Shark Antibody Variable Domains Rigidify Upon Affinity Maturation-Understanding the Potential of Shark Immunoglobulins as Therapeutics

Abstract

Sharks and other cartilaginous fish are the phylogenetically oldest living organisms that have antibodies as part of their adaptive immune system. As part of their humoral adaptive immune response, they produce an immunoglobulin, the so-called immunoglobulin new antigen receptor (IgNAR), a heavy-chain only antibody. The variable domain of an IgNAR, also known as V _NAR , binds the antigen as an independent soluble domain. In this study, we structurally and dynamically characterized the affinity maturation mechanism of the germline and somatically matured (PBLA8) V _NAR to better understand their function and their applicability as therapeutics. We observed a substantial rigidification upon affinity maturation, which is accompanied by a higher number of contacts, thereby contributing to the decrease in flexibility. Considering the static x-ray structures, the observed rigidification is not obvious, as especially the mutated residues undergo conformational changes during the simulation, resulting in an even stronger network of stabilizing interactions. Additionally, the simulations of the V _NAR in complex with the hen egg-white lysozyme show that the V _NAR antibodies evidently follow the concept of conformational selection, as the binding-competent state already preexisted even without the presence of the antigen. To have a more detailed description of antibody-antigen recognition, we also present here the binding/unbinding mechanism between the hen egg-white lysozyme and both the germline and matured V _NAR s. Upon maturation, we observed a substantial increase in the resulting dissociation-free energy barrier. Furthermore, we were able to kinetically and thermodynamically describe the binding process and did not only identify a two-step binding mechanism, but we also found a strong population shift upon affinity maturation toward the native binding pose.

Keywords: VNAR; affinity maturation; binding interfaces; binding mechanisms; conformational selection; encounter complex; shark antibodies.

FIGURE 1

Structural comparison of an immunoglobulin (IgG) structure with an IgG new antigen receptor (IgNAR). The structure of a V_NAR with its unique binding site geometry is depicted next to the IgNAR.

FIGURE 2

Sequence alignment of the 13-point mutations and structural comparison of the ancestral and matured V_NAR domains are illustrated and color coded, respectively.

FIGURE 3

Free energy surfaces and Markov-state models of the apo naive and matured V_NAR CDR3 and CDR1 loops. (A) Free energy surface of the naive V_NAR CDR3 and CDR1 loops, the starting X-ray structure (PDB accession code: 2I27) is depicted as a black dot. (B) Results of the Markov-state models with the respective macrostate probabilities. The thickness of the arrows denotes the transition timescale and the width of the surrounding circle represents the state population. (C) Free energy surface of the matured V_NAR CDR3 and CDR1 loops projected into the same coordinate system as the naive V_NAR and the starting crystal structure is also illustrated as a black dot (PDB accession code: 2I24). (D) Agreement with the obtained free energy surface only one macrostate.

FIGURE 4

Free energy surfaces and Markov-state models of the complexed naive and matured V_NAR CDR3 and CDR1 loops. (A) Free energy surface of the naive V_NAR, the starting x-ray structure (PDB accession code: 2I26) is depicted as a black dot. (B) Results of the Markov-state models with the respective macrostate probabilities. The thickness of the arrows denotes the transition timescale and the width of the surrounding circle represents the state population. (C) Free energy surface of the matured V_NAR CDR3 and CDR1 loops projected into the same coordinate system as the naive V_NAR and the starting crystal structure is also illustrated as a black dot (PDB accession code: 2I25). (D) Agreement with the obtained free energy landscape two macrostates and again the transition timescales and state populations are represented by the thickness of the arrows and the width of the circles, respectively.

FIGURE 5

Visualization of the salt bridges and hydrogen bond interactions between lysozyme and the naive and matured V_NAR domains represented as flare plots. The residues of the antibody are colored in blue, while the antigen is depicted in green. The color-coding corresponds to the structure illustrated next to the flare plots. The CDR3 loop is colored red and the CDR1 loop is depicted in yellow. The mutated residues are shown in bold.

FIGURE 6

Free energy surfaces, Markov-state models, and interaction energies within the obtained three metastable states (complex, encounter and unbound). (A) Free energy surfaces of the observed unbinding pathways of the naive and matured V_NAR domains. The Markov-state model is depicted in panel (B) showing the state probabilities and transition timescales between the three identified states. (C) Average electrostatic and Van der Waals interactions at the binding interface of the antibody and the antigen for the complex, encounter, and unbound state.

FIGURE 7

Schematic summary and representation of the binding pathway. Upon affinity maturation, the native complex state becomes the most probable state, while the encounter complex is favored in the binding pathway of the naive antibody.