Structural Basis for Capping Protein Sequestration by Myotrophin (V-1)

Abstract

Capping protein (CP) is a ubiquitously expressed, heterodimeric 62-kDa protein that binds the barbed end of the actin filament with high affinity to block further filament elongation. Myotrophin (V-1) is a 13-kDa ankyrin repeat-containing protein that binds CP tightly, sequestering it in a totally inactive complex in vitro. Here, we elucidate the molecular interaction between CP and V-1 by NMR. Specifically, chemical shift mapping and intermolecular paramagnetic relaxation enhancement experiments reveal that the ankyrin loops of V-1, which are essential for V-1/CP interaction, bind the basic patch near the joint of the ␣ tentacle of CP shown previously to drive most of the association of CP with and affinity for the barbed end. Consistently, site-directed mutagenesis of CP shows that V-1 and the strong electrostatic binding site for CP on the barbed end compete for this basic patch on CP. These results can explain how V-1 inactivates barbed end capping by CP and why V-1 is incapable of uncapping CP-capped actin filaments, the two signature biochemical activities of V-1. Capping protein (CP)3 is a ubiquitously expressed 62-kDa ␣/␤ heterodimer that binds the barbed end of the actin filament with high affinity (K d ϭ 0.1 nM) (1) to prevent further actin monomer association and dissociation, thereby limiting the extent of filament elongation in vivo (2, 3). In actin-based motility, such as that occurring in lamellipodia, new filaments are nucleated by the Arp2/3 complex to create a dendritic actin network at the leading edge. Biochemical, cell biological, and modeling studies all indicate that rapid filament capping by CP is required to sustain a dendritic network that is sufficiently branched to provide the motive force required for leading edge extension (4 -7). Consistent with its central role in actin network assembly, CP is one of only five proteins required for the reconstitution of actin-based motility in vitro (4,5,8), and cells lacking CP have profound deficiencies in actin cytoskeleton assembly (9 -13).Determination of the CP crystal structure led to the "tentacles" model of barbed end capping by CP (14). The two structurally homologous CP subunits form a central ␤-sheet that includes the bulk of the protein core, above which there are two antiparallel ␣-helices, one belonging to each subunit (14). At the end of these helices, each subunit contains a C-terminal "tentacle" which, on CP␣, is composed of an unstructured region punctuated in the middle by a short 4-residue helix, and on CP␤, it is composed of a longer amphipathic helix that protrudes from the protein core (Fig. 1). Based on crystallographic evidence, it was proposed that these C-terminal tentacles are flexible in solution, allowing them to bind and cap the barbed end. Extensive mutational studies in yeast (15) and vertebrate (1) CP that focused on the tentacles provided strong support for the tentacles model of capping. Specifically, deletion of the ␣ tentacle decreased the affinity of CP for the barbed end by 6,000-fold and its on-rate by 20-...

V-1 regulates capping protein activity in vivo

“…2B shows the binding isotherm we obtained, which yielded an affinity of ∼85 nM for the 1:1 complex. This value is comparable to the values obtained for the mouse proteins, which ranged between ∼20 and ∼50 nM (23,27,28). To confirm that D.d.…”

“…The second direct CP regulator, V-1 or myotrophin, is a ∼13-kDa ankyrin-repeat protein that binds CP 1:1 with an affinity of ∼20 nM to render CP incapable of binding the barbed end (i.e., V-1 sequesters CP) (27). Structural studies of mouse V-1 have shown that it abrogates CP's barbed end capping activity in a steric fashion by occupying CP's main barbed end interaction site (23,28). Like CARMIL, V-1 is expressed throughout most of the Eukaryotic kingdom.…”

“…Structural studies of mouse V-1 have shown that it abrogates CP's barbed end capping activity in a steric fashion by occupying CP's main barbed end interaction site (23,28). Like CARMIL, V-1 is expressed throughout most of the Eukaryotic kingdom.…”

Capping protein regulatory cycle driven by CARMIL and V-1 may promote actin network assembly at protruding edges

“…A subsequent allosteric change in CP caused by ternary complex formation then drives V-1 dissociation, yielding the CP:CAH3 complex. Importantly, the binding sites on the surface of CP for CAH3 and V-1 are nonoverlapping, consistent with the possibility that these three proteins form a ternary complex (17)(18)(19)(20)(21).…”

Regulators of actin filament barbed ends at a glance

“…Capping-protein-interacting proteins achieve this in two ways (see Edwards et al, 2014 for a review). First, some proteins such as myotrophin (also known as protein V1), which is also present at micromolar amounts, sequester free capping protein by binding to its actin-binding site with high affinity (K d =20 nM, denoted K V , Box 1; Bhattacharya et al, 2006;Zwolak et al, 2010). Thus, the concentration of free capping protein available for barbed-end capping is buffered to 5-50 nM by myotrophin (see poster).…”