A crystallographic study reveals the structural basis for regulation by two different inhibitors of the actin capping protein, a critical factor controlling actin-driven cell motility.
The actin capping protein (CP) tightly binds to the barbed end of actin filaments, thus playing a key role in actin-based lamellipodial dynamics. V-1 and CARMIL proteins directly bind to CP and inhibit the filament capping activity of CP. V-1 completely inhibits CP from interacting with the barbed end, whereas CARMIL proteins act on the barbed end-bound CP and facilitate its dissociation from the filament (called uncapping activity). Previous studies have revealed the striking functional differences between the two regulators. However, the molecular mechanisms describing how these proteins inhibit CP remains poorly understood. Here we present the crystal structures of CP complexed with V-1 and with peptides derived from the CP-binding motif of CARMIL proteins (CARMIL, CD2AP, and CKIP-1). V-1 directly interacts with the primary actin binding surface of CP, the C-terminal region of the α-subunit. Unexpectedly, the structures clearly revealed the conformational flexibility of CP, which can be attributed to a twisting movement between the two domains. CARMIL peptides in an extended conformation interact simultaneously with the two CP domains. In contrast to V-1, the peptides do not directly compete with the barbed end for the binding surface on CP. Biochemical assays revealed that the peptides suppress the interaction between CP and V-1, despite the two inhibitors not competing for the same binding site on CP. Furthermore, a computational analysis using the elastic network model indicates that the interaction of the peptides alters the intrinsic fluctuations of CP. Our results demonstrate that V-1 completely sequesters CP from the barbed end by simple steric hindrance. By contrast, CARMIL proteins allosterically inhibit CP, which appears to be a prerequisite for the uncapping activity. Our data suggest that CARMIL proteins down-regulate CP by affecting its conformational dynamics. This conceptually new mechanism of CP inhibition provides a structural basis for the regulation of the barbed end elongation in cells.
Actin is a ubiquitous eukaryotic protein that polymerizes into bidirectional filaments and plays essential roles in a variety of biological processes, including cell division, muscle contraction, neuronal development, and cell motility. The actin capping protein (CP) tightly binds to the fast-growing end of the filament (the barbed end) to block monomer association and dissociation at this end, thus acting as an important regulator of actin filament dynamics in cells. Using X-ray crystallography, we present the atomic structures of CP in complex with fragments of two inhibitory proteins, V-1 and CARMIL, to compare the modes of action of these two regulators. The structures demonstrate that V-1 directly blocks the actin-binding site of CP, thereby preventing filament capping, whereas CARMIL functions in a very different manner. Detailed comparison of several CP structures revealed that CP has two stable domains that are continuously twisting relative to each other. CARMIL peptides were found to bind across the two domains of CP on a surface distinct from its actin binding sites. We propose that CARMIL peptides attenuate the binding of CP to actin filaments by suppressing the twisting movement required for tight barbed end capping. Our comparative structural studies therefore have revealed substantial insights in the variety of mechanisms by which different actin regulatory factors function.