Conformational Chaperones for Structural Studies of Membrane Proteins Using Antibody Phage Display with Nanodiscs

Abstract

SUMMARY A major challenge in membrane biophysics is to define the mechanistic linkages between a protein’s conformational transitions and its function. We describe a novel approach to stabilize transient functional states of membrane proteins in native-like lipid environments allowing for their structural and biochemical characterization. This is accomplished by combining the power of antibody Fab-based phage display selection with the benefits of embedding membrane protein targets in lipid-filled nanodiscs. In addition to providing a stabilizing lipid environment, nanodiscs afford significant technical advantages over detergent-based formats. This enables the production of a rich pool of high performance Fab binders that can be used as crystallization chaperones, as fiducial markers for single particle cryo-EM and as probes of different conformational states. Moreover, nanodisc generated Fabs can be used to identify detergents that best mimic native membrane environments for use in biophysical studies.

The structural basis for activation of voltage sensor domains in an ion channel TPC1

“…As a basis for understanding the voltage-dependent activation mechanism and its modulation by Ca 2+ -ions, we determined the structure of AtTPC1 DDE by cryo-EM (Figure 1a, Figure S2, S3, S4, Table S1). We employed saposin A nanoparticles (30) to reconstitute AtTPC1 DDE into a membrane environment, and an antibody Fab made against AtTPC1 (CAT06/H12) (31) to facilitate particle alignment (32) (Figures S5, S6, See Supplemental Discussion).…”

“…To generate Fabs, the efficiency of biotinylation of AtTPC1 WT -E3D1 nanodiscs was evaluated by pull-down on streptavidin-coated magnetic beads (Promega). Library sorting steps were performed using Fab Library E (DNA kindly provided by S. Koide(47)) based on previous protocols(31, 48). Six independent phage library sorting experiments were performed against biotinylated AtTPC1 WT -E3D1 nanodiscs in 2 buffers containing 20 mM Hepes pH 7.3, 200 mM NaCl, 5% glycerol, 1% BSA, and either 1 mM CaCl 2 or 1 mM EGTA.…”

Synthetic single domain antibodies for the conformational trapping of membrane proteins

“…Pure in vitro binder selections operate independent of target toxicity and sequence conservation, allow for a wide range of selection conditions including low-affine ligands to trap targets in desired conformations and are also significantly faster than selections depending on a slow immune response. Despite of these advantages over in vivo procedures, successful in vitro selections against membrane proteins are limited to a few specialized labs 5,[28][29][30][31][32][33] .…”

“…Conformation-specific binders raised against membrane proteins have the ability to manipulate cells directly at the cell surface and are exquisite tools for basic science and drug discovery [1][2][3] . However, binder selection against this difficult class of target proteins is challenging [4][5][6] . A particularly successful method relies on the immunization of camelids for the pre-enrichment of B-cells that encode targetspecific nanobodies, the variable domains of heavy-chain-only antibodies 6 .…”

A review of antibody-based therapeutics targeting G protein-coupled receptors: an update

“…Recent developments designed to enhance the ability for antibody discovery and functional screening include increasing cell surface expression and methods for detection of GPCRexpressing cell lines [21,22]; access to new generation detergents such as calixarenes [23], GPCR stabilization for improved expression [24,25], enhanced thermostability or conformation [26], the implementation of lipoparticles [27], magnetic proteoliposomes [28] or virus-like particles [29], spherical-supported bilayer lipid membranes [30] and the generation of nanodiscs [31] have all added to the arsenal of antigen formats that can be applied not only in the generation of antibodies but also associated downstream discovery processes and compliment established tool reagents, such as whole cells or membranes over-expressing the target of interest. The effectiveness of DNA immunization expression constructs or GPCR-expressing cells can be enhanced using adjuvants [32,33] with inventive selection strategies [34] and affinity maturation cell-based approaches devised, for example, CHO cell display libraries of single-chain variable fragments The typical structure for each major GPCR family is presented where the blue horizontal lines represent the lipid bilayer of the cell membrane and numbers 1-7 each represents a GPCR helix or individual transmembrane (TM) domain.…”