Constraining enzyme conformational change by an antibody leads to hyperbolic inhibition.

TitleConstraining enzyme conformational change by an antibody leads to hyperbolic inhibition.
Publication TypeJournal Article
Year of Publication2011
AuthorsOyen, D., V. Srinivasan, J. Steyaert, and J. N. Barlow
JournalJ Mol Biol
Volume407
Issue1
Pagination138-48
Date Published2011 Mar 18
ISSN1089-8638
KeywordsAllosteric Regulation, Animals, Binding Sites, Camelids, New World, Catalysis, Enzyme Inhibitors, Kinetics, Models, Molecular, Niacinamide, Peptide Library, Protein Binding, Protein Conformation, Single-Chain Antibodies, Tetrahydrofolate Dehydrogenase
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.

DOI10.1016/j.jmb.2011.01.017
Alternate JournalJ. Mol. Biol.
PubMed ID21238460