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

摘要

Sharks and other cartilaginous fish are the phylogenetically oldest living organisms which have antibodies as part of their adaptive immune system. As part of their humoral adaptive immune response, they produce an immunoglobulin, the so-called new antigen receptor (IgNAR), a heavy-chain only antibody. The variable domain of an IgNAR, also known as VNAR, binds the antigen as an independent soluble domain. In this study we structurally and dynamically characterize the affinity maturation mechanism of a germline and somatically matured (PBLA8) VNAR. We observe a substantial rigidification upon affinity maturation, which is accompanied by higher number of contacts, thereby contributing to the decrease in flexibility. By just 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 VNAR in complex with the hen-egg white lysozyme show that the VNAR antibodies clearly follow the concept of conformational selection, as the binding competent state already pre-existed 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 VNARs. Upon maturation we observe a substantial increase in the resulting dissociation free energy barrier. Furthermore, we are able to kinetically and thermodynamically describe the binding process and do not only identify a two-step binding mechanism, but we also find a strong population shift upon affinity maturation towards the native binding pose.