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Crystal structure of arginine-bound lysosomal transporter SLC38A9 in the cytosol-open state

Nature Structural & Molecular Biology volume 25, pages 522–527 (2018)Cite this article

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Abstract

Recent advances in understanding intracellular amino acid transport and mechanistic target of rapamycin complex 1 (mTORC1) signaling shed light on solute carrier 38, family A member 9 (SLC38A9), a lysosomal transporter responsible for the binding and translocation of several essential amino acids. Here we present the first crystal structure of SLC38A9 from Danio rerio in complex with arginine. As captured in the cytosol-open state, the bound arginine was locked in a transitional state stabilized by transmembrane helix 1 (TM1) of drSLC38A9, which was anchored at the groove between TM5 and TM7. These anchoring interactions were mediated by the highly conserved WNTMM motif in TM1, and mutations in this motif abolished arginine transport by drSLC38A9. The underlying mechanism of substrate binding is critical for sensitizing the mTORC1 signaling pathway to amino acids and for maintenance of lysosomal amino acid homeostasis. This study offers a first glimpse into a prototypical model for SLC38 transporters.

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Fig. 1: Overall architecture and the luminal gating network of arginine-bound drSLC38A9.
Fig. 2: Arginine bound in the cytosol-open conformation of drSLC38A9.
Fig. 3: Arginine binding of drSLC38A9 stabilized by TM1a anchored by a hydrophobic box.
Fig. 4: Comparative models of selected SNAT family members.

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Acknowledgements

We thank D. Cawley for development and production of monoclonal antibodies. We thank K. Rajashankar and the staff at NECAT for their support with X-ray data collection. We thank D. Casio and J. Hattne for discussions over X-ray data collection and structural determination. We thank L. Shao and S. Liu for critical reading of the manuscript. This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the US National Institutes of Health (P41 GM103403). The Pilatus 6 M detector on the 24-ID-C beamline is funded by an NIH-ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract DE-AC02-06CH11357. Research in the Gonen laboratory is funded by the Howard Hughes Medical Institute.

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Author notes

  1. These authors contributed equally: Hsiang-Ting Lei, Jinming Ma.

Authors and Affiliations

  1. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA

    Hsiang-Ting Lei, Jinming Ma, Silvia Sanchez Martinez & Tamir Gonen

  2. Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, CA, USA

    Jinming Ma & Tamir Gonen

  3. Departments of Physiology and Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA

    Tamir Gonen

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Contributions

H.-T.L., J.M. and T.G. designed the project. H.-T.L. and J.M. performed experiments, including protein preparation, antibody screening, crystallization and data collection. H.-T.L. performed model building and refinement. S.S.M. and H.-T.L. performed the radioligand uptake assay. H.-T.L., J.M. and T.G. participated in data analysis and figure preparation. H.-T.L., J.M. and T.G. wrote the manuscript.

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Correspondence to Tamir Gonen.

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Integrated Supplementary Information

Supplementary Figure 1 Overall structure of drSLC38A9.

a, Central panel, side view in plane of lysosomal membrane. The position of the Fab fragment bound on the luminal side is shown by a gray triangle above the luminal loops. Left panel, cytosolic view showing the vestibule at the cytosolic side. Right panel, luminal view. At the luminal side, residues on TM1b, loop 5–6 and loop 7–8 are grouped in cluster 1 (W128, K131, Q132, R344, E411, P415) and those in TM6a, TM10 and TM11 are grouped in cluster 2 (P348, G491, R495, N542, Q546). An enlarged window from the luminal view encompasses luminal gating cluster 1 and cluster 2. b, The electron density of the determined asymmetric unit of drSLC38A9–Fab crystals. Each asymmetric unit contained two drSLC38A9 molecules (labeled SLC38A9 mol 1 and mol 2) as well as two Fab fragments (labeled Fab mol 1 and Fab mol 2). c,d, Examples of the quality of the electron density maps in the determined structure with fitting of the model of drSLC38A9–Fab.

Supplementary Figure 2 Crystal packing and asymmetric unit of the drSLC38A9–Fab complex.

a, Crystal packing showing the drSLC38A9–Fab complex lattice. Fab fragments (gray) stack tightly along the crystallographic b axis and are connected by drSLC38A9 (cyan) layers in the crystallographic ac plane in a propeller-like head-to-side manner. One asymmetric unit is selected to show the building block that is composed of two Fab (orange) and two drSLC38A9 (red) molecules. b, Interactions between drSLC38A9 and adjacent Fab fragments. One drSLC38A9 (red) makes contacts with four other molecules. The biologically functional contact is between the luminal loops of drSLC38A9 (red) and the complementarity-determining regions (CDRs) of the Fab (orange). The three other contacts, which appear to be crystal contacts and nonspecific, occur between loop 2–3 (red) and TM5 (blue 1) and between TM3, TM10 (red) and a groove shaped from the two adjacent Fab fragments (green 2 and yellow 3).

Supplementary Figure 3 Superposition of drSLC38A9 (colored) and AdiC (3L1L) (translucent yellow).

TM3, TM4, TM8 and TM9 are used in the superimposition. The location of the bound arginine in drSLC38A9 is shown by the dashed oval. Structural alignment was performed in PyMOL. AdiC to drSLC38A9; r.m.s. deviation = 3.57 Å, Cealign for 72 residues.

Supplementary Figure 4 Elution profile of the ΔN-SLC38A9–Fab assembly.

a, The solid line represents the elution trace of the ΔN-SLC38A9–Fab complex and unbound Fab. The dashed line is the elution trace of pure ΔN-SLC38A9. An apparent peak shift was observed for the formation of the ΔN-SLC38A9–Fab complex. b, Fractions selected in the red box in a were sampled and analyzed by SDS–PAGE before being pooled and concentrated for crystallization.

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Lei, HT., Ma, J., Sanchez Martinez, S. et al. Crystal structure of arginine-bound lysosomal transporter SLC38A9 in the cytosol-open state. Nat Struct Mol Biol 25, 522–527 (2018). https://doi.org/10.1038/s41594-018-0072-2

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