On the tryptophan residue of smooth muscle myosin that responds to binding of nucleotide.
Initially, we asked which (of 10) smooth muscle myosin head residues responds to MgADP or MgATP binding with enhanced fluorescence emission (Trp-441 and Trp-512 were leading candidates)? To decide, we prepared sham-mutated smooth muscle heavy meromyosin (HMM), W441F HMM, and W512F HMM. On adding MgATP, emission of wild-type and W441F HMMs increased by 25-27%, but that of W512F HMM by 5%. So, in myosin, 512 is the "sensitive Trp." Unexpectedly, properties of W512F HMM [elevated Ca(2+)-ATPase, depressed EDTA (K(+))-ATPase, no regulation of its basal or actin-activated Mg(2+)-ATPase by phosphorylation of its "regulatory" light chain, limited actin activation, and inability to move actin filaments in a motility assay] are strikingly like those of smooth muscle myosin reacted at Cys-717 with thiol reagent. From crystallography-based [Houdusse, A., Kalabakis, V. N., Himmel, D., Szent-Györgyi, A. G. & Cohen, C. (1999) Cell 97, 459-470] simulations, we found that in wild-type HMM with MgADP added, Trp-512 is in a "hydrophobic pocket," but that pocket becomes distorted in W512F HMM. We think that there is a "path of influence" from 512 to 717 to the active site. We suggest that the mutational changes at 512 are transmitted along this path to Cys-717, where they induce changes similar to those caused by reacting wild-type HMM with thiol reagent.
Onishi
H
H
Department of Structural Analysis, National Cardiovascular Center Research Institute, Fujishiro-dai, Suita, Osaka 565-8565, Japan. honishi@ri.ncvc.go.jp
Konishi
K
K
Fujiwara
K
K
Hayakawa
K
K
Tanokura
M
M
Martinez
H M
HM
Morales
M F
MF
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D009130:Muscle, Smooth / Q000378:metabolism*; D016296:Mutagenesis; D009218:Myosins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D009218:Myosins