Conformation of actin subunits at the barbed and pointed ends of F-actin with and without capping proteins

Abstract

Advances in cryo-electron microscopy have made possible the determination of structures of the barbed and pointed ends of F-actin, both in the absence and the presence of capping proteins that block subunit exchange. The conformation of the two exposed protomers at the barbed end resembles the "flat" conformation of protomers in the middle of F-actin. The barbed end changes little upon binding of CapZ, which in turn undergoes a major conformational change. At the pointed end, however, protomers have the "twisted" conformation characteristic of G-actin, whereas tropomodulin binding forces a flat conformation upon the second subunit. The structures provide a mechanistic understanding for the asymmetric addition/dissociation of actin subunits at the ends of F-actin and open the way to future studies of other regulators of filament end dynamics.

Keywords: actin; structural biology.

Fig. 1:. Conformation of actin subunits at the barbed and pointed ends of F-actin.

(A-B) Representative cryo-EM micrograph of actin polymerized in the absence (A) or the presence (B) of capping proteins. (C) Merged cryo-EM reconstructions of the free pointed (top) and barbed (bottom) ends of F-actin. Terminal protomers at the free pointed end have a G-actin-like, twisted conformation (pink), whereas protomers at the free barbed end have an F-actin-like, flat conformation (gray). The long- and short-pitch helices of F-actin as indicated by yellow and cyan traces, respectively. (D) Two views of the CapZ heterodimer (CapZα, blue, CapZβ, cyan) bound to the barbed end. CapZ undergoes a major conformational change upon binding, but the barbed end changes little as a result of this interaction. (E) Two views of Tmod (lime) bound to the pointed end. Tmod binds at the interface between the first three protomers at the pointed end and forces protomer-2 into an F-actin-like, flat conformation (gray). (F) The structures depicted in parts C-E demonstrate that subunits in F-actin have different conformations depending on whether they are in middle or at the ends of F-actin. These structural differences correlate with kinetic differences in the association/dissociation constants of subunits at the filament ends. Only the main pathway at equilibrium is depicted, with ATP-actin adding preferentially to the barbed end and ADP-actin dissociating from the pointed end (Pollard, 1986). CapZ and Tmod inhibit subunit exchange at the barbed and pointed ends, respectively.