The Effects of Smooth Muscle Calponin on the Strong and Weak Myosin Binding Sites of F-actin

Mohammed El-Mezgueldi,Steven B Marston

doi:10.1074/jbc.271.45.28161

Abstract

We have investigated the mechanism of inhibition of the actomyosin MgATPase by the smooth muscle protein calponin. We have shown previously the specific interaction of calponin with Glu334 of actin (EL-Mezgueldi, M., Fattoum, A., Derancourt, J., and Kassab, R. (1992) J. Biol. Chem. 267, 15943-15951). This residue is within the sequence 332-334, which has been proposed to be an important part of the strong myosin binding site (Rayment, I., Holden, H. M., Whittaker, M., Yohn, C. B., Lorenz, M., Holmes, K. C., and Milligan, R. A. (1993) Science 261, 58-65). Therefore, we suggested that calponin will affect the strong binding actin-myosin interaction. To test this hypothesis we have investigated the effect of calponin on the strong binding of S-1.MgAMP-PNP (5'-adenylyl imidodiphosphate) and on the weak binding of S-1.MgADP.Pi to actin. We found that an inhibitory concentration of calponin decreased the binding of S-1. MgAMP-PNP to actin but had no effect on the binding of S-1.MgADP.Pi. Similar results were obtained with skeletal muscle and smooth muscle S-1. In competition experiments calponin was found to displace S-1. MgAMP-PNP and S-1.MgADP but not S-1.MgADP.Pi from the actin filament. S-1 displaced calponin from actin in the rigor state, in the presence of MgADP, and in the presence of MgAMP-PNP. We conclude that calponin inhibits the actin activated S-1 ATPase by blocking a strong S-1 binding site on actin and does not block the weak binding site.

Highlights

Three actin-binding proteins, troponin I, caldesmon, and calponin, have been identified as capable of inhibiting the activation of myosin ATPase by muscle thin filaments [1,2,3]
We found that an inhibitory concentration of calponin decreased the strong binding of S-11⁄7MgAMP-PNP to actin
To define whether the shared site on actin corresponds only to the strong myosin binding site or includes the weak binding site we investigated the relationship between calponin inhibition and the binding of S-11⁄7MgADP1⁄7Pi to actin

Summary

Introduction

Three actin-binding proteins, troponin I, caldesmon, and calponin, have been identified as capable of inhibiting the activation of myosin ATPase by muscle thin filaments [1,2,3]. In contrast Lys and the NH2 terminus of actin, proposed to be part of the weak binding site of the myosin head (S-1), have been excluded from calponin binding [11] From these structural studies (summarized in Fig. 7) it is reasonable to suggest that calponin would inhibit the strong binding actin-myosin interaction and that calponin and the S-1 myosin head would compete for a common binding site on actin. To test this hypothesis we have investigated the effect of calponin on the binding of S-1 to actin in the absence or the presence of MgAMP-PNP, MgADP, or MgATP. We conclude that calponin binding to actin affects the strong binding of myosin to actin but has no effect on the weak binding

Methods

Results

Conclusion