the entire set of helices from TM1 through TM12 together adopt a pseudosymmetric fold enclosing a central cavity.\nWe have revised the text for clarity. The revised text now reads as follows (lines 116–119 in the revised manuscript, highlighted in yellow):\n“TM1–TM12 forms the transmembrane core enclosing a central substrate-binding cavity, which is the hallmark feature of the APC superfamily37, and excluding the auxiliary helices ATM1 and ATM2 outside. (Supplementary Fig. 2a).”\n- Line 119: ‘1.644 Å’ – two digits after the comma are enough\nA: We thank you for this correction. The value has been rounded accordingly, and the text now reads “1.64 Å” in the revised manuscript.\nThe revised text now reads as follows (lines 121–124 in the revised manuscript, highlighted in yellow):\n“The transmembrane core of mCAT can be well superimposed with its prokaryotic homolog, GkApcT13, with an RMSD of 1.64 Å for 338 Cα atoms on (Supplementary Fig. 2a), while ATM1 and ATM2 are only conserved within the CAT family, as well as in another two CAT-associated proteins, SLC7A4 and SLC7A1438.”\n-Include a full sequence alignment of various homologues, also highlighting the conservation of the two extra TM’s. Do the authors have any idea about the functional role of these TM’s? Can they be removed?\nA: We sincerely thank you for raising this insightful point. In the revised manuscript, we have added a full-length sequence alignment of multiple homologues (Fig. L7, now shown in revised Supplementary Fig. 3), with conserved regions clearly annotated, including the two additional transmembrane segments (ATM1 and ATM2). The alignment indicates that these helices are well conserved among CAT1 orthologs across species, but not within the broader CAT or SLC7 family.\nAs you correctly noted, the precise functional contribution of these helices remains unclear. Given their peripheral location relative to the core transport domain, they are unlikely to be directly involved in substrate translocation, but they may contribute to membrane localization, structural stability, or regulatory interactions. This raises an intriguing possibility about their role in CAT1 biology, which we believe will benefit from closer examination in the future.\nFig. L7 (revised Supplementary Fig. 3)\n- Figure 2A: Please show the density for ornithine as a panel figure; state the map type and threshold. After checking the provided map, I’m not fully convinced whether the ligand can be unambiguously assigned to ornithine. Without prior knowledge from the bacterial/LAT1 and 2 transporter work, it is very unlikely to assign this density to the ligand density. A comparison to an apo structure would be very helpful. In addition, functional data on binding relevant mutants and/or MD simulations could further strengthen the hypothesis that this density could be assigned to ornithine. The section on the ornithine binding mode needs to be rephrased, and statements like ‘..exhibited a strong fit, …’ should be avoided\nA: We sincerely thank you for these thoughtful and constructive suggestions. In the revised manuscript, Fig. 2 has been updated to include a panel displaying the ornithine density (Fig. L5, mesh, contour level 2 σ), allowing readers to directly evaluate the ligand assignment.\nTo further support the assignment, we carried out molecular dynamics simulations. Across three independent 100-ns replicates, ornithine remained stably accommodated within the binding pocket, with an average RMSD of ~1.5 Å relative to the initial model (Fig. L2, left). In addition, guided by structural modeling, we introduced alanine substitutions at potential polar contact sites (S44, Y257, S343, and S347). Electrophysiological recordings revealed that, with the exception of S44A (consistent with backbone-mediated interaction), mutations at the other three positions led to a significant reduction in transport efficiency (Fig. L2, middle and right). Together, these results strengthen the plausibility of assigning the observed density to ornithine.\nWe also carefully revised the text in this section to avoid overly absolute wording such as “exhibited a strong fit” and have rephrased the description more cautiously. We appreciate your advice, which has helped us to improve both the presentation and the scientific rigor of this part of the manuscript.\nThe revised text now reads as follows (lines 133–136 in the revised manuscript, highlighted in yellow):\n“This density could be fitted with an ornithine molecule (Fig. 2b), and three independent 100-ns molecular dynamics simulations showed that the modeled ligand remained stable (average RMSD ~1.5 Å), supporting our assignment of ornithine (Figs. 2b, c).”\nFig. L5 (revised Fig. 2b)\nFig. L2 (revised Figs. 2c-e)\n- In supplementary figure 2, a comparison of the binding sites of different homologues bound to different AS is stated. However, the residues forming the binding sites and interactions with the AS are neither shown nor stated. This makes a comparison very difficult.\nA: Thank you for pointing this out. In the revised Supplementary Fig. 2, we now explicitly annotate the residues that form the substrate-binding sites and their interactions with the assigned ligands in each homologue (Fig. L1). This addition makes the comparison clearer and facilitates direct evaluation of the similarities and differences across transporters.\nFig. L1 (revised Fig. 3g (left) and Supplementary Fig. 8a (right))\n- Line 149: remove one ‘its’\nA: We apologize for this oversight and have corrected the expression in the revised manuscript. The revised text now reads as follows (lines 155–158 in the revised manuscript, highlighted in yellow):\n“In GkApcT, E115 together with Y116 and S321 directly coordinates the arginine guanidinium group (Fig. 2f and Supplementary Fig. 2d), with E115 acting as a protonation site for H⁺-coupled transport, while D237 stabilizes the TM3–TM6 interface via a water-mediated bridge rather than direct substrate contact13.”\n- Line 155: What is Extended Data Fig. 3?\nA: We are sorry for the mistake in the original submission. The text has been revised, and the incorrect Extended Data Fig. 3 has been replaced with a direct citation to the reference.\nThe revised text now reads as follows (lines 159–161 in the revised manuscript, highlighted in yellow):\n“Notably, the replacement of the glutamate (E115) with a serine at position 120 abolishes protonation, suggesting CAT1 functions as a membrane-potential-driven and proton-independent transporter38.”\n- Line 178: To my knowledge, LAT1 is a transporter and not a channel\nA: We thank you for pointing this out. You are correct that LAT1 is a transporter rather than a channel. In our original submission, the wording was inaccurate — our intention was to refer to the gating mechanism of LAT1, not to imply that LAT1 functions as a channel. We have revised the text accordingly (lines 165–168) to avoid this ambiguity.\nThe sentence now reads as follows:\n“These findings are consistent with those for LAT1, in which the equivalent residue N258 (equivalent to D263 in mCAT1) does not directly coordinateneutral amino acid substrates but instead acts as a structural switch regulating access to the translocation pathway, a role essential for transport dynamics18.”\n- Line 179: Why is the protein AdiC mentioned here? It has not been introduced before\nA: We appreciate you pointing this out. AdiC is a well-characterized bacterial arginine/agmatine antiporter from E. coli, which has been extensively studied as a structural and mechanistic model within the APC transporter family. In the revised manuscript, we now introduce AdiC in the Introduction (second paragraph, lines 45–50, highlighted text) to provide the necessary context before it is discussed in the Results section.\n“Structural and functional studies of SLC7 family members, including the bacterial proton-dependent transporter GkApcT13 and AdiC14, the heteromeric amino acid transporter b[0,+]AT115-17, and the neutral amino acid transporters LAT118,19 and LAT220,21, have demonstrated that most SLC7 proteins require association with a heavy chain subunit from the SLC3 family for proper biogenesis and function.”\n- Line 185-187: Transition to receptor-bound chapter missing: 4 well-characterized examples of what?? I guess you mean receptor interaction. In this context, I would also mention the MFSD2A–SYNC2 complex\nA: We thank you for this constructive suggestion. You are correct that our original wording was ambiguous. What we intended to describe were well-characterized viral receptor–ligand complexes. To improve the transition, we revised the manuscript at the beginning of Result 4 (mCAT1 is a FrMLV receptor to specifically recognize its RBD domain), we introduced the context more clearly, while also including the MFSD2A–SYNC2 complex as you suggested (lines 199–204, highlighted text):\n“mCAT1 functions dually as a cationic amino acid transporter and as the entry receptor for FrMLV. To delineate the molecular basis of this viral recognition, we performed a structural analysis of the mCAT1/frRBD complex, thereby extending structural insights into viral receptor–ligand recognition, which have so far been obtained only for a limited number of well-characterized examples, including ASCT2–RD11444, NTCP–HBV45,46, NPC1–Ebola47, MFSD6–EV-D6848,49, and MFSD2A–SYNC250.”