The allosteric mechanism induced by protein kinase A (PKA) phosphorylation of dematin (band 4.9).
Dematin (band 4.9) is an F-actin binding and bundling protein best known for its role within red blood cells, where it both stabilizes as well as attaches the spectrin/actin cytoskeleton to the erythrocytic membrane. Here, we investigate the structural consequences of phosphorylating serine 381, a covalent modification that turns off F-actin bundling activity. In contrast to the canonical doctrine, in which phosphorylation of an intrinsically disordered region/protein confers affinity for another domain/protein, we found the converse to be true of dematin: phosphorylation of the well folded C-terminal villin-type headpiece confers affinity for its intrinsically disordered N-terminal core domain. We employed analytical ultracentrifugation to demonstrate that dematin is monomeric, in contrast to the prevailing view that it is trimeric. Next, using a series of truncation mutants, we verified that dematin has two F-actin binding sites, one in the core domain and the other in the headpiece domain. Although the phosphorylation-mimicking mutant, S381E, was incapable of bundling microfilaments, it retains the ability to bind F-actin. We found that a phosphorylation-mimicking mutant, S381E, eliminated the ability to bundle, but not bind F-actin filaments. Lastly, we show that the S381E point mutant caused the headpiece domain to associate with the core domain, leading us to the mechanism for cAMP-dependent kinase control of dematin's F-actin bundling activity: when unphosphorylated, dematin's two F-actin binding domains move independent of one another permitting them to bind different F-actin filaments. Phosphorylation causes these two domains to associate, forming a compact structure, and sterically eliminating one of these F-actin binding sites.
Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts 02118.
Lin
L
Chen
Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts 02118.
Jeffrey W
JW
Brown
Department of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 2010, Australia.
Yee-Foong
YF
Mok
Department of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 2010, Australia.
Danny M
DM
Hatters
Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts 02118. Electronic address: cjmck@bu.edu.
C James
CJ
McKnight
D008841:Actin Cytoskeleton / Q000737:chemistry; D000199:Actins / Q000737:chemistry; D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry*; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011499:Protein Processing, Post-Translational; D014461:Ultracentrifugation
D000199:Actins; C044805:DMTN protein, human; D008840:Microfilament Proteins; D017868:Cyclic AMP-Dependent Protein Kinases