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

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 Glu 334 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-1⅐MgAMP-PNP (5-adenylyl imidodiphosphate) and on the weak binding of S-1⅐MgADP⅐P i to actin. We found that an inhibitory concentration of calponin decreased the binding of S-1⅐MgAMP-PNP to actin but had no effect on the binding of S-1⅐MgADP⅐P i . Similar results were obtained with skeletal muscle and smooth muscle S-1. In competition experiments calponin was found to displace S-1⅐MgAMP-PNP and S-1⅐MgADP but not S-1⅐MgADP⅐P i 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.

3-D image reconstruction of reconstituted smooth muscle thin filaments containing calponin: Visualization of interactions between F-actin and Calponin

“…Although the content of calponin in vivo is considerable ($one calponin: seven actin monomers; Lehman & Kaminer, 1984;Takahashi et al, 1986;Lehman, 1991), most recent biochemical studies suggest that still higher levels of calponin are needed to inhibit actomyosin ATPase (see Gimona & Small, 1996) and that this inhibition may be low at physiological salt concentrations under in vitro conditions (EL- Mezgueldi et al, 1996). In addition, whereas calponin is approximately equimolar with tropomyosin in vivo, inhibition of actomyosin ATPase by calponin is independent of tropomyosin in vitro (Winder & Walsh, 1990).…”

Regulation of Shortening Velocity by Calponin in Intact Contracting Smooth Muscles

“…The three dimensional recon-struction of the actin-calponin structure demonstrates that calponin appears to contact with actin near both the N-terminus and residues close to the C-terminus (12). It has been suggested that the interaction of calponin with the glutamate residue at the C-terminus of actin molecule inhibits weak to strong transition of the actin-myosin binding, and thus play a role in the reduction of crossbridge cycling rate (13). Exogenous calponin inhibits both maximum shortening velocity (14,15) and isometric tension (16 -18) in permeabilized smooth muscle.…”

Simultaneous Interaction of Actin With a‐Actinin and Calponin

“…As mentioned earlier, calponin and a -actinin occupy overlapping sites on actin monomers and their spatial organization on actin laments is very similar (6)(7)(8)(9). In addition, both calponin and a -actinin contain one or two calponin homology domains (CH domains) that, at least in the case of a -actinin, are directly involved in the binding of these ABPs to actin (7,17).…”

“…Indeed, the a -actinin-binding sequences on actin are located within residues 112-125 (or 87-96) and residues 360-372 (or 348-355) of actin subdomain 1 (6,7). The biochemical data indicate that three sequences of actin monomer (residues 1-226, 326-355, and 373-375) are implicated in calponin binding (8). Three-dimensional reconstruction of actin laments containing calponin shows that calponin contacts residues 349-352 of one actin monomer and residues 92-95 (and 43-48) on the axially neighboring actin monomer (9).…”