Actin assembly and disassembly are important for directing the shape and movement of cells and tissues during normal development and physiological function as well as for aiding in the motility of some pathogens upon infection.1, 2 Actin filaments grow and shrink by gaining and losing monomeric subunits from their two ends – barbed and pointed. Actin filaments near membranes can push on membranes by growth from free barbed ends, following nucleation by Arp2/3 complex. Actin and Arp2/3-based assembly and motility can require contributions from actin capping protein (CP), ADF/cofilin, profilin and thymosin.2, 3 Regulation of filament polymerization and depolymerization via actin capping protein (CP) and its binding partners is essential to provide the force and energy for movements of many cellular membranes, including vesicles and the plasma membrane.

CP is expressed in a wide range of organisms, from budding yeast to humans.4 In vertebrates, CP is as an α/β heterodimer, and each subunit occurs as three distinct isoforms. The α1, α2, and α3 isoforms are expressed from three different genes.5 The β isoforms are all expressed from one gene and differ by alternative splicing.6 α3 and β3 are expressed exclusively in male germ cells.7, 8 α1and α2 are expressed in a wide variety of cells and tissues, at different ratios.9 The β1 isoform is expressed in striated muscle and is located at Z-lines, where it binds the barbed end of the sarcomeric thin filament.6 CP purified from skeletal muscle contains the β1 isoform, which is often called “CapZ” because of its Z-line localization.10 The β2 isoform is predominantly expressed in non-muscle cells and tissues; β2 is also expressed in striated muscle cells, and it does not localize to Z lines.6, 11 The molecular basis for their differing localization in striated muscle is not known.

The CP heterodimer adopts a mushroom-like shape.4 The mushroom cap is comprised of interlaced β sheets and α helices. The mushroom stalk protrudes perpendicularly from the bottom of the cap surface and is composed of α helices (Figure 1). The top surface of the mushroom cap binds to filament barbed ends, preventing association and dissociation of actin subunits.4

A number of biomolecules bind directly to CP and regulate the interaction of CP with barbed ends. Those molecules comprise polyphosphoinositides, the protein V-1/myotrophin, and a diverse set of proteins with CP-interacting (CPI) motifs, including the CARMIL, CKIP and WASHCAP (FAM21) protein families.4, 15, 16 In cytoplasm, CP and V-1 are both present at high concentration in micromolar quantities, they both diffuse freely, and they bind tightly to each other with nanomolar affinity.17 In contrast, CPI-motif proteins are present in far smaller amounts, and they are generally targeted to specific membrane locations.15, 16 The effect of CPI-motif binding to CP includes weakening the binding affinity of CP for V-1.18, 19 These observations raised the possibility, proposed by Hammer and colleagues,18 that CPI-motif proteins activate CP locally at a membrane by promoting dissociation of the CP inhibitor V-1. The binding of CP to V-1, F-actin, and CPI motifs, are linked molecular processes.19 V-1 binds directly to the cap region of CP and sterically blocks the site required for binding to actin-filament barbed ends.17, 18, 20, 21, 22 Structures of co-complexes show that the binding sites for F-actin and V-1 overlap extensively, but not completely.19 In contrast, the binding sites for CPI-motif proteins are instead located in the stalk region of CP and are spatially different from those for F-actin and V-1 (Figure 1).15, 16, 21, 22 Therefore, CPI-motif proteins inhibit the binding of CP to actin filament barbed ends and to V-1 by an allosteric mechanism.15, 16, 21, 22

The physical basis of the allosteric regulation and linkage between these two distinct sites was investigated with hydrogen–deuterium exchange mass spectrometry (HDX-MS).23 The HDX-MS study revealed that the solvent accessibility of CP was altered at both binding sites when either a CPI-motif peptide or V-1 was added to CP in solution. The findings indicate that interaction with either ligand can induce conformational changes to sites within CP that are distant from the ligand binding surface. Available crystal structures of co-complexes show only slight differences in the conformations of CP when complexed with a CPI-motif peptide or V-121, 24, 25; however, rather large biochemical effects are observed when CP is bound to either ligand.18, 19, 21 Together, these findings are consistent with linked changes in the conformation and/or the structural dynamics of the two sites. The atomistic details of the linkage are not clear because the spatial resolution of HDX-MS is limited to proteolytic peptides, and crystal structures do not capture potential dynamics within different states.

Here, to advance our understanding of the allosteric linkage mechanism that regulates CP function, we sought evidence at the atomic level for changes in the conformation or dynamics of CP in solution, by comparing free CP (Apo-CP) with CP complexed with either a CPI-motif peptide or V-1. We chose to use CP composed of the α1 and β2 isoforms because this is commonly found in non-muscle cells. We performed confocal single-molecule Förster resonance energy transfer (FRET) of molecules in solution, which allows one to quantify conformational changes in the protein alone and in complex with regulators. We complemented the experimental results with molecular dynamics (MD) analyses of the conformation of CP in those settings, to provide an atomistic description of the structural changes identified by single-molecule FRET.