Basis of Negative Cooperativity in Two-Sided Monobody Block of Fluc

Abstract

Fluc type fluoride channels are constructed as anti-parallel homo-dimers with two-fold symmetry parallel to the membrane plane. They are blocked by a set of nanomolar-affinity fibronectin-domain monobodies originally selected from phage-display libraries that bind chemically equivalent epitopes present on both ends of the channel. Kinetic analysis of single-channel recordings made with monobody on both sides of the membrane shows substantial negative cooperativity (∼1.7 kcal/mol) between the two blocking sites. The nature of this negative cooperativity is not known. Direct monobody-binding assays, single-channel recordings of a Fluc channel homolog and mutagenesis of charged residues E27Q and E79Q indicate that the basis of negative cooperativity is electrostatic repulsion across the channel between neighboring monobodies. The charge-reduced monobody exhibits a novel trimolecular blocking behavior and independent blocking in the doubly occupied state.