A FERM domain autoregulates Drosophila myosin 7a activity
| Autor: | Yi Yang, Matt L. Walker, Michael Chen, James R. Sellers, Peter J. Knight, Verl Siththanandan, Thomas G. Baboolal, Michelle Peckham |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2009 |
| Předmět: |
Protein Folding
Myosin light-chain kinase Insecta Protein Conformation macromolecular substances Biology Myosins SH3 domain Cell Line Myosin head Protein structure Transduction Genetic Myosin Animals Drosophila Proteins Actin Coiled coil Adenosine Triphosphatases Multidisciplinary FERM domain Dose-Response Relationship Drug Biological Sciences Molecular biology Actins Protein Structure Tertiary Kinetics Myosin VIIa Biophysics Protein Binding |
| Popis: | Full-length Drosophila myosin 7a (myosin 7a-FL) has a complex tail containing a short predicted coiled coil followed by a MyTH4-FERM domain, an SH3 domain, and a C-terminal MyTH4-FERM domain. Myosin 7a-FL expressed in Sf9 cells is monomeric despite the predicted coiled coil. We showed previously that Subfragment-1 (S1) from this myosin has MgATPase of V max ≈ 1s −1 and K ATPase ≈ 1 μM actin. We find that myosin 7a-FL has V max similar to S1 but K ATPase ≈ 30 μM. Thus, at low actin concentrations (5 μM), the MgATPase of S1 is fully activated, whereas that of myosin 7a-FL is low, suggesting that the tail regulates activity. Electron microscopy of myosin 7a-FL with ATP shows the tail is tightly bent back against the motor domain. Myosin 7a-FL extends at either high ionic strength or without ATP, revealing the motor domain, lever, and tail. A series of C-terminal truncations show that deletion of 99 aa (the MyTH7 subdomain of the C-terminal FERM domain) is sufficient to abolish bending, and the K ATPase is then similar to S1. This region is highly conserved in myosin 7a. We found that a double mutation in it, R2140A-K2143A, abolishes bending and reduces K ATPase to S1 levels. In addition, the expressed C-terminal FERM domain binds actin with K d ≈ 30 μM regardless of ATP, similar to the K ATPase value for myosin 7a-FL. We propose that at low cellular actin concentrations, myosin 7a-FL is bent and inactive, but at high actin concentrations, it is unfolded and active because the C-terminal FERM domain binds to actin. |
| Databáze: | OpenAIRE |
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