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Phenylglyoxal reveals phosphorylation-dependent difference in the conformation of Acanthamoeba myosin II active site
1Department of Muscle Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland. jolanta@nencki.gov.pl
Abstract:
Acanthamoeba myosin II is regulated in an unique way by phosphorylation of three serine residues located within nonhelical tailpiece of the rod domain. Phosphorylation inhibits functions associated with the NH2-terminal motor domain, i.e., actin-activated activity and ability to move actin filaments. Number of data indicate functional communication between these distant domains. In this work, effect of modification of arginine residues with phenylglyoxal on the Ca2+-ATPase activity and susceptibility to endoproteinase ArgC cleavage of monomeric phospho- and dephosphomyosin II has been investigated. Upon the phenylglyoxal treatment the activity of dephosphomyosin II was decreasing faster that the activity of phosphomyosin. The modification also affected the proteolytic fragmentation of phospho- and dephosphomyosin II: the cleavage of heavy chain was further inhibited for phosphomyosin and enhanced for dephosphomyosin with a concomitant exposure of an additional cleavage site within the head domain. No difference in the quantity of modified arginines was observed. These results indicate a difference between the conformation of active sites of phospho- and dephosphomyosin II.
Insights
Phosphorylation uniquely regulates Acanthamoeba myosin II. Modifying arginine residues with phenylglyoxal revealed distinct conformational differences between phosphorylated and dephosphorylated myosin II states, impacting enzymatic activity and protein structure.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Acanthamoeba myosin II is regulated by phosphorylation of serine residues in its tailpiece.
- Phosphorylation inhibits motor domain functions, suggesting domain communication.
- The role of arginine residues in myosin II regulation is not fully understood.
Purpose of the Study:
- To investigate the effect of arginine modification on Acanthamoeba myosin II.
- To analyze changes in Ca2+-ATPase activity and susceptibility to endoproteinase ArgC cleavage.
- To compare the conformations of monomeric phosphorylated and dephosphorylated Acanthamoeba myosin II.
Main Methods:
- Chemical modification of arginine residues using phenylglyoxal.
- Assay of Ca2+-ATPase activity.
- Proteolytic digestion with endoproteinase ArgC.
- Analysis of fragmentation patterns.
Main Results:
- Phenylglyoxal treatment decreased dephosphomyosin II activity faster than phosphomyosin II.
- Modification differentially affected proteolytic fragmentation, inhibiting cleavage in phosphomyosin II and enhancing it in dephosphomyosin II.
- An additional cleavage site was exposed in the head domain of dephosphomyosin II.
- No difference was observed in the quantity of modified arginine residues.
Conclusions:
- Arginine modification reveals distinct conformational states between phospho- and dephosphomyosin II.
- These conformational differences impact enzymatic activity and protein structure.
- The study highlights the complex regulation of Acanthamoeba myosin II.