\nWe believe these changes clarify the transition to the receptor-bound section and improve the overall flow of the Results.\n- Lines 194-197: I recommend not discussing a 1Å shift in a 3.7 Å structure. Furthermore, the authors discuss reorientations of sidechains (e.g. E123’), but this side chain is poorly defined in the EM map\nA: We thank you for this valuable suggestion. We agree with your assessment, and in the revised manuscript we have deleted this part of the text to avoid over-interpretation from regions with limited map definition.\n- Lines 205 – 222: The authors describe several interactions between mCAT1 and frRBD. However, the positions of some of the mentioned side chains are only poorly defined in the EM map (e.g., E237, R511, …), so it is very speculative whether these residues, for example, form a hydrogen or not. I advise checking the validity of the position of the mentioned residues, which are supposed to be involved in various interactions.\nA: We sincerely thank you for this important observation. Following your advice, we have carefully revised the description of the interface interactions and weakened the wording to avoid over-interpretation from poorly defined side chains. For example, the text now reads (lines 218–231, highlighted text):\n“Y94′ is positioned near K222, while T129′ and N137′ approach E237, and N133′ lies adjacent to E221, forming polar interactions. R131′ is also located close to E221 and E237, suggesting electrostatic contributions that may further stabilize this region. Patch 2 is characterized by an extended hydrogen-bonding network between frRBD and mCAT1 (Fig. 3c). Here, S114′ and D120′ of frRBD interact with T321 and G236 of mCAT1. The carbonyl of C117′ is positioned near N232, D120′ is oriented toward the backbone amide of Y235, and W136′ lies close to the backbone amide of G236. These interdigitating contacts likely constitute a stabilizing core of the interface. Patch 3 comprises additional polar contacts, primarily involving potential salt bridges (Fig. 3d). R119′ of VRA-H2 is situated near E60 of TM1, while E123′ is positioned adjacent to K234 of ECL3. Additionally, T126′ of Loop is oriented toward R511 of ECL6, suggesting a hydrogen-bonding H2H3 interaction. In addition to these patches, aromatic stacking may further stabilize the interface: P104′ and W136′of frRBD are positioned for CH/π52 or π–π interactions with Y235 and F224 of mCAT1 (Fig. 3e).”\nIn addition, to complement these structural observations, we performed functional validation experiments. In pseudovirus infection assays, alanine substitutions of interface residues such as R131′, N133′, W136′, R119′, E123′, and T126′ (as well as the double mutant W136′/P104′) all led to a noticeable reduction of viral entry efficiency (Fig. L1, also see the revised Fig. 3g), indicating that these residues contribute to mCAT1 engagement. Moreover, binding assays with purified RBD variants (R131′, N133′, and R119′) showed moderately reduced affinities toward mCAT1 (Fig. L1, also see the revised Supplementary Fig. 8a). Together, these results support the functional relevance of these side chains despite their weaker densities in the EM map.\nWe believe that this combination of more cautious structural interpretation and complementary functional assays strengthens the robustness of our conclusions.\nFig. L1 (revised Fig. 3g (left) and Supplementary Fig. 8a (right))\n- Lines 222 – 232: The authors used a computational tool to dissect the strength of the binding interface between mCAT1 and frRBD. Although I agree that it is not necessary to mutate all residues potentially involved in the interaction, but I recommend testing at least some of the computational predictions experimentally. This only requires the generation of a few mutants and testing them in binding assays.\nA: We are grateful for this constructive recommendation. Following your suggestion, we generated a panel of frRBD interface mutants (R119′, E123′, T126′, R131′, N133′, W136′,and the double mutant W136′/P104′) and evaluated their effects in pseudovirus infection assays (Fig. L1, also see the revised Fig. 3g). Each of these substitutions led to a detectable reduction in viral entry efficiency, confirming their contribution to mCAT1 engagement.\nIn addition, we purified several RBD variants (Y94′, R131′, N133′, and R119′) and carried out BLI binding experiments (Fig. L1, also see the revised Supplementary Fig. 8a), which revealed moderately reduced affinities relative to the wild-type RBD. These functional data provide experimental evidence supporting the importance of the identified residues in stabilizing the mCAT1/frRBD interface.