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

Adam Zwolak,Ikuko Fujiwara,John A Hammer,Nico Tjandra

doi:10.1074/jbc.m110.135848

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 alpha 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.

Highlights

The results shown here explain why V-1 cannot uncap previously capped barbed ends
We demonstrated that CP mutations that reduce its in the peak intensities of residues in the ␤ tentacle upon V-1 affinity for V-1 reduce its affinity for the barbed end, pro- binding, indicating no change in their dynamics
Kd values between V-1/CP were calculated from fluorescence anisotropy data, and those for CP binding to barbed ends were calculated from pyrene actin fluorescence experiments

Summary

Introduction

The results shown here explain why V-1 cannot uncap previously capped barbed ends. In much the same way V-1 is able to bind and occlude the high affinity binding site of CP for the barbed end, and the barbed end occludes the V-1-binding site on CP, rendering V-1 unable to bind and uncap the barbed end. Our results explain the two signature biochemical activities of V-1 as follows: its ability to completely sequester CP, and its inability to remove CP from the barbed end. We showed that both V-1 and the barbed end compete for many of the same residues on the basic patch of CP, and binding of either V-1 or the barbed end to CP renders the other unable to bind. The atomic coordinates for the 10 lowest energy structures of the CP-V-1 complex have been deposited in the Protein Data Bank

Results

Discussion

Conclusion