Lys-65 and Glu-168 are the residues for carbodiimide-catalyzed cross-linking between the two heads of rigor smooth muscle heavy meromyosin.
We have previously demonstrated that the two heads of chicken gizzard heavy meromyosin (HMM) in a rigor complex with rabbit skeletal F-actin could be cross-linked by the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Here, we report the location of the cross-linked sites in the amino acid sequence of the HMM heavy chain. One of the cross-linked residues was identified as Glu-168 by sequencing the CN1.CN6 cross-linked peptide containing residues 1-77 (CN1) and 164-203 (CN6). This site is located close to the ATP-binding site of HMM. Since the other site was further into the amino acid sequence of CN1, another cross-linked peptide corresponding to residues 53-66 and 145-182 was isolated from the 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide-treated acto-tryptic gizzard HMM digested further by other proteolytic enzymes. The amino acid sequence of this peptide and its cyanogen bromide fragment indicated that the cross-linking occurred between Glu-168 and Lys-65. Our results suggests that these two amino acid side chains are in contact with each other in the acto-gizzard HMM rigor complex and participate in the electrostatic interaction between the two HMM heads bound to F-actin. Based on the head-to-head contact, we propose a three-dimensional model for the attachment of gizzard HMM heads to F-actin.
Onishi
H
H
Department of Structural Analysis, National Cardiovascular Center Research Institute, Osaka, Japan.
Maita
T
T
Matsuda
G
G
Fujiwara
K
K
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D003432:Cross-Linking Reagents; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000378:metabolism*; D005895:Gizzard, Avian / Q000378:metabolism; D005971:Glutamates; D018698:Glutamic Acid; D008239:Lysine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism; D015879:Myosin Subfragments / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D011817:Rabbits
D000199:Actins; D003432:Cross-Linking Reagents; D005971:Glutamates; D015879:Myosin Subfragments; D010446:Peptide Fragments; D018698:Glutamic Acid; D008239:Lysine; D005022:Ethyldimethylaminopropyl Carbodiimide