Determinants of the assembly and function of antibody variable domains

Eva Maria Herold,Martin Zacharias,Christine John,Stephan Kremser,Sabrina Deubler,Johannes Buchner,Benedikt Weber,Jonathan Eras,Carolin Berner

doi:10.1038/s41598-017-12519-9

Eva Maria Herold, Martin Zacharias + Show 7 more

Open Access

https://doi.org/10.1038/s41598-017-12519-9

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Abstract

The antibody Fv module which binds antigen consists of the variable domains VL and VH. These exhibit a conserved ß-sheet structure and comprise highly variable loops (CDRs). Little is known about the contributions of the framework residues and CDRs to their association. We exchanged conserved interface residues as well as CDR loops and tested the effects on two Fvs interacting with moderate affinities (KDs of ~2.5 µM and ~6 µM). While for the rather instable domains, almost all mutations had a negative effect, the more stable domains tolerated a number of mutations of conserved interface residues. Of particular importance for Fv association are VLP44 and VHL45. In general, the exchange of conserved residues in the VL/VH interface did not have uniform effects on domain stability. Furthermore, the effects on association and antigen binding do not strictly correlate. In addition to the interface, the CDRs modulate the variable domain framework to a significant extent as shown by swap experiments. Our study reveals a complex interplay of domain stability, association and antigen binding including an unexpected strong mutual influence of the domain framework and the CDRs on stability/association on the one side and antigen binding on the other side.

Highlights

In the humoral immune response, antigen recognition is mediated by immunoglobulins, by the N-terminal variable domains of the light chain (VL) and of the heavy chain (VH) which associate non-covalently to form the so-called Fv-fragment
The application of variable domains from different species and subclasses should reveal to which extent the mutation-associated effects are conserved. Their sequences contain all the conserved residues identified by Wang and coworkers except for an alanine at position in MAK33 VL, which is exchanged to serine and a glycine at position in MAK VH, which is an arginine in MAK33
All wt domains studied show a typical ß-sheet structure with the exception of MAK33 VL, which exhibits a characteristic shape in agreement with previous studies in which we investigated the folding pathway of the protein[24] and its amyloidogenic variants[25,26,27] (Fig. 2a and b, Fig. 3a and b)

Summary

Introduction

In the humoral immune response, antigen recognition is mediated by immunoglobulins, by the N-terminal variable domains of the light chain (VL) and of the heavy chain (VH) which associate non-covalently to form the so-called Fv-fragment. Additional experimental studies addressed the influence of the exchange of particular conserved amino acids on the association of VH and VL via the stabilities of covalently linked scFv and Fab fragments[14,15,16,17,18] While these studies lay the groundwork, we are still far from a detailed and comprehensive understanding of the organization of the Fv interface. We focused in the analysis on the isolated variable domains and the direct influence of point mutations on their interaction, and not on Fab or scFv fragments as in previous studies[12,16,18] to draw conclusions concerning their stabilities and antigen binding properties. Our results on the effects of mutations on domain structure, stability, association and antigen binding together with CDR exchange experiments reveal complex relationships between structural and functional properties within the VL and VH domains

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