\n- Figure 4c, d: Please indicate the time frame for the on-reaction and the off-reaction; add corresponding fit; start at 0 s. The authors do not state what conditions under reducing conditions mean. This information is missing, and how can they be sure that the disulfide bond is reduced? One could also mutate one of the Cys residues to a Ser and check for binding.\nA: We sincerely thank the reviewer for this constructive comment. In line with your suggestion, we have revised our experiments and removed the binding analysis under reducing conditions. To directly probe the role of the conserved disulfide bond, we generated a C309S mutant of mCAT1. Bio-layer interferometry (BLI) assays demonstrated that the C309S mutation led to more than an order-of-magnitude reduction in binding affinity compared with wild-type mCAT1, with the x-axis of the sensorgrams now set to start at 0 s (Fig. L3). In addition, pseudovirus infection assays confirmed that C309S partially impaired viral entry (Fig. L3).These results indicate that the conserved disulfide bond contributes substantially to stabilizing the mCAT1/frRBD interaction, although it is not the sole determinant of binding.\nFig. L3 (revised Figs. 4d-e)\n- The authors show a very simplified Table 1 (B-factors, Validation scores, … missing)\nA: We greatly appreciate your careful review and for pointing out this omission. In the revised manuscript, we have expanded Table 1 to include B-factors, validation scores, and other standard cryo-EM statistics, in line with community reporting practices (Table L1). We believe this provides a more complete overview of the structural data quality.\nTable 1 Statistics of cryo-EM data collection, processing, and model refinement.\n(EMDB: EMD-63995)\n(PDB: 9UAT )\nData collection and processing\nEM equipment Krios (Thermo Scientific)\nVoltage (kV) 300\nDetector Gatan K3 Summit\nEnergy filter Gatan GIF Quantum, 20 eV slit\nElectron dose (e-/Å2) 50\nPixel size (Å) 1.087\nDefocus range (μm) -1.2 ~ -2.2\nNumber of collected micrographs 2,402\nSample mCAT1/frRBD\n3D Reconstruction\nSoftware cryoSPARC\nNumber of used particles 106,963\nResolution (Å) 3.65\nSymmetry C1\nRefinement\nModel resolution (Å)\n3.9\n(FSC threshold = 0.5)\nSoftware Phenix\nCell dimensions\na=b=c (Å) 278.272\nα=β=γ (˚) 90\nModel composition\nAtoms 11613 (Hydrogen 5788)\nProtein residues 767\nLigands (Orn) 1\nB factors (Å2)\nProtein 78.73\nLigand (Orn) 66.20\nR.m.s. deviations\nBond lengths (Å) 0.002\nBond angles (°) 0.498\nValidation\nMolProbity score 1.44\nCaBLAM outliers 0.7%\nCC (Mask) 0.71\nClash score 6.08\nRotamers outliers (%) 2.74\nRamachandran plot\nFavored (%) 97.45\nAllowed (%) 2.55\nDisallowed (%) 0\nTable L1 (revised Table 1)"}, {"round": 2, "reviewer_comments": {"reviewer_2": "The manuscript of Lingyun Xia et al. about structural/functional ornithine transport and virus receptor characterization of\nCAT1 has been improves substantially after this revision. Now the functional studies of relevant transporter residues to\nsupport the conclusions related to substrate (ornithine) interaction and virus interaction (i.e., currents associated to amino\nacid transport, affinity measurements of CAT1-frRBD of FrMLV virus, even MD analysis to crosscheck interaction of residues\nwith the substrate ornithine). When analysing the impact of missense mutations it is necessary to check whether loss-of-\nfunction of the studied mutants is not the consequence of compromised expression of the mutated transporter and/or the\nsurface expression in the cell system used to measure the transporter activity. The authors show expression by the\nfluorescence of WT and mutated versions of CAT1-GFP (Supp. Fig. 4). This is a semi-quantitative information. Western Blot\nanalysis gives a quantitative expression to compare WT and the mutated versions of the studied CAT1. Moreover, the same\ndata shown in Supp. Fig 4 could be used to show semi-quantitative expression of the different variants of CAT1-GFP at the\ncell membrane. The present format of Supp. Fig. 4 does not allows seeing the expression of the CAT1-GFP variants at the\ncell membrane. Please, show a larger magnification of the cells. It looks like these studies will support the present\nconclusions but it is necessary to show this information.\nThe authors have also addressed whether the ECL3-ECL4 disulphide bridge has an impact on CAT1 transport function and\nwhether SU BRD blocks CAT1 transport function. The fact that this bridge is not essential for CAT1-RBD and CAT1\ntransport, and that CAT1-RBD interaction does not block transport does not decreases the relevance of the manuscript.", "reviewer_3": "The revised manuscript “Structural insights into cationic amino acid transport and viral receptor engagement by CAT1” by\nXia et al. shows improvements over the earlier version. Additional experimental data have been included, the structural\nmodel has been updated, and the data are now interpreted more carefully. However, a few points still require clarification.