1. Structural Biology and Molecular Biophysics
Download icon

Measuring the sequence-affinity landscape of antibodies with massively parallel titration curves

  1. Rhys M Adams
  2. Thierry Mora  Is a corresponding author
  3. Aleksandra M Walczak  Is a corresponding author
  4. Justin B Kinney  Is a corresponding author
  1. École Normale Supérieure, France
  2. Cold Spring Harbor Laboratory, United States
Research Article
  • Cited 33
  • Views 4,103
  • Annotations
Cite this article as: eLife 2016;5:e23156 doi: 10.7554/eLife.23156

Abstract

Despite the central role that antibodies play in the adaptive immune system and in biotechnology, much remains unknown about the quantitative relationship between an antibody's amino acid sequence and its antigen binding affinity. Here we describe a new experimental approach, called Tite-Seq, that is capable of measuring binding titration curves and corresponding affinities for thousands of variant antibodies in parallel. The measurement of titration curves eliminates the confounding effects of antibody expression and stability that arise in standard deep mutational scanning assays. We demonstrate Tite-Seq on the CDR1H and CDR3H regions of a well-studied scFv antibody. Our data shed light on the structural basis for antigen binding affinity and suggests a role for secondary CDR loops in establishing antibody stability. Tite-Seq fills a large gap in the ability to measure critical aspects of the adaptive immune system, and can be readily used for studying sequence-affinity landscapes in other protein systems.

Data availability

The following data sets were generated
    1. Adams RM
    2. Kinney JB
    3. Mora T
    4. Walczak AM
    (2016) Saccharomyces cerevisiae high-throughput titration curves
    Publicly available at the NCBI BioProject database (accession no: PRJNA344711).

Article and author information

Author details

  1. Rhys M Adams

    Laboratoire de Physique Théorique, École Normale Supérieure, Paris, France
    Competing interests
    The authors declare that no competing interests exist.
  2. Thierry Mora

    Laboratoire de Physique Statistique, École Normale Supérieure, Paris, France
    For correspondence
    tmora@lps.ens.fr
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5456-9361
  3. Aleksandra M Walczak

    Laboratoire de Physique Théorique, École Normale Supérieure, Paris, France
    For correspondence
    awalczak@lpt.ens.fr
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2686-5702
  4. Justin B Kinney

    Simons Center for Quantitative Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, United States
    For correspondence
    jkinney@cshl.edu
    Competing interests
    The authors declare that no competing interests exist.

Funding

European Research Council (StG n. 306312)

  • Rhys M Adams
  • Thierry Mora
  • Aleksandra M Walczak

Simons Center for Quantitative Biology

  • Justin B Kinney

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Jesse D Bloom, Fred Hutchinson Cancer Research Center, United States

Publication history

  1. Received: November 10, 2016
  2. Accepted: December 27, 2016
  3. Accepted Manuscript published: December 30, 2016 (version 1)
  4. Accepted Manuscript updated: January 3, 2017 (version 2)
  5. Version of Record published: January 26, 2017 (version 3)

Copyright

© 2016, Adams et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 4,103
    Page views
  • 779
    Downloads
  • 33
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Benjamin C McIlwain et al.
    Research Article Updated

    Fluc family fluoride channels protect microbes against ambient environmental fluoride by undermining the cytoplasmic accumulation of this toxic halide. These proteins are structurally idiosyncratic, and thus the permeation pathway and mechanism have no analogy in other known ion channels. Although fluoride-binding sites were identified in previous structural studies, it was not evident how these ions access aqueous solution, and the molecular determinants of anion recognition and selectivity have not been elucidated. Using x-ray crystallography, planar bilayer electrophysiology, and liposome-based assays, we identified additional binding sites along the permeation pathway. We used this information to develop an oriented system for planar lipid bilayer electrophysiology and observed anion block at one of these sites, revealing insights into the mechanism of anion recognition. We propose a permeation mechanism involving alternating occupancy of anion-binding sites that are fully assembled only as the substrate approaches.

    1. Structural Biology and Molecular Biophysics
    Piotr Kolata, Rouslan G Efremov
    Research Article

    Respiratory complex I is a multi-subunit membrane protein complex that reversibly couples NADH oxidation and ubiquinone reduction with proton translocation against trans-membrane potential. Complex I from Escherichia coli is among the best functionally characterized complexes, but its structure remains unknown, hindering further mechanistic studies to understand the enzyme coupling mechanism. Here we describe the single particle cryo-electron microscopy (cryo-EM) structure of the entire catalytically active E. coli complex I reconstituted into lipid nanodiscs. The structure of this mesophilic bacterial complex I displays highly dynamic connection between the peripheral and membrane domains. The peripheral domain assembly is stabilized by unique terminal extensions and an insertion loop. The membrane domain structure reveals novel dynamic features. Unusual conformation of the conserved interface between the peripheral and membrane domains suggests an uncoupled conformation of the complex. Considering constraints imposed by the structural data we suggest a new simple hypothetical coupling mechanism for the molecular machine.