The putative actin-binding interface of myosin is separated by a large cleft that extends into the base of the nucleotide binding pocket, suggesting that it may be important for mediating the nucleotide-dependent changes in the affinity for myosin on actin. We have genetically engineered a truncated version of smooth muscle myosin containing the motor domain and the essential light chain-binding region (MDE), with a single tryptophan residue at position 425 (F425W-MDE) in the actin-binding cleft. Steady-state fluorescence of F425W-MDE demonstrates that Trp-425 is in a more solvent-exposed conformation in the presence of MgATP than in the presence of MgADP or absence of nucleotide, consistent with closure of the actin-binding cleft in the strongly bound states of MgATPase cycle for myosin. Transient kinetic experiments demonstrate a direct correlation between the rates of strong actin binding and the conformation of Trp-425 in the actin-binding cleft, and suggest the existence of a novel conformation of myosin not previously seen in solution or by x-ray crystallography. Thus, these results directly demonstrate that: 1) the conformation of the actin-binding cleft mediates the affinity of myosin for actin in a nucleotide-dependent manner, and 2) actin induces conformational changes in myosin required to generate force and motion during muscle contraction.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology