An unusual transduction pathway in human tonic smooth muscle myosin.
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.
Halstead
Miriam F
MF
Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota, USA.
Ajtai
Katalin
K
Penheiter
Alan R
AR
Spencer
Joshua D
JD
Zheng
Ye
Y
Morrison
Emma A
EA
Burghardt
Thomas P
TP
D000255:Adenosine Triphosphate / Q000737:chemistry; D016367:Baculoviridae / Q000378:metabolism; D001665:Binding Sites; D017931:DNA Primers / Q000737:chemistry; D020224:Expressed Sequence Tags; D006801:Humans; D008954:Models, Biological; D008956:Models, Chemical; D009130:Muscle, Smooth / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009218:Myosins / Q000737:chemistry / Q000378:metabolism*; D010649:Phenylalanine / Q000737:chemistry; D011487:Protein Conformation; D020033:Protein Isoforms; D011506:Proteins / Q000737:chemistry; D015398:Signal Transduction
D017931:DNA Primers; D020033:Protein Isoforms; D011506:Proteins; D010649:Phenylalanine; D000255:Adenosine Triphosphate; D009218:Myosins
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