Crystal Structures of Human TBC1D1 and TBC1D4 (AS160) RabGTPase-activating Protein (RabGAP) Domains Reveal Critical Elements for GLUT4 Translocation
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We have solved the x-ray crystal structures of the RabGAP domains of human TBC1D1 and human TBC1D4 (AS160), at 2.2 and 3.5 Å resolution, respectively. Like the yeast Gyp1p RabGAP domain, whose structure was solved previously in complex with mouse Rab33B, the human TBC1D1 and TBC1D4 domains both have 16 α-helices and no β-sheet elements. We expected the yeast Gyp1p RabGAP/mouse Rab33B structure to predict the corresponding interfaces between cognate mammalian RabGAPs and Rabs, but found that residues were poorly conserved. We further tested the relevance of this model by Ala-scanning mutagenesis, but only one of five substitutions within the inferred binding site of the TBC1D1 RabGAP significantly perturbed catalytic efficiency. In addition, substitution of TBC1D1 residues with corresponding residues from Gyp1p did not enhance catalytic efficiency. We hypothesized that biologically relevant RabGAP/Rab partners utilize additional contacts not described in the yeast Gyp1p/mouse Rab33B structure, which we predicted using our two new human TBC1D1 and TBC1D4 structures. Ala substitution of TBC1D1 Met(930), corresponding to a residue outside of the Gyp1p/Rab33B contact, substantially reduced catalytic activity. GLUT4 translocation assays confirmed the biological relevance of our findings. Substitutions with lowest RabGAP activity, including catalytically dead RK and Met(930) and Leu(1019) predicted to perturb Rab binding, confirmed that biological activity requires contacts between cognate RabGAPs and Rabs beyond those in the yeast Gyp1p RabGAP/mouse Rab33B structure.
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The TBC1D7 apo structure reveals an architecture highly conserved with other TBC domain proteins, with little conformational difference in comparison to the TSC1-complexed structures.
“The apo structure (Fig. 5D) reveals an architecture highly conserved with other TBC domain proteins (Fischer et al., 2016; Fukuda, 2011; Park et al., 2011), with little conformational difference in comparison to the TSC1-complexed structures.”
From Signal integration in the (m)TORC1 growth pathway (2018) · cites this paper as Park et al., 2011
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- Authors
- Park, S.-Y., Jin, W., Woo, J. R., & Shoelson, S. E.
- Affiliations
- Joslin Diabetes Center (US)
- Corresponding author
- Shoelson, Steven E.
- Venue
- Journal of Biological Chemistry
- Publisher
- Elsevier BV
- Published
- 2011-05
- Volume
- 286
- Issue
- 20
- Pages
- 18130-18138
- ISSN
- 0021-9258 (ISSN-L)
- PMC
- PMC3093885
- OpenAlex
- W2010974202
- Cited by
- 25
- References
- 22
- Citations by year
- 2024: 3 · 2023: 1 · 2020: 2 · 2019: 3 · 2018: 2 · 2017: 3 · 2016: 2 · 2015: 1 · 2014: 3 · 2013: 2 · 2012: 2
- FWCI
- 1.23
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Journal of Biological Chemistry (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- U.S. Department of Energy (DEAC-02-98CH10886, -AC02-98 CH 10886, DE-AC02-)
- Joslin Diabetes Center (P30DK36836)
- National Institutes of Health (DE-AC02-98CH10886, P30 DK36836, R01 DK51729, DK 36836)
- National Institute of Diabetes and Digestive and Kidney Diseases (R01 DK 43123, R01-DK-51729, R01DK051729, P30-DK36836, F32 DK77485, R01DK043123, F32DK077485, P30DK036836)
- National Institute of Biomedical Imaging and Bioengineering (P30EB009998)
- Brookhaven National Laboratory (AC-02-98CH10886, DE-AC02- 98CH10886)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Receptor Mechanisms and Signaling
- Keywords
- TBC1D1, TBC1D4, AS160, Rab GTPase-activating protein, GLUT4 translocation, RAB33B, catalytic efficiency, alpha-helical domain, skeletal muscle glucose transport
- MeSH terms
- Animals, Humans, Models, Molecular, Structure-Activity Relationship, Protein Structure, Secondary, Protein Structure, Tertiary, Crystallography, X-Ray, GTPase-Activating Proteins, rab GTP-Binding Proteins, Saccharomyces cerevisiae Proteins, Glucose Transporter Type 4, Mice
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