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
  4. Justin B Kinney
  1. École Normale Supérieure, France
  2. Cold Spring Harbor Laboratory, United States
Research Article
  • Cited 37
  • Views 4,492
  • 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
    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
    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

  • 4,492
    Page views
  • 824
    Downloads
  • 37
    Citations

Article citation count generated by polling the highest count across the following sources: Scopus, Crossref, 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
    Alena Kroupova et al.
    Research Article

    RtcB enzymes are RNA ligases that play essential roles in tRNA splicing, unfolded protein response, and RNA repair. In metazoa, RtcB functions as part of a five-subunit tRNA ligase complex (tRNA-LC) along with Ddx1, Cgi-99, Fam98B and Ashwin. The human tRNA-LC or its individual subunits have been implicated in additional cellular processes including microRNA maturation, viral replication, DNA double-strand break repair and mRNA transport. Here we present a biochemical analysis of the inter-subunit interactions within the human tRNA-LC along with crystal structures of the catalytic subunit RTCB and the N-terminal domain of CGI-99. We show that the core of the human tRNA-LC is assembled from RTCB and the C-terminal alpha-helical regions of DDX1, CGI-99, and FAM98B, all of which are required for complex integrity. The N-terminal domain of CGI-99 displays structural homology to calponin-homology domains, and CGI-99 and FAM98B associate via their N-terminal domains to form a stable subcomplex. The crystal structure of GMP-bound RTCB reveals divalent metal coordination geometry in the active site, providing insights into its catalytic mechanism. Collectively, these findings shed light on the molecular architecture and mechanism of the human tRNA ligase complex, and provide a structural framework for understanding its functions in cellular RNA metabolism.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Jing Li et al.
    Research Article

    Integrin conformational ensembles contain two low-affinity states, bent-closed and extended-closed, and an active, high-affinity, extended-open state. It is widely thought that integrins must be activated before they bind ligand; however, one model holds that activation follows ligand binding. As ligand-binding kinetics are not only rate limiting for cell adhesion but also have important implications for the mechanism of activation, we measure them here for integrins α4β1 and α5β1 and show that the low-affinity states bind substantially faster than the high-affinity state. On and off-rates are similar for integrins on cell surfaces and as ectodomain fragments. Although the extended-open conformation's on-rate is ~20-fold slower, its off-rate is ~25,000-fold slower, resulting in a large affinity increase. The tighter ligand-binding pocket in the open state may slow its on-rate. Low affinity integrin states not only bind ligand more rapidly, but are also more populous on the cell surface than high affinity states. Thus, our results suggest that integrin binding to ligand may precede, rather than follow, activation by 'inside-out signaling'.