Actin is a key component in all eukaryotic cells and plays an essential role in a wide range of cellular processes, such as migration, endocytosis, cytokinesis and generation of contraction[1–4]. Actin monomers (G-actin) are able to polymerize into filamentous actin (F-actin) resulting in polar helical structures. The two ends of the filament exhibit distinct biochemical properties and are differentiated as “barbed” and “pointed”, so named after the arrowhead appearance when filaments are decorated with myosin S1. Filament barbed ends dominate the dynamics of filament assembly due to higher association and dissociation rates for actin monomers compared to the pointed ends[3, 7, 8]. Furthermore, since the filament barbed end is preferred for actin monomer addition, whereas net disassembly is favoured at its counterpart, it is being referred to as the fast growing end (pointed end = slow growing end).
In living cells the actin cytoskeleton is in a state of rapid dynamics. Remodelling of the actin cytoskeleton is crucial in terms of inducing changes in cell shape, motility and adhesion and requires strict regulation, both temporally and spatially, thus enabling the cell to function in a controlled manner[4, 9]. This is achieved by a vast number of specialized proteins that bind to actin, thereby modulating actin filament organization and turnover in response to the changing needs of the cell[10, 11]. Actin-binding proteins are able to fulfil a large variety of tasks including the control of actin assembly and disassembly as well as regulating filament branching and bundling to help arrange actin filaments into higher order structures. They can be categorized into proteins which bind to actin monomers, filamentous actin or both. While actin monomer binding proteins control the amount and availability of monomers for polymerization, proteins that bind filamentous actin are involved, among others, in barbed and pointed end capping, filament severing, and filament crosslinking.
Capping protein (CP) is an F-actin binding protein and blocks actin filament elongation and turnover by preventing the addition of new monomers at the fast growing end. Binding of CP to actin filaments occurs with high affinity (Kd < 1 nM) and 1:1 stoichiometry. Two major variants of CP have been determined: a cytoplasmic form that is also termed Cap32/34 (32 = β- and 34 = α-subunit;) and an isoform found in the Z-discs of skeletal muscles that is often called CapZ[14, 15]. CP is a heterodimeric protein composed of an α- and a β-subunit, both having molecular masses in the range of 30–36 kDa. The protein is expressed in all eukaryotic organisms and the subunits exhibit high sequence similarity across the eukaryotic tree of life.
Vertebrates usually express three conserved isoforms of each of the α- and β-subunit[16–18] as opposed to invertebrates, plants, and lower eukaryotes, which in general contain single isoforms of each subunit. The vertebrate α-subunit isoforms are encoded by different genes, whereas the β-subunits arise by alternative splicing from a single gene[16, 17]. One isoform of both the α- and β-subunits is specifically expressed in germ cells (α3, β3), while the remaining ones (α1, α2 and β1, β2) are somatically expressed at varying ratios in different cell types and tissues. β1 is the predominant isoform in muscle cells. In contrast, β2 is mainly expressed in non-muscle tissues. The β isoforms are not able to rescue each others’ function and are thus believed to fulfil different biochemical and cellular tasks. On the other hand, there is little indication of specific functions for the α isoforms.
Vertebrates contain two somatic variants of CP. The sarcomeric variant, which is being referred to as CapZ throughout this manuscript, includes the β1 isoform and is positioned at the Z-discs of striated muscles. CapZ is proposed to help attaching actin filament barbed ends to the Z-discs and to prevent the thin filaments from growing into the adjacent sarcomere, thus serving as a key element in thin filament assembly and regulation within the Z-disc. By contrast, the cytoplasmic variant, which comprises the β2 isoform, is found at the contact sites of actin with membranes, where it is believed to play an essential role in the dendritic nucleation model. In this model activation of the Arp2/3 complex results in a branched network of actin filaments thereby generating new barbed ends, which are primarily oriented towards the cell membrane. As actin subunits are added to the newly created filament ends the membrane is pushed forward. By capping these ends over time, the growing filaments are kept short and branched, which stabilizes the filament network and sustains the propulsive force for leading edge elongation of migrating cells. In addition, actin assembly is restricted to the new barbed ends near the plasma membrane, thus enabling rapid and directed extension of the cell front.
Several molecules are able to modulate the barbed end capping activity of CP by either binding directly to the protein or through association with filament barbed ends and thereby inhibiting CP from binding. Polyphosphoinositides (PPIs), such as phosphatidylinositol-4,5-bisphosphate (PIP2)[24–26] and the proteins CARMIL and V-1 were found to directly associate with CP and to inhibit its capping activity. The crystal structures of CapZ (chicken α1/β1) in complex with CARMIL and V-1, respectively, were recently reported[29, 30]. However, to date no high resolution structure of CP bound to PIP2 exists. One possible role of PIP2, an important component of the plasma membrane and one of the most potent signalling lipids, might be to facilitate membrane movement of highly motile cells, such as those of Dictyostelium discoideum, through inhibition of actin filament capping by CP near the membrane, thus allowing rapid protrusion of the cell edge. Computational docking studies predict that PIP2 interacts with a set of three highly conserved basic residues in close proximity to the α-subunit’s C-terminus. Two of these basic residues are critical for actin filament capping. Such an interaction would therefore prevent for steric reasons the ability of CP to associate with the actin filament.
The crystal structure of CapZ (chicken α1/β1) has provided valuable insight into the atomic architecture of CP found at the Z-discs of skeletal muscles. However, until now a high-resolution structure of the cytoplasmic variant is not available. By characterizing the atomic structure of Cap32/34 from the cellular slime mold Dictyostelium discoideum as a model for cytoplasmic CP and comparing it to that of CapZ, we aimed to elucidate structural and functional differences between the two CP isoforms. This allowed us to shed light on potential interaction sites with muscle and non-muscle specific components, respectively.