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Phosphorylation-dependent structural changes in the regulatory light chain domain of smooth muscle heavy meromyosin
1Department of Biochemistry and Biophysics, Washington State University, Pullman, Washington 99164-4660, USA.
Abstract:
Smooth muscle heavy meromyosin, a double-headed proteolytic fragment of myosin lacking the COOH-terminal two-thirds of the tail, has been shown previously to be regulated by phosphorylation. To examine phosphorylation-dependent structural changes near the head-tail junction, we prepared five well regulated heavy meromyosins containing single-cysteine mutants of the human smooth muscle regulatory light chain labeled with the photocross-linking reagent, benzophenone-iodoacetamide. For those mutants that generated cross-links, only one type of cross-linked species was observed, a regulatory light chain dimer. Irradiated mutants fell into two classes. First, for Q15C, A23C, and wild type (Cys-108), a regulatory light chain dimer was formed for dephosphorylated but not thiophosphorylated heavy meromyosin. These data provide direct chemical evidence that in the dephosphorylated state, Gln-15, Ala-23, and Cys-108 on one head are positioned near (within 8.9 A) the regulatory light chain of the partner head and that thiophosphorylation abolishes proximity. This behavior was also observed for the Q15C mutant on a truncated heavy meromyosin lacking both catalytic domains. For the actin-heavy meromyosin complex, cross-links were formed in both de- and thiophosphorylated states. S59C and T134C mutants were in a second mutant class, where regulatory light chain dimers were not detected in dephosphorylated or thiophosphorylated heavy meromyosin, suggesting positions outside the region of interaction of the regulatory light chains.
Insights
Phosphorylation regulates smooth muscle heavy meromyosin structure. Dephosphorylated states show regulatory light chain proximity, which thiophosphorylation disrupts, revealing key structural changes near the head-tail junction.
Area of Science:
- Biochemistry
- Molecular Biology
- Muscle Physiology
Background:
- Smooth muscle heavy meromyosin (HMM) is a myosin fragment regulated by phosphorylation.
- Understanding phosphorylation-dependent structural changes in HMM is crucial for muscle function.
Purpose of the Study:
- To investigate structural changes near the head-tail junction of smooth muscle HMM in response to phosphorylation.
- To identify specific residues involved in these phosphorylation-dependent structural rearrangements.
Main Methods:
- Preparation of five well-regulated HMMs with single-cysteine mutants of the human smooth muscle regulatory light chain.
- Labeling mutants with benzophenone-iodoacetamide and subsequent photocross-linking.
- Analysis of cross-linked species, specifically regulatory light chain dimers, using dephosphorylated and thiophosphorylated HMM.
Main Results:
- Two classes of mutants were identified based on cross-linking patterns.
- Class 1 (Q15C, A23C, wild type Cys-108) formed regulatory light chain dimers in dephosphorylated but not thiophosphorylated HMM, indicating proximity changes.
- Class 2 (S59C, T134C) did not form dimers, suggesting their positions are outside the interaction region.
Conclusions:
- Thiophosphorylation abolishes the proximity of specific regulatory light chain residues (Gln-15, Ala-23, Cys-108) to the partner head's regulatory light chain.
- These findings provide direct chemical evidence for phosphorylation-induced structural rearrangements near the head-tail junction of smooth muscle HMM.