Research Background and Problem Statement

Hereditary Folate Malabsorption (HFM) is a rare autosomal recessive disorder characterized by impaired intestinal absorption of folates and hindered transport across the choroid plexus into cerebrospinal fluid. This disease is caused by inactivating mutations in the gene encoding the Proton-Coupled Folate Transporter (PCFT-SLC46A1). Understanding the impact of these mutations on the structure and function of PCFT is crucial for elucidating the pathophysiology of HFM.

Recently, high-resolution structures of chicken PCFT (Gallus Gallus PCFT, GPCFT) were obtained using cryo-electron microscopy, sharing 58% sequence homology with human PCFT (Human PCFT, HPCFT). This provides new opportunities to study functional-deficient mutations in HPCFT. Previous studies primarily relied on homology modeling based on templates of other SLC transporters, which were insufficient for detailed structural change analysis. Therefore, researchers aimed to explore the specific effects of these mutations on the structure and function of HPCFT through molecular dynamics simulations and investigate the mechanisms by which compensatory mutations restore function.

Paper Source and Author Information

This paper was authored by Prithviraj Nandigrami, I. David Goldman, and Andras Fiser, who are affiliated with the Department of Systems & Computational Biology, Department of Biochemistry, and Departments of Medicine, Oncology, and Molecular Pharmacology at Albert Einstein College of Medicine. The paper was published in the Journal of Biological Chemistry, received on August 4, 2024, revised on January 17, 2025, and has the DOI: https://doi.org/10.1016/j.jbc.2025.108280.

Research Workflow and Experimental Methods

1. Homology Modeling and Molecular Dynamics Simulations

1.1 Model Construction

Researchers built homology models of HPCFT based on the cryo-EM structures of GPCFT (PDB ID: 7BC7 and 7BC6) using the Modeller program. The GPCFT structures included both inhibitor-bound and inhibitor-free forms, with an all-atom root mean square deviation (RMSD) of less than 0.5 Å between them. All models were optimized, including supplementation of missing atoms, terminal capping, and disulfide bond modeling.

1.2 Molecular Dynamics Simulations

All models were embedded in a phospholipid bilayer and simulated in a water box. Simulation conditions included the TIP3P water model, a KCl salt concentration of 0.15 M, periodic boundary conditions (PBCs), and Particle Mesh Ewald (PME) approximation for long-range interactions. Each model underwent at least 1.5 microseconds of simulation, generating approximately 10,000 representative snapshots for subsequent analysis.

2. Structural Feature Monitoring

2.1 Pore Size Changes

Researchers used the HOLE program to calculate the average pore radius of various HPCFT variants to assess changes in pore size. Results showed that all single-point mutations leading to HPCFT dysfunction (e.g., F392V, S196L, F392D) significantly increased the pore radius (p-values = 0.0001, 0.0025, 0.0007, respectively). Introducing compensatory mutations (e.g., F392V/S196L, F392D/G305L) restored the pore radius to near wild-type levels (p-values = 0.242 and 0.335).

2.2 Conformational Dynamics

To evaluate the impact of different mutations on TM helix conformations, researchers analyzed the conformational clustering of TM4 and TM10. Wild-type and functional double mutants (e.g., F392M, F392V/S196L, F392D/G305L) exhibited fewer conformational clusters (3.5-5), while dysfunctional single mutants showed higher diversity (3-4 times more). Additionally, the dominant conformation in dysfunctional mutants accounted for only 29-33% of total conformations, indicating greater structural disorder.

2.3 Solvent Accessible Surface Area Changes

Researchers assessed changes in solvent accessible surface area (SASA). Dysfunctional single mutants (e.g., F392V, S196L, F392D) showed significant reductions in SASA (p-value < 0.0001), whereas introducing compensatory mutations or retaining functional single mutants (e.g., F392M) resulted in SASA levels similar to the wild-type (p-values = 0.3242, 0.7364, 0.0063).

2.4 Secondary Structure Content Changes

Using the DSSP program, researchers calculated the secondary structure content of each TM helix. Results indicated a significant reduction in helical structure content (10-20%) for all TM helices, which was restored upon introducing compensatory mutations.

2.5 Residue Contact Analysis

Using the InterCAAT program, researchers analyzed long-range contacts between Phe392 and other residues. Functional loss mutations (e.g., F392V, F392D) led to a significant decrease in long-range contacts, which were restored by introducing compensatory mutations.

3. Analysis of Other Mutants

Researchers also analyzed another dysfunctional mutant, D109A, located in a different region. They found it similarly resulted in pore enlargement (increase of 1.0 Å) and reduced solvent accessibility, consistent with findings from F392V.

Key Research Findings

1. Loss-of-Function Mutations Lead to Pore Enlargement and Structural Instability

Studies revealed that loss-of-function mutations (e.g., F392V, S196L, F392D) cause significant pore enlargement, accompanied by destabilization of inner-core TM helices and reduced solvent accessibility. These changes disrupt the stability of the folate substrate translocation pathway, leading to loss of transport function.

2. Compensatory Mutations Restore Function

Introducing compensatory mutations (e.g., F392V/S196L, F392D/G305L) restored the pore radius to near wild-type levels, improved structural stability of inner-core TM helices, and enhanced solvent accessibility, thereby restoring transport function.

3. Impact of D109A Mutation

The D109A mutation, located in a different region, similarly led to pore enlargement and reduced solvent accessibility, further confirming the general impact of these mutations on HPCFT structure and function.

Conclusion and Significance

1. Scientific Value

This study reveals the specific mechanisms by which loss-of-function mutations lead to pore enlargement and structural instability in HPCFT, as well as the mechanisms by which compensatory mutations restore function. These findings deepen our understanding of HPCFT’s structure and function and provide theoretical foundations for developing therapeutic strategies for HFM.

2. Application Value

The results can aid in developing novel drugs, particularly personalized treatment plans for HFM patients. Moreover, the molecular dynamics simulation methods used in this study can be applied to research on other members of the SLC superfamily of transporters, offering new insights into the molecular mechanisms of related diseases.

3. Research Highlights

Important Discoveries

• Revealed the specific mechanisms by which loss-of-function mutations lead to pore enlargement and structural instability.
• Discovered the mechanisms by which compensatory mutations restore function.

Methodological Innovations

• Used cryo-EM structures of GPCFT as templates to build high-quality homology models of HPCFT.
• Conducted detailed analyses of the impact of mutations on HPCFT structure and function using molecular dynamics simulations.

Uniqueness

• Studied a rare disease, HFM, which has important clinical implications.
• Combined multiple experimental methods (molecular dynamics simulations, solvent accessible surface area analysis, secondary structure content analysis) to comprehensively analyze the effects of mutations on HPCFT.

This study provides new perspectives on understanding loss-of-function mutations and their compensatory mutations in HPCFT, offering significant scientific and practical value.