\n1.) The authors argue that ‘we reprocessed the cryo-EM data, which improved the final map quality to 3.65 Å and …’.\nHowever, based on Supplementary Fig. 1 and Table 1, I cannot see any differences in the cryo-EM workflow (particle\nnumbers, data processing, resolution, FSCs, etc.) compared to the previous version. While the structure model has been\nimproved, the EM-map remains unchanged (except for some post-processing efforts). This needs clarification.\n2.) Figure 2b: Which map type is shown here? Is this the postprocessed DeepEMhancer map? Please clarify.\n3.) Figure 2e: At which membrane potential do the authors compare the evoked currents of the different mutants? For such a\ncomparison, is it not essential that the expression levels of the different mutants are unchanged compared to WT?\nPlease provide this information and data.\n4.) Figure 3g/Supplementary figure 7: Pseudovirus entry assay. The figure legend does not match the figure. ‘Bars show\nmean normalized infection relative to WT frRBD (set to 1); error bars, s.e.m., n values are indicated above the bars. …’ . Y-\naxis shows absolute values of ‘something’ and n-values are not stated. I recommend normalizing the data and showing\nsomething like an infection relative to WT. With this, one can hopefully convince a reader; at the moment, I find this difficult to\nfollow and understand. Overall, this ‘entry assay’ requires a better explanation for the reader on what has been done and\nwhat is shown in the graphs. Also, the figure legend of supplementary figure 7 is confusing (where is the band\ncorresponding to the surface subunit (SU) as described?)\n5.) Supplementary figure 8: The authors argue that the frRBD variants show a reduced binding affinity to mCAT1. To me,\nthis is unclear. This would require an analysis of the experimental data (fit to a 1:1 binding model). Since the experiment has\nonly been done at a single concentration, the results will have a substantial error, but at least one can compare numbers.\nAt the moment, it is hard to make any judgement.\n6.) Table 1 still contains sections, likely originating from crystallographic times (e.g. cell dimensions). Please update to the\ncurrent standards.", "reviewer_4": "I have been brought in as a reviewer to assess the technical aspects of the molecular dynamics simulations. It appears that\nthe authors utilized molecular dynamics simulations to assess whether or not ornithine was stable within the binding pocket;\nhowever it is unclear exactly what is being simulated. For example, was the whole mCAT1-orithine complex simulated or\njust local residues? I am also worried about the simulation run time of 100 ns. 100 ns isn't a lot of time for a bound ligand to\nmove freely between different states, and I find it important to extend this or at least rationalize why 100 ns was chosen.\nIn sum, I would like to see the authors explain:\n- Exactly what they simulated in terms of residues and the molecule (bound ligand). This should be explicit in the methods\nsection.\n- How big their simulation box is and the number of atoms/residues involved. This should be explicit in the methods section.\n- Discussion om the timescale of binding/unbinding of ornithine. This will help in concluding if a 100ns run time is\nreasonable or not."}, "author_response": "REVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe manuscript of Lingyun Xia et al. about structural/functional ornithine transport and virus receptor characterization of CAT1 has been improved substantially after this revision. Now the functional studies of relevant transporter residues to support the conclusions related to substrate (ornithine) interaction and virus interaction (i.e., currents associated to amino acid transport, affinity measurements of CAT1-frRBD of FrMLV virus, even MD analysis to crosscheck interaction of residues with the substrate ornithine). When analysing the impact of missense mutations it is necessary to check whether loss-of-function of the studied mutants is not the consequence of compromised expression of the mutated transporter and/or the surface expression in the cell system used to measure the transporter activity. The authors show expression by the fluorescence of WT and mutated versions of CAT1-GFP (Supp. Fig. 4). This is a semi-quantitative information. Western Blot analysis gives a quantitative expression to compare WT and the mutated versions of the studied CAT1. Moreover, the same data shown in Supp. Fig 4 could be used to show semi-quantitative expression of the different variants of CAT1-GFP at the cell membrane. The present format of Supp. Fig. 4 does not allow seeing the expression of the CAT1-GFP variants at the cell membrane. Please, show a larger magnification of the cells. It looks like these studies will support the present conclusions but it is necessary to show this information.