Constraining Enzyme Conformational Change by an Antibody Leads to Hyperbolic Inhibition

Abstract

Although it has been known for many years that antibodies display properties characteristic of allosteric effectors, the molecular mechanisms responsible for these effects remain poorly understood. Here, we describe a single-domain antibody fragment (nanobody) that modulates protein function by constraining conformational change in the enzyme dihydrofolate reductase (DHFR). Nanobody 216 (Nb216) behaves as a potent allosteric inhibitor of DHFR, giving rise to mixed hyperbolic inhibition kinetics. The crystal structure of Nb216 in complex with DHFR reveals that the nanobody binds adjacent to the active site. Half of the epitope consists of residues from the flexible Met20 loop. This loop, which ordinarily oscillates between occluded and closed conformations during catalysis, assumes the occluded conformation in the Nb216-bound state. Using stopped flow, we show that Nb216 inhibits DHFR by stabilising the occluded Met20 loop conformation. Surprisingly, kinetic data indicate that the Met20 loop retains sufficient conformational flexibility in the Nb216-bound state to allow slow substrate turnover to occur.

Regulation ofβ2-Adrenergic Receptor Function by Conformationally Selective Single-Domain Intrabodies

“…Indeed, nanobodies have been described that allosterically activate or inhibit the catalytic function of specific enzymes (Saerens et al, 2004;Barlow et al, 2009;Oyen et al, 2011). Furthermore, a nanobody that inhibits Clostridium botulinum neurotoxin proteases maintained its antagonist properties when expressed as an intrabody in neuronal cells (Tremblay et al, 2010).…”

Biotechnological applications of recombinant single-domain antibody fragments

“…Such allosteric control of the enzyme activity raised hyperbolic inhibition kinetics and induced a conformational rearrangement of the whole enzyme structure [181]. Structural data were also necessary to explain the allosteric inhibition mechanism of the botulinum neurotoxin induced by another VHH [85].…”

Discovery and characterization of olokizumab

“…It has also been suggested that antibodies induce conformational changes, causing functional modification of target proteins (both inhibition and enhancement) 40 . However, only a few studies 41 , 42 have so far provided direct evidence of such conformational changes. Our study has shown that the conformational structure of IL-6 in complex with olokizumab is substantially different to that of IL-6 bound to gp130, 6 which may provide additional rationale for the potent anti-inflammatory effects of olokizumab.…”