Development of "Plug and Play" Fiducial Marks for Structural Studies of GPCR Signaling Complexes by Single-Particle Cryo-EM
Abstract
"Universal" synthetic antibody (sAB)-based fiducial marks have been generated by customized phage display selections to facilitate the rapid structure determination of G protein-coupled receptor (GPCR) signaling complexes by single-particle cryo-electron microscopy (SP cryo-EM). sABs were generated to the two major G protein subclasses: trimeric G_i and G_s, as well as mini-G_s, and were tested to ensure binding in the context of their cognate GPCRs. Epitope binning revealed that multiple distinct epitopes exist for each G(αβγ) protein. Several Gβγ-specific sABs, cross-reactive between trimeric G_i and G_s, were identified suggesting they could be used across all subclasses in a "plug and play" fashion. sABs were also generated to a representative of another class of GPCR signaling partner, G protein receptor kinase 1 (GRK1) and evaluated further, supporting the generalizability of the approach. EM data suggested that the subclass-specific sABs provide effective single and dual fiducials for multiple GPCR signaling complexes.
Additional Information
© 2019 Elsevier Ltd. Received 14 June 2019, Revised 26 August 2019, Accepted 7 October 2019, Available online 25 October 2019. We thank Shohei Koide for providing the DNA of phage Library E and Satchal K. Erramilli for helpful discussion. This work was supported by NIH grants GM117372 (to A.A.K.), GM127710 (to H.E.X.) and 1R35GM128641-01 (to C.Z.). We also acknowledge support from Pfizer (to A.A.K.) and Jay and Betty Van Andel Foundation (to H.E.X.). Data and Code Availability: Molecular dynamics simulation system parameters and trajectories are available upon request. Author Contributions: Conceptualization, P.D., S.M., H.E.X., and A.A.K.; Methodology, P.D. and S.M.; Investigation, P.D., S.M., X.G., Y.K., P.W.d.W., L.W., and Y.Z.; Writing – Original Draft, P.D., S.M., and A.A.K.; Writing – Review & Editing, P.D., S.M., and A.A.K.; Visualization, P.D.; Supervision, K.M., C.Z., H.E.X., and A.A.K.; Funding Acquisition, C.Z., H.E.X., and A.A.K. The authors declare no competing interests.Attached Files
Supplemental Material - 1-s2.0-S0969212619303454-mmc1.pdf
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Additional details
- Eprint ID
- 100419
- Resolver ID
- CaltechAUTHORS:20191223-145547096
- GM117372
- NIH
- GM127710
- NIH
- 1R35GM128641-01
- NIH
- Pfizer
- Jay and Betty Van Andel Foundation
- Created
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2019-12-23Created from EPrint's datestamp field
- Updated
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2021-11-16Created from EPrint's last_modified field