The N-terminal Lobes of Both Regulatory Light Chains Interact with the Tail Domain in the 10 S-inhibited Conformation of Smooth Muscle Myosin

Abstract

In the presence of ATP, unphosphorylated smooth muscle myosin can form a catalytically inactive monomer that sediments at 10 Svedbergs (10 S). The tail of 10 S bends into thirds and interacts with the regulatory domain. ADP-P i is "trapped" at the active site, and consequently the ATPase activity is extremely low. We are interested in the structural basis for maintenance of this off state. Our prior photocross-linking work with 10 S showed that tail residues 1554 -1583 are proximal to position 108 in the C-terminal lobe of one of the two regulatory light chains (Olney, J. J., Sellers, J. R., and Cremo, C. R. (1996) J. Biol. Chem. 271, 20375-20384). These data suggested that the tail interacts with only one of the two regulatory light chains. Here we present data, using a photocross-linker on position 59 on the N-terminal lobe of the regulatory light chain (RLC), demonstrating that both regulatory light chains of a single molecule can cross-link to the light meromyosin portion of the tail. Mass spectrometric data show four specific cross-linked regions spanning residues 1428 -1571 in the light meromyosin portion of the tail, consistent with cross-linking two RLC to one light meromyosin. In addition, we find that position 59 can cross-link internally to residues 42-45 within the same RLC subunit. The internal cross-link only forms in 10 S and not in unphosphorylated heavy meromyosin (lacking the light meromyosin), suggesting a structural rearrangement within the RLC attributed to the interaction of the tail with the head.

Direct evidence for functional smooth muscle myosin II in the 10S self-inhibited monomeric conformation in airway smooth muscle cells

“…This evidence however is insufficient to directly demonstrate that 10S exists, as other forms of SMM, such as small filaments would likely behave in a similar manner. We characterized Peptide A that destabilized the 10S conformation by a mechanism that is consistent with the extensive structural information about the head-tail interaction in 10S SMM (8,24,25) (Fig. 4A).…”

“…4A) derived from the sequence of the SMM HC immediately N-terminal of the head-tail junction (position of the invariant proline) including the RLC binding motif. We have previously shown that in the 10S conformation both the N-and C-terminal lobes of the RLC interact with Bend 2 in the light meromyosin (LMM) region of the SMM tail (24,25) (Fig. 4A).…”

The kinesin-1 motor protein is regulated by a direct interaction of its head and tail

“…Sample preparation and trypsin digestion procedures for mass spectrometry and mass spectral data analysis were essentially as described (18). Some of the digests were applied to a C18 PepMap 100 column (Dionex) and chromatographed using a gradient from 2% acetonitrile, 0.1% formic acid, to 80% acetonitrile, 0.08% formic acid on an UltiMate 3000 Nano LC System (Dionex) attached to a Probot microfraction collector (LC Packings) spotting sample directly to a MALDI plate in 7 mg/ml ␣-cyano-4-hydroxy-cinnamic acid supplemented with 2% (wt/wt) ammonium citrate in 75% acetonitrile.…”

“…Internal standards were insulin B-chain and angiotensin 1-7 clip. The sequence of the dimeric head ϩ G234A ϩ S188C cross-linked to the tail construct containing residues 823-944 was confirmed by tandem mass spectrometry as described (18).…”

“…The sequence of the cross-linked product of the dimeric headϩG234AϩS188C and the dimeric tail containing residues 823-944 was confirmed by sequencing using tandem mass spectrometry as described in ref. 18. All other cross-linked products occurred in ''families,'' that is, several similar masses were identified that result from slight variations in the tryptic digest pattern.…”