High-affinity actin-binding nebulin fragments influence the ActoS1 complex

Human nebulin fragments, NA3 and NA4, corresponding to individual superrepe ats display high-affinity interactions with individual actin protomers in c osedimentation and solid-phase binding assays. Stoichiometric analysis of n ebulin fragment-induced actin polymerization and inhibition of actin-activa ted S1 ATPase indicate that one superrepeat influences multiple actin molec ules along the F-actin filament, consistent with a combination of strong an d weak interactions of nebulin over the length of the actin filament. The m echanisms by which human nebulin fragments affect the interaction between a ctin and myosin S1 are studied by fluorescence quenching, polarization, and resonance energy transfer. We show that, under strong binding conditions, premixing actin with the NA3 prior to adding myosin subfragment 1 (S1) inhi bits the rate of actoS1 association. The nebulin fragments, NA3 and NA4, ca used little effect on the extent of actoS1 binding at equilibrium but did a lter the nature of the complex as evidenced by an increase in the resonance energy transfer efficiencies between S1 and actin in the absence of ATP. T he addition of low concentrations of ATP rapidly dissociates the strong-bin ding actoS1 irrespective of the presence or absence of nebulin fragment. In terestingly, the strongly bound state reforms rapidly after S1 hydrolyzes a ll available ATP. These observations are consistent with the notion that ne bulin might contribute to optimizing the alignment of actomyosin interactio ns and inhibit suboptimal actomyosin contacts.