Development of an antibody fragment that stabilizes GPCR/G-protein complexes

Nat Commun. 2018 Sep 13;9(1):3712. doi: 10.1038/s41467-018-06002-w.

Abstract

Single-particle cryo-electron microscopy (cryo-EM) has recently enabled high-resolution structure determination of numerous biological macromolecular complexes. Despite this progress, the application of high-resolution cryo-EM to G protein coupled receptors (GPCRs) in complex with heterotrimeric G proteins remains challenging, owning to both the relative small size and the limited stability of these assemblies. Here we describe the development of antibody fragments that bind and stabilize GPCR-G protein complexes for the application of high-resolution cryo-EM. One antibody in particular, mAb16, stabilizes GPCR/G-protein complexes by recognizing an interface between Gα and Gβγ subunits in the heterotrimer, and confers resistance to GTPγS-triggered dissociation. The unique recognition mode of this antibody makes it possible to transfer its binding and stabilizing effect to other G-protein subtypes through minimal protein engineering. This antibody fragment is thus a broadly applicable tool for structural studies of GPCR/G-protein complexes.

Publication types

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

MeSH terms

  • Antibodies, Monoclonal / chemistry
  • Binding Sites
  • Cryoelectron Microscopy
  • Crystallography, X-Ray
  • GTP-Binding Proteins / chemistry*
  • Humans
  • Immunoglobulin Fragments / chemistry*
  • Macromolecular Substances / chemistry
  • Nucleotides / chemistry
  • Protein Binding
  • Protein Domains
  • Protein Engineering / methods
  • Protein Structure, Secondary
  • Receptors, G-Protein-Coupled / chemistry*
  • Rhodopsin / chemistry
  • Signal Transduction

Substances

  • Antibodies, Monoclonal
  • Immunoglobulin Fragments
  • Macromolecular Substances
  • Nucleotides
  • Receptors, G-Protein-Coupled
  • Rhodopsin
  • GTP-Binding Proteins