Molecular basis of tail-anchored integral membrane protein recognition by the cochaperone Sgt2
The targeting and insertion of tail-anchored (TA) integral membrane proteins (IMPs) into the correct membrane is critical for cellular homeostasis. The fungal protein Sgt2, and its human homolog SGTA, is the entry point for clients to the guided entry of tail-anchored protein (GET) pathway, which targets endoplasmic reticulum-bound TA IMPs. Consisting of three structurally independent domains, the C terminus of Sgt2 binds to the hydrophobic transmembrane domain (TMD) of clients. However, the exact binding interface within Sgt2 and molecular details that underlie its binding mechanism and client preference are not known. Here, we reveal the mechanism of Sgt2 binding to hydrophobic clients, including TA IMPs. Through sequence analysis, biophysical characterization, and a series of capture assays, we establish that the Sgt2 C-terminal domain is flexible but conserved and sufficient for client binding. A molecular model for this domain reveals a helical hand forming a hydrophobic groove approximately 15 Å long that is consistent with our observed higher affinity for client TMDs with a hydrophobic face and a minimal length of 11 residues. This work places Sgt2 into a broader family of TPR-containing cochaperone proteins, demonstrating structural and sequence-based similarities to the DP domains in the yeast Hsp90 and Hsp70 coordinating protein, Sti1.
© 2021 The Authors. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Received 17 December 2020, Revised 4 February 2021, Available online 19 February 2021. We thank D. G. VanderVelde for assistance with NMR data collection; S. Mayo for providing computing resources; S. Shan, H. J. Cho, Y. Liu, and members of the Clemons lab for support and discussion. We thank J. Mock and A. M. Thinn for comments on the article. Author contributions: K.-F. L., M. Y. F., S. M. S., and W. M. C. designed the experiments and wrote the article. K.-F. L. performed all NMR and CD experiments. K.-F. L. and M. Y. F. performed the pull-down assays. S. M. S. created the computational model of Sgt2-C. K.-F. L. and S. M. S. identified Sgt2-C as an STI1 domain. All the authors have reviewed and approved the article. Funding and additional information: This work was supported by the National Institutes of Health grants GM105385 and GM097572 (to W. M. C.), NIH/National Research Service Award Training Grant GM07616 (to S. M. S. and M. Y. F.), and a National Science Foundation Graduate Research fellowship Grant 1144469 (to S. M. S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Data availability: All data is provided in the article
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