\nThe authors have also addressed whether the ECL3-ECL4 disulphide bridge has an impact on CAT1 transport function and whether SU BRD blocks CAT1 transport function. The fact that this bridge is not essential for CAT1-RBD and CAT1 transport, and that CAT1-RBD interaction does not block transport does not decrease the relevance of the manuscript.\nA1: Thank you for the positive comment on our last revised manuscript and especially the valuable suggestion regarding the expression level of CAT1 mutants. We have now included representative fluorescence microscopy images at higher magnification in the revised Supplementary Fig. 4, which clearly show the plasma membrane localization of CAT1–GFP and its mutants. In addition, we have provided the original fluorescence images as source data to ensure transparency. Moreover, we have performed western blot analysis using an anti-Flag to quantitatively compare the expression levels of WT and all mutants in the same expression system. The results confirm that all mutants are expressed at comparable levels to the wild-type protein, indicating that the observed functional differences are not due to altered expression or membrane localization. These new data are now described in the revised Methods and Results sections and support our previous conclusions.\nSupplementary Fig. 4 Expression of mCAT1–eGFP and interface mutants in HEK293T cells.\nReviewer #2 (Remarks to the Author):\nThe revised manuscript “Structural insights into cationic amino acid transport and viral receptor engagement by CAT1” by Xia et al. shows improvements over the earlier version. Additional experimental data have been included, the structural model has been updated, and the data are now interpreted more carefully. However, a few points still require clarification.\nQ1: The authors argue that ‘we reprocessed the cryo-EM data, which improved the final map quality to 3.65 Å and …’. However, based on Supplementary Fig. 1 and Table 1, I cannot see any differences in the cryo-EM workflow (particle numbers, data processing, resolution, FSCs, etc.) compared to the previous version. While the structure model has been improved, the EM-map remains unchanged (except for some post-processing efforts). This needs clarification.\nA1: We apologize for the misleading wording in the previous version. As you correctly pointed out, the cryo-EM data reprocessing did not significantly improve the overall map quality. Therefore, we keep using the earlier EM map. We have, however, re-refined the structural model against this map and obtained an improved model.\nQ2: Figure 2b: Which map type is shown here? Is this the postprocessed DeepEMhancer map? Please clarify.\nA2: Yes, the map shown in Fig. 2b is the post-processed map generated by DeepEMhancer. We have now clearly indicated this information in the figure legend.\nWe have modified the figure legend as follows:\n“The cryo-EM density (post-processed by DeepEMhancer) is rendered as a blue mesh contoured at the display threshold used in ChimeraX (level 2.0).”\nQ3: Figure 2e: At which membrane potential does the authors compare the evoked currents of the different mutants? For such a comparison, is it not essential that the expression levels of the different mutants are unchanged compared to WT? Please provide this information and data.\nA3: As shown in Fig. 2e (and Supplementary Fig. 11b), the evoked currents of the wild-type and mutant CAT1 proteins were compared at a membrane potential of –80 mV. We have explicitly indicated this voltage in the updated figure legend for clarity. All constructs (WT and mutants) were fused with a N-terminal Flag tag and a C-terminal eGFP. We have added fluorescence quantification data and western blot (Supplementary Fig. 4, shown here below), which demonstrate that the expression levels of the mutants are comparable to those of the wild-type protein. These results confirm that the observed current differences are not due to altered expression.\nSupplementary Fig. 4 Expression of mCAT1–eGFP and interface mutants in HEK293T cells.