An Unusual Transduction Pathway in Human Tonic Smooth Muscle Myosin

Abstract

The motor protein myosin binds actin and ATP, producing work by causing relative translation of the proteins while transducing ATP free energy. Smooth muscle myosin has one of four heavy chains encoded by the MYH11 gene that differ at the C-terminus and in the active site for ATPase due to alternate splicing. A seven-amino-acid active site insert in phasic muscle myosin is absent from the tonic isoform. Fluorescence increase in the nucleotide sensitive tryptophan (NST) accompanies nucleotide binding and hydrolysis in several myosin isoforms implying it results from a common origin within the motor. A wild-type tonic myosin (smA) construct of the enzymatic head domain (subfragment 1 or S1) has seven tryptophan residues and nucleotide-induced fluorescence enhancement like other myosins. Three smA mutants probe the molecular basis for the fluorescence enhancement. W506+ contains one tryptophan at position 506 homologous to the NST in other myosins. W506F has the native tryptophans except phenylalanine replaces W506, and W506+(Y499F) is W506+ with phenylalanine replacing Y499. W506+ lacks nucleotide-induced fluorescence enhancement probably eliminating W506 as the NST. W506F has impaired ATPase activity but retains nucleotide-induced fluorescence enhancement. Y499F replacement in W506+ partially rescues nucleotide sensitivity demonstrating the role of Y499 as an NST facilitator. The exceptional response of W506 to active site conformation opens the possibility that phasic and tonic isoforms differ in how influences from active site ATPase propagate through the protein network.

Human Tonic and Phasic Smooth Muscle Myosin Isoforms Are Unresponsive to the Loop 1 Insert

“…A new protein purification protocol was developed to better remove contaminants potentially perturbing quantitative fluorescence increments and applied to all constructs. We found that WT sSMA and sSMB proteins both have a significant fluorescence increment of ~18% with ATP binding to the active site as reported previously for sSMA and where we showed that the increment is not due to the NST at W506 [ 8 ]. Mutant sSMA-W506 and the new mutant construct sSMB-W506 have 6 of 7 tryptophans replaced with phenylalanine or methionine.…”

“…Short S1 has ELC but not RLC binding sites and the molecule is a monomer containing MHC and ELC. Without RLC, short S1 is always “on” for actin activation [ 8 ].…”

“…The i7 deletion inhibition of Pi release suggests influencing regions of myosin beyond the active site and near switch 2 where it can also affect the relay helix and the NST. We proposed that the i7 deletion in SMA influences the energy transduction machinery in the relay helix because our human short S1 SMA construct, sSMA, lacked NST sensitivity to ATP hydrolysis [ 8 ]. sSMA is the only myosin isoform known to lack NST sensitivity to ATP hydrolysis.…”

Myosin dynamics on the millisecond time scale

“…The simulation identifies the Y503 phenol side group and backbone amide groups from W510, E511, K709, and G710 as the main electron exchange partners. We have recently directly confirmed the W510 and Y503 phenol side group interaction leading to W510 fluorescence intensity perturbation using site directed mutagenesis [86].…”

“…The simulation identifies the Y503 phenol side group and backbone amide groups from W510, E511, K709, and G710 as the main electron exchange partners. The influence of the Y503 phenol side group on W510 fluorescence sensitivity to nucleotide or trapped nucleotide analogs was recently confirmed experimentally using site directed mutagenesis of a smooth muscle myosin construct [86]. K SV shows acrylamide quenching is most effective in apo S1 and least effective in the presence of nucleotide or trapped nucleotide analogs reflecting maximal and minimal W510 ASA in the apo and trapped nucleotide analog S1.…”