Cryo-EM structures of the human glutamine transporter SLC1A5 (ASCT2) in the outward-facing conformation
- Pfizer Inc, United States
- University of Oxford, United Kingdom
Abstract
Alanine-serine-cysteine transporter 2 (ASCT2, SLC1A5) is the primary transporter of glutamine in cancer cells and regulates the mTORC1 signaling pathway. The SLC1A5 function involves finely tuned orchestration of two domain movements that include the substrate-binding transport domain and the scaffold domain. Here, we present cryo-EM structures of human SLC1A5 and its complex with the substrate, L-glutamine in an outward-facing conformation. These structures reveal insights into the conformation of the critical ECL2a loop which connects the two domains, thus allowing rigid body movement of the transport domain throughout the transport cycle. Furthermore, the structures provide new insights into substrate recognition, which involves conformational changes in the HP2 loop. A putative cholesterol binding site was observed near the domain interface in the outward-facing state. Comparison with the previously determined inward-facing structure of SCL1A5 provides a basis for a more integrated understanding of substrate recognition and transport mechanism in the SLC1 family.
Data availability
All the cryo-EM data were deposited to the Protein Data Bank (PDB ID: 6MP6, 6MPB) and the EMDB (EMD-9187, EMD-9188) for immediate release upon publication.
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SLC1A5_cKM4012Protein Data Bank, 6MP6.
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SLC1A5_cKM4012_L_GlnProtein Data Bank, 6MPB.
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SLC1A5_cKM4012Electron Microscopy Data Bank, EMD-9187.
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SLC1A5_cKM4012_L_GlnElectron Microscopy Data Bank, EMD-9188.
Article and author information
Author details
Funding
Y.D. and E.P.C. are members of the SGC, (Charity ref: 1097737) funded by AbbVie, Bayer Pharma AG, Boehringer Ingelheim, the Canada Foundation for Innovation, Genome Canada, GlaxoSmithKline, Janssen, Lilly Canada, Merck & Co., Novartis, the Ontario Ministry of Economic Development and Innovation, Pfizer, São Paulo Research Foundation-FAPESP and Takeda, as well as the Innovative Medicines Initiative Joint Undertaking ULTRA-DD grant 115766 and the Wellcome Trust106169/Z/14/Z.
Copyright
© 2019, Yu et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Cryo-EM structures of the human glutamine transporter SLC1A5 (ASCT2) in the outward-facing conformation
eLife 8:e48120.
https://doi.org/10.7554/eLife.48120
Further reading
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- Structural Biology and Molecular Biophysics
Under physiological conditions, proteins continuously undergo structural fluctuations on different timescales. Some conformations are only sparsely populated, but still play a key role in protein function. Thus, meaningful structure–function frameworks must include structural ensembles rather than only the most populated protein conformations. To detail protein plasticity, modern structural biology combines complementary experimental and computational approaches. In this review, we survey available computational approaches that integrate sparse experimental data from electron paramagnetic resonance spectroscopy with molecular modeling techniques to derive all-atom structural models of rare protein conformations. We also propose strategies to increase the reliability and improve efficiency using deep learning approaches, thus advancing the field of integrative structural biology.
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- Structural Biology and Molecular Biophysics
In eukaryotes, RNAs transcribed by RNA Pol II are modified at the 5′ end with a 7-methylguanosine (m7G) cap, which is recognized by the nuclear cap binding complex (CBC). The CBC plays multiple important roles in mRNA metabolism, including transcription, splicing, polyadenylation, and export. It promotes mRNA export through direct interaction with a key mRNA export factor, ALYREF, which in turn links the TRanscription and EXport (TREX) complex to the 5′ end of mRNA. However, the molecular mechanism for CBC-mediated recruitment of the mRNA export machinery is not well understood. Here, we present the first structure of the CBC in complex with an mRNA export factor, ALYREF. The cryo-EM structure of CBC-ALYREF reveals that the RRM domain of ALYREF makes direct contact with both the NCBP1 and NCBP2 subunits of the CBC. Comparing CBC-ALYREF with other cellular complexes containing CBC and/or ALYREF components provides insights into the coordinated events during mRNA transcription, splicing, and export.
