Development of "Plug and Play" Fiducial Marks for Structural Studies of GPCR Signaling Complexes by Single-Particle Cryo-EM

Structure. 2019 Dec 3;27(12):1862-1874.e7. doi: 10.1016/j.str.2019.10.004. Epub 2019 Oct 25.

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 Gi and Gs, as well as mini-Gs, 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 Gi and Gs, 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.

Keywords: G proteins; GPCR; GRK (G protein receptor kinase); SP cryo-EM; affinity maturation; interface energetics; phage display; synthetic antibodies; universal fiducial mark.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Antibodies / chemistry*
  • Antibodies / genetics
  • Antibodies / metabolism
  • Antibody Specificity
  • Binding Sites
  • Cloning, Molecular
  • Cryoelectron Microscopy
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • G-Protein-Coupled Receptor Kinase 1 / chemistry*
  • G-Protein-Coupled Receptor Kinase 1 / genetics
  • G-Protein-Coupled Receptor Kinase 1 / metabolism
  • GTP-Binding Protein alpha Subunits, Gi-Go / chemistry*
  • GTP-Binding Protein alpha Subunits, Gi-Go / genetics
  • GTP-Binding Protein alpha Subunits, Gi-Go / metabolism
  • GTP-Binding Protein alpha Subunits, Gs / chemistry*
  • GTP-Binding Protein alpha Subunits, Gs / genetics
  • GTP-Binding Protein alpha Subunits, Gs / metabolism
  • Gene Expression
  • Genetic Vectors / chemistry
  • Genetic Vectors / metabolism
  • Humans
  • Kinetics
  • Models, Molecular
  • Mutation
  • Peptide Library*
  • Protein Binding
  • Protein Conformation, alpha-Helical
  • Protein Conformation, beta-Strand
  • Protein Interaction Domains and Motifs
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Thermodynamics

Substances

  • Antibodies
  • Peptide Library
  • Recombinant Proteins
  • G-Protein-Coupled Receptor Kinase 1
  • GRK1 protein, human
  • GTP-Binding Protein alpha Subunits, Gi-Go
  • GTP-Binding Protein alpha Subunits, Gs