An Aromatic Cap Seals the Substrate Binding Site in an ECF-Type S Subunit for Riboflavin
Open accessAbstract
ECF transporters are a family of active membrane transporters for essential micronutrients, such as vitamins and trace metals. Found exclusively in archaea and bacteria, these transporters are composed of four subunits: an integral membrane substrate-binding subunit (EcfS), a transmembrane coupling subunit (EcfT), and two ATP-binding cassette ATPases (EcfA and EcfA'). We have characterized the structural basis of substrate binding by the EcfS subunit for riboflavin from Thermotoga maritima, TmRibU. TmRibU binds riboflavin with high affinity, and the protein-substrate complex is exceptionally stable in solution. The crystal structure of riboflavin-bound TmRibU reveals an electronegative binding pocket at the extracellular surface in which the substrate is completely buried. Analysis of the intermolecular contacts indicates that nearly every available substrate hydrogen bond is satisfied. A conserved aromatic residue at the extracellular end of TM5, Tyr130, caps the binding site to generate a substrate-bound, occluded state, and non-conservative mutation of Tyr130 reduces the stability of this conformation. Using a novel fluorescence binding assay, we find that an aromatic residue at this position is essential for high-affinity substrate binding. Comparison with other S subunit structures suggests that TM5 and Loop5-6 contain a dynamic, conserved motif that plays a key role in gating substrate entry and release by S subunits of ECF transporters.
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The current lack of annotation of M. mycoides homologue Q6MS70 is likely because the prediction relies on structural similarity to ECF-type riboflavin uptake proteins from T. maratima and S. aureus, which have sub-2 Å rmsd but less than 22% sequence identity.
“The current lack of annotation of the M. mycoides homologue Q6MS70 is likely because our annotation prediction builds strongly on structural similarity to ECF-type riboflavin uptake proteins from T. maratima61 and S. aureus,62 which have sub-2 Å rmsd’s to the predicted MMSYN1_0877 structure but amino acid sequence identities of less than 22%.”
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- Authors
- Karpowich, N. K., Song, J., & Wang, D.-N.
- Affiliations
- NYU Langone Health (US)
- New York University (US)
- Corresponding author
- Karpowich, Nathan K.
- Wang, Da-Neng
- Venue
- Journal of Molecular Biology
- Publisher
- Elsevier BV
- Published
- 2016-07
- Volume
- 428
- Issue
- 15
- Pages
- 3118-3130
- ISSN
- 0022-2836 (ISSN-L)
- PMC
- PMC4975955
- OpenAlex
- W2431012988
- Cited by
- 16
- References
- 62
- Citations by year
- 2026: 1 · 2025: 2 · 2024: 1 · 2023: 2 · 2022: 3 · 2021: 2 · 2020: 2 · 2019: 1 · 2018: 1 · 2017: 1
- FWCI
- 0.54
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Journal of Molecular Biology (acceptedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institutes of Health (R01-GM093825, R01-MH083840, R01-DA019676, R01-DK053973, R01-DK099023, F32-HL091618)
- American Heart Association
- Topics
- Health Sciences › Medicine › Oncology › Drug Transport and Resistance Mechanisms
- Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Keywords
- riboflavin transport, substrate binding site, Thermotoga maritima, crystal structure, substrate affinity, hydrogen bonding, membrane transporters
- MeSH terms
- Adenosine Triphosphatases, Bacterial Proteins, Binding Sites, Models, Molecular, Protein Conformation, Riboflavin, Vitamins, Crystallography, X-Ray, ATP-Binding Cassette Transporters, Thermotoga maritima, Protein Subunits, Membrane Transport Proteins
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