\nQ4: Figure 3g/Supplementary figure 7: Pseudovirus entry assay. The figure legend does not match the figure. ‘Bars show mean normalized infection relative to WT frRBD (set to 1); error bars, s.e.m., n values are indicated above the bars. …’ . Y-axis shows absolute values of ‘something’ and n-values are not stated. I recommend normalizing the data and showing something like an infection relative to WT. With this, one can hopefully convince a reader; at the moment, I find this difficult to follow and understand. Overall, this ‘entry assay’ requires a better explanation for the reader on what has been done and what is shown in the graphs. Also, the figure legend of supplementary figure 7 is confusing (where is the band corresponding to the surface subunit (SU) as described?)\nA4: We are grateful for your insightful suggestions and have carefully addressed it. The pseudoviruses were generated by co-transfecting the MLV Env expression plasmid together with the pNL4-3.Luc.R⁻E⁻ backbone (HIV-derived, carrying a luciferase reporter gene but lacking functional Env). The resulting pseudoviruses incorporate MLV Env on their envelope, in which the mature Env is cleaved into the surface (SU) and transmembrane (TM) subunits. The internal luciferase reporter enables quantitative measurement of infection efficiency. Virus input was normalized to Env expression, and the pseudoviruses were used to infect receptor-expressing cells (counted and seeded under identical conditions). Higher infection efficiency results in stronger luciferase signals. Each experiment was independently repeated three times, with four technical replicates per condition. Detailed experimental procedures are now described in the revised Methods section. The previously presented data corresponded to one representative experiment out of three biological replicates, with four replicates in each group and plotted in log₁₀ scale (the previously submission already included all raw data). Following your valuable suggestion, we have revised Fig. 3g. For each independent experiment, the mean of four parallel samples was calculated. The averaged values from three independent experiments were then normalized to the overall mean of the wild-type control across the three experiments (set to 100%), thereby presenting the relative effects of the mutants more clearly (see below). In addition, we have corrected the legend of Supplementary Fig. 7: the SU band was not visible because the Flag tag is located at the C-terminus of Env (TM region). The erroneous statement has been removed.\nFig. 3g Pseudovirus entry assay for selected frRBD alanine variants targeting interface residues.\nQ5: Supplementary figure 8: The authors argue that the frRBD variants show a reduced binding affinity to mCAT1. To me, this is unclear. This would require an analysis of the experimental data (fit to a 1:1 binding model). Since the experiment has only been done at a single concentration, the results will have a substantial error, but at least one can compare numbers. At the moment, it is hard to make any judgement.\nA5: We appreciate the reviewer’s insightful comment. Indeed, as noted, the BLI screening was performed at a single concentration (100 nM), which limits the determination of precise kinetic constants. To better illustrate the binding behavior, we have performed individual curve fitting to a 1:1 binding model for each sensorgram. Although quantitative affinity estimation is not possible under this condition, the fitted curves support the same qualitative trend observed in the raw binding responses, confirming that the mutants weaken the interaction. The revised figure legend and Methods section have been updated accordingly.\nSupplementary Fig. 8 Bio-layer interferometry analysis of frRBD binding to mCAT1\nQ6: Table 1 still contains sections, likely originating from crystallographic times (e.g. cell dimensions). Please update to the current standards.\nA6: Thanks for pointing out this issue. We have revised Table 1 to cryo-EM style and removed all crystallography-specific parameters (e.g., cell dimensions).\nTable 1 Statistics of cryo-EM data collection, processing, and model refinement.