Abstract 11055: Determination of the Critical Residues Responsible for Cardiac Myosin Binding Protein C Interaction With Actin and Their Structural Importance
Rationale: Myosin binding protein C’s (MyBP-C) interaction with the actin thin filament has been supported by numerous biochemical and modeling studies but the data are contradictory with respect to the actual domains mediating the proteins’ interaction. We adopted an independent genetic approach to systematically define cardiac MyBP-C’s (cMyBP-C) critical domain(s) and specific residues responsible for actin binding.
Objectives: To define cMyBP-C’s structural domains that are necessary and sufficient to mediate interactions with actin and the head region of the myosin heavy chain (S2-MyHC) and determine the structural significance of these interactions.
Methods and Results: We systemically defined the actin binding site(s) using genetically-based yeast-2-hybrid experiments. We confirmed that cMyBP-C’s C1 and m domains interact with actin and at the same time, cMyBP-C’s m domain interacts with S2-MyHC. We were able to identify the critical actin binding residues within both C1 and m domains that are essential for actin binding. We also were able to distinguish them from the critical residues in the m domain that were responsible for mediating S2-MyHC binding. We also used the yeast-3-hybrid system to determine whether cMyBP-C’s binding to actin influenced its ability to bind to S2-MyHC and tested the converse as well. To define the structural significance of actin/cMyBP-C interaction, we silenced the endogenous cMyBP-C in neonatal rat cardiomyocytes using cMyBP-C siRNA and replaced the endogenous protein with cMyBP-C containing a mutated actin binding site. The mutated cMyBP-C failed to incorporate normally into the sarcomere and disrupted normal sarcomeric structure.
Conclusions: Residues responsible for actin and S2-MyHC binding were defined and shown to be present in both distinct and partially overlapping domains. Expression of an actin binding-deficient cMyBP-C resulted in abnormal cytosolic distribution of the protein, indicating that interaction with actin is essential for formation and/or maintenance of normal cMyBP-C sarcomeric distribution.
- © 2012 by American Heart Association, Inc.