Myosin binding protein C (MyBP-C) is a
thick filament protein involved in the regulation of
muscle contraction.
Mutations in the gene for MyBP-C are the second most frequent cause of
hypertrophic cardiomyopathy. MyBP-C binds to
myosin with two
binding sites, one at its
C-terminus and another at its
N-terminus. The
N-terminal binding site, consisting of
immunoglobulin domains C1 and C2 connected by a flexible linker, interacts with the S2 segment of
myosin in a phosphorylation-regulated manner. It is assumed that the function of MyBP-C is to act as a tether that fixes the S1
heads in a resting position and that
phosphorylation releases the S1
heads into an active state. Here, we report the structure and binding properties of domain C1. Using a combination of
site-directed mutagenesis and
NMR interaction experiments, we identified the
binding site of domain C1 in the immediate vicinity of the S1-S2 hinge, very close to the light chains. In addition, we identified a
zinc binding site on domain C1 in close proximity to the S2
binding site. Its
zinc binding affinity (K(d) of approximately 10-20 microM) might not be sufficient for a physiological effect. However, the familial hypertrophic cardiomyopathy-related
mutation of one of the
zinc ligands,
glutamine 210 to
histidine, will significantly increase the binding affinity, suggesting that this
mutation may affect S2 binding. The close proximity of the C1
binding site to the hinge, the light chains and the S1
heads also provides an explanation for recent observations that (a) shorter fragments of MyBP-C unable to act as a tether still have an effect on the
actomyosin ATPase and (b) as to why the
myosin head positions in
phosphorylated wild-type mice and MyBP-C
knockout mice are so different: Domain C1 bound to the S1-S2 hinge is able to manipulate S1
head positions, thus influencing force generation without tether. The potentially extensive extra interactions of C1 are expected to keep it in place, while
phosphorylation dislodges the C1-C2 linker and domain C2. As a result, the
myosin heads would always be attached to a tether that has phosphorylation-dependent length regulation.
