Central helix role in the contraction‐relaxation switching mechanisms of permeabilized skeletal and smooth muscles with genetic manipulation of calmodulin

Abstract

A prominent common feature of calmodulin and troponin structures is the unusually long central helix which separates the two lobes, each containing two Ca 2 +-binding sites. To study the role of certain highly conserved residues in the helix in the contraction-relaxation switching mechanism in muscle, we measured the CaZ+-activated force of permeabilized skeletal and smooth muscles with three genetically manipulated forms of calmodulin. Mutated calmodulin was made to substitute for troponin-C in vertebrate skeletal fiber. The mutants had 1-4 deletions in the conserved cluster (positions 81 84) in the solvent-exposed region of the central helix, which also substantially shortened the helix. The force of the maximally activated fiber was found to be diminished only with the mutant in which the entire cluster Ser-81 to Glu-84 (CaMzl81 84) was deleted. All such deletions were found to be completely ineffective in blocking the Ca2+-switching process in smooth muscle strips. The results show for the first time that at least a part of the highly conserved four-residue cluster in the central helix is critical for the contraction mechanism of striated muscle. Further, the possibility is raised that the reduced length of the central helix may be a determining factor in the CaZ+-switching mechanism in fasttwitch muscle. These findings combined with the results on smooth muscle indicate diversity in the structure-function specifications for the central helix of calmodulin for different target proteins.

Crystallographic snapshots of initial steps in the collapse of the calmodulin central helix

“…Several studies have been carried out on various mutant variants of the CaM central helix, including mutation or deletion of the acidic residues (Craig et al, 1987;Persechini et al, 1989Persechini et al, , 1991Gulati et al, 1990;VanBerkum et al, 1990;Raghunathan et al, 1993;Medvedeva et al, 1995Medvedeva et al, , 1999Tabernero et al, 1997). In general, the outcome has been CaM that has been functional but with a lower affinity towards target proteins.…”

Calcium-dependent mechanisms of regulation of smooth muscle contraction

“…Further investigations in this area will focus primarily on the molecular details of the mechanism, e.g., studies of the structure-function relations of MLCK and calmodulin, using site-directed mutagenesis. This work is already well underway (see Bagchi et al 1989;Persechini et al 1989;Gulati et al 1990), but there is clearly much to be done. The molecular genetic approach must be complemented by three-dimensional structure determinations; the X-ray crystal structure of CaM has been determined (Babu et al 1988); crystallographic analysis of MLCK and its complex with ca2+-calmodulin will be a major advance.…”

Solution secondary structure of calcium‐saturated troponin C monomer determined by multidimensional heteronuclear NMR spectroscopy

“…This model has strong support from several lines of evidence, including NMR (Levine et al, 1977(Levine et al, , 1978Evans et al, 1980;Gagne et al, 1994) and site-directed mutagenesis (Fujimori et al, 1990;Grabarek et al, 1990;Gusev et al, 1991;Pearlstone et al, 1992aPearlstone et al, , 1992b. There have been several studies of TnC that have focused on the function of the central helix (Reinach & Karlsson, 1988;Xu & Hitchcock-DeGregori, 1988;Gulati et al, 1990Gulati et al, , 1993Dobrowolski et al, 1991;Babu et al, 1993;Wang et al, 1993). These studies have served to illustrate that the central helix of TnC is essential for TnC function, and that the helix is not as rigid as suggested by the crystal structure, although the long helix is extended in the model of the complex between TnC and TnI recently proposed from small-angle X-ray scattering (Olah & Trewhella, 1994).…”