Mechanistic analysis of allosteric and non-allosteric effects arising from nanobody binding to two epitopes of the dihydrofolate reductase of Escherichia coli

Abstract

Although allosteric effector antibodies are used widely as modulators of receptors and enzymes, experimental analysis of their mechanism remains highly challenging. Here, we investigate the molecular mechanisms of allosteric and non-allosteric effector antibodies in an experimentally tractable system, consisting of single-domain antibodies (nanobodies) that target the model enzyme dihydrofolate reductase (DHFR) from Escherichia coli. A panel of thirty-five nanobodies was isolated using several strategies to increase nanobody diversity. The nanobodies exhibit a variety of effector properties, including partial inhibition, strong inhibition and stimulation of DHFR activity. Despite these diverse effector properties, chemical shift perturbation NMR epitope mapping identified only two epitope regions: epitope α is a new allosteric site that is over 10Å from the active site, while epitope β is located in the region of the Met20 loop. The structural basis for DHFR allosteric inhibition or activation upon nanobody binding to the α epitope was examined by solving the crystal structures of DHFR in complex with Nb113 (an allosteric inhibitor) and Nb179 (an allosteric activator). The structures suggest roles for conformational constraint and altered protein dynamics, but not epitope distortion, in the observed allosteric effects. The crystal structure of a β epitope region binder (ca1698) in complex with DHFR is also reported. Although CDR3 of ca1698 occupies the substrate binding site, ca1698 displays linear mixed inhibition kinetics instead of simple competitive inhibition kinetics. Two mechanisms are proposed to account for this apparent anomaly. Evidence for structural convergence of ca1698 and Nb216 during affinity maturation is also presented.

Nanobodies as Probes for Protein Dynamics in Vitro and in Cells

“…Signals of the residues located at the contact surface with the nanobody show intensity loss, or chemical shift change, depending on the nanobody binding parameters. Crystal structures of several nanobody complexes with dihydrofolate reductase showed good agreement between NMR mapping and the x-ray structures (73).…”

Stability of isolated antibody-antigen complexes as a predictive tool for selecting toxin neutralizing antibodies

“…Abs that stabilize one conformational state over another may be common 51,52,53,54,55 . The MOA may be rarely recognized since, in these cases, no notable conformational changes would be observed upon Ab binding and alternate conformations of the antigen may elude structural determination.…”

“…The structure of the complex was solved by molecular replacement using Phaser 62 . The structures of RTA (PDB 1J1M), 38 RTA1-33/44-198 (PDB 4IMV), 21 a sdAb (PDB 4FHB), 51 and the structure of the A3C8 sdAb (PDB 5SV4) were used as search models. Model-building was performed using Coot 63,64 .…”

Antibody Fragments Defining Biologically Relevant Conformations of Target Proteins

“…This interesting observation opens up new, generalizable avenues for antibody-based inhibitors of enzymes and receptors [58]. Of particular interest to this review, the same group reported earlier [59] that allosteric effects, brought about by binding of antibody fragments, were due to conformational constraint and altered protein dynamics rather than epitope distortion, suggesting that the antibody fragments were locking out naturally sampled conformations rather than imposing unnatural perturbation.…”