Myosin binding protein C interaction with actin: characterization and mapping of the binding site.
Myosin binding protein C (MyBPC) is a multidomain protein associated with the thick filaments of striated muscle. Although both structural and regulatory roles have been proposed for MyBPC, its interactions with other sarcomeric proteins remain obscure. The current study was designed to examine the actin-binding properties of MyBPC and to define MyBPC domain regions involved in actin interaction. Here, we have expressed full-length mouse cardiac MyBPC (cMyBPC) in a baculovirus system and shown that purified cMyBPC binds actin filaments with an affinity of 4.3 ± 1.1 μM and a 1:1 molar ratio with regard to an actin protomer. The actin binding by cMyBPC is independent of protein phosphorylation status and is not significantly affected by the presence of tropomyosin and troponin on the actin filament. In addition, cMyBPC-actin interaction is not modulated by calmodulin. To determine the region of cMyBPC that is responsible for its interaction with actin, we have expressed and characterized five recombinant proteins encoding fragments of the cMyBPC sequence. Recombinant N-terminal fragments such as C0-C1, C0-C4, and C0-C5 cosediment with actin in a linear, nonsaturable manner. At the same time, MyBPC fragments lacking either the C0-C1 or C0-C4 region bind F-actin with essentially the same properties as full-length protein. Together, our results indicate that cMyBPC interacts with actin via a single, moderate affinity site localized to the C-terminal region of the protein. In contrast, certain basic regions of the N-terminal domains of MyBPC may act as small polycations and therefore bind actin via nonspecific electrostatic interactions.
Department of Physiology, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA. rybakova@physiology.wisc.edu
Inna N
IN
Rybakova
Marion L
ML
Greaser
Richard L
RL
Moss
D008841:Actin Cytoskeleton / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000199:Actins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D010449:Peptide Mapping; D010766:Phosphorylation / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D000099163:Myosin Binding Protein C
D000199:Actins; D002147:Calmodulin; D002352:Carrier Proteins; D000099163:Myosin Binding Protein C