\n(EMDB: EMD-63995)\n(PDB: 9UAT )\nData collection and processing\nEM equipment Krios (Thermo Scientific)\nVoltage (kV) 300\nDetector Gatan K3 Summit\nEnergy filter Gatan GIF Quantum, 20 eV slit\nElectron dose (e-/Å2) 50\nPixel size (Å) 1.087\nDefocus range (μm) -1.2 ~ -2.2\nNumber of collected micrographs 2,402\nSample mCAT1/frRBD\n3D Reconstruction\nSoftware cryoSPARC\nBox size (pixel) 256\nNumber of initial picked particles 1,530,071\nNumber of used particles 106,963\nResolution (Å) 3.65\n(FSC threshold = 0.143)\nSymmetry C1\nRefinement\nInitial model used Predicted by AlphaFold2\nModel resolution (Å) 3.9\n(FSC threshold = 0.5)\nSoftware Phenix\nModel composition\nAtoms 11,613 (Hydrogen 5,788)\nProtein residues 767\nLigands (Orn) 1\nB factors (Å2)\nProtein 78.73\nLigand (Orn) 66.20\nR.m.s. deviations\nBond lengths (Å) 0.002\nBond angles (°) 0.498\nValidation\nMolProbity score 1.44\nCaBLAM outliers (%) 0.70\nCC (Mask) 0.71\nClash score 6.08\nRotamers outliers (%) 2.74\nRamachandran plot\nFavored (%) 97.45\nAllowed (%) 2.55\nDisallowed (%) 0\nReviewer #3 (Remarks to the Author):\nI have been brought in as a reviewer to assess the technical aspects of the molecular dynamics simulations. It appears that the authors utilized molecular dynamics simulations to assess whether or not ornithine was stable within the binding pocket; however it is unclear exactly what is being simulated. For example, was the whole mCAT1-orithine complex simulated or just local residues? I am also worried about the simulation run time of 100 ns. 100 ns isn't a lot of time for a bound ligand to move freely between different states, and I find it important to extend this or at least rationalize why 100 ns was chosen.\nIn sum, I would like to see the authors explain:\nQ1: Exactly what they simulated in terms of residues and the molecule (bound ligand). This should be explicit in the methods section.\nA1: We simulated the mCAT1–ornithine complex. The frRBD domain was not included in the simulated system, as it is located far from the binding pocket and does not contribute to ligand interactions. We have now added more detailed information about the composition of the simulated system in the Methods section Molecular Dynamics Simulations.\nQ2: How big their simulation box is and the number of atoms/residues involved. This should be explicit in the methods section.\nA2: The simulation box dimensions are 92.7 × 83.8 × 98.8 Å3, containing a total of 68270 atoms. This information has been added to the Methods section Molecular Dynamics Simulations.\nQ3: Discussion om the timescale of binding/unbinding of ornithine. This will help in concluding if a 100ns run time is reasonable or not.\nA3: We thank the reviewer for raising this important point. We have now added a discussion on the timescale to the main text (lines 139–144).\nOur simulations were initiated from the cryo-EM-derived bound state. As the reviewer implies, the complete unbinding of a substrate from a transport protein is a rare event that typically occurs on the millisecond timescale or longer, as supported by previous studies (Nature Communications 11, 5016 (2020); Nature Communications 9, 230 (2018); Nature 518, 68-73 (2015)). A 100 ns simulation is therefore orders of magnitude too short to observe such an unbinding event, and it was not our goal to do so.\nInstead, the purpose of our 100 ns simulations was to assess the stability of the bound conformation within the binding pocket. Given this objective, a 100 ns timescale is entirely appropriate and standard practice (Nature. 2024 Sep;633(8029):473-479; Nat Struct Mol Biol. 2023 Jul;30(7):1023-1032). Our analysis confirmed that the ornithine ligand maintains a stable pose with low RMSD throughout all replicates, which validates the stability of the binding mode we observed in the cryo-EM structure."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have now satisfactorily addressed my concerns, and the manuscript has been strongly improved compared to the earlier versions.", "reviewer_3": "I'm happy with their justifications for what the authors did and don't have further concerns.", "reviewer_1": "The manuscript of Lingyun Xia et al. about structural/functional ornithine transport and virus receptor characterization of CAT1 has addressed the only point that I raised in this run of revision, also raised by reviewer 2: WB expression and semi-quantitative expression by immunofluorescence of mCAT1-eGFP in human cells of the point mutations of mCAT1 studied in this work. The data shows similar total expression of the mutated versions by WB and immunofluorescence data support that dramatic reduction of the transport function of the mutants studied is not the consequence of defective expression.\nThus, in relation to the only point raised by me in this run of revision, I am satisfied with the new incorporated data and support publication of this work in Nat Commun."}, "author_response": "A: We thank you for your positive evaluation and support for publication. We are pleased that the additional data fully addressed your concern.\nA: We appreciate your supportive comments and are glad that all your concerns have been satisfactorily resolved.\nA: We thank you for your positive feedback and are pleased that you have no further concerns."}]

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