| 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. CP is required for actin-based motility and can be regulated in two main ways. CP can be sequestered in a totally inactive complex in vitro by myotrophin/V-1, a 13 kDa, ankyrin repeat-containing protein. Alternatively, CP can be removed from the barbed end by CARMIL in a process known as "uncapping". Here I elucidate the molecular interactions responsible for sequestering and uncapping by nuclear magnetic resonance (NMR). Specifically, chemical shift mapping and intermolecular paramagnetic relaxation enhancement experiments reveal that the ankyrin loops of V-1 bind the basic patch near the joint of CP's alpha tentacle, that was shown previously to drive most of CP's association with and affinity for the barbed end. Consistently, site-directed mutagenesis of CP shows that the strong electrostatic binding site for CP on the barbed end and V-1 compete for this basic patch on CP. These results can explain how V-1 completely inactivates barbed end capping by CP and why V-1 is incapable of uncapping CP-capped actin filaments, its two signature biochemical activities. Likewise, the structure of CP bound to CAH3a/b, the minimal sufficient uncapping domain of CARMIL, determined in this study offers explanation of the signature activities of CAH3: its ability to uncap previously capped filaments and the weak capping activity of the CP: CAH3 complex. Specifically, I show that the CAH3a sub-domain is required for its high affinity, electrostatic interaction with a previously unidentified site on CP, opposite its actin binding surface. This interaction orients the CAH3b sub-domain, required for potent anti-CP activity, directly adjacent to the basic patch on CP, disrupting its interaction with the barbed end. Determination of the mechanisms of V-1 and CAH3a/b binding to CP together represent two paradigms for CP regulation where disruption of the interaction of CP's basic patch with the barbed end is a common regulatory theme. |
