Contributions of the Complementarity Determining Regions to the Thermal Stability of a Single-Domain Antibody

Nikhil Bayya,Ellen R Goldman,Eugene A Permyakov,George P Anderson,Dan Zabetakis

doi:10.1371/journal.pone.0077678

Nikhil Bayya, Ellen R Goldman + Show 3 more

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https://doi.org/10.1371/journal.pone.0077678

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Abstract

Single domain antibodies (sdAbs) are the recombinantly-expressed variable domain from camelid (or shark) heavy chain only antibodies and provide rugged recognition elements. Many sdAbs possess excellent affinity and specificity; most refold and are able to bind antigen after thermal denaturation. The sdAb A3, specific for the toxin Staphylococcal enterotoxin B (SEB), shows both sub-nanomolar affinity for its cognate antigen (0.14 nM) and an unusually high melting point of 85°C. Understanding the source of sdAb A3’s high melting temperature could provide a route for engineering improved melting temperatures into other sdAbs. The goal of this work was to determine how much of sdAb A3’s stability is derived from its complementarity determining regions (CDRs) versus its framework. Towards answering this question we constructed a series of CDR swap mutants in which the CDRs from unrelated sdAbs were integrated into A3’s framework and where A3’s CDRs were integrated into the framework of the other sdAbs. All three CDRs from A3 were moved to the frameworks of sdAb D1 (a ricin binder that melts at 50°C) and the anti-ricin sdAb C8 (melting point of 60°C). Similarly, the CDRs from sdAb D1 and sdAb C8 were moved to the sdAb A3 framework. In addition individual CDRs of sdAb A3 and sdAb D1 were swapped. Melting temperature and binding ability were assessed for each of the CDR-exchange mutants. This work showed that CDR2 plays a critical role in sdAb A3’s binding and stability. Overall, results from the CDR swaps indicate CDR interactions play a major role in the protein stability.

Highlights

Single-domain antibodies are small recombinantlyproduced binding elements derived from the heavy-chain-only antibodies produced by camelids and sharks [1,2,3,4]
SdAb A3 was selected from a library of phage-displayed sdAbs derived from an immunized llama and shows high affinity and specificity for Staphylococcal enterotoxin B (SEB) [10,11]
As in conventional variable heavy domains, there are four highlyconserved framework regions alternating with highly-variable complementarity determining regions (CDRs) which embody the specific binding interaction of the antigen-antibody complex

Summary

Introduction

Single-domain antibodies (sdAbs) are small recombinantlyproduced binding elements derived from the heavy-chain-only antibodies produced by camelids and sharks [1,2,3,4]. SdAb A3 was selected from a library of phage-displayed sdAbs derived from an immunized llama and shows high affinity and specificity for Staphylococcal enterotoxin B (SEB) [10,11]. In this study we compare sdAb A3 to both low-melting sdAb D1 (50°C) and moderate-melting sdAb C8 (60°C), whose sequences are shown in Figure 1 for comparison Both sdAb D1 and sdAb C8 have binding specificity for ricin which can be used to distinguish functional activity from sdAb A3 [12,13,14]

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