(Received for publication, July 24, 1995; and in revised form, August 25, 1995)
From the
Regulation of the ATPase activity of smooth and non-muscle myosin II involves reversible phosphorylation of the regulatory light chain (RLC). The RLC from skeletal muscle myosin (skRLC) is unable to confer regulation (myosin is locked in an inactive state) to smooth muscle myosin when substituted for the endogenous smooth RLC (smRLC). Studies of chimeric light chains comprised of the N- or C-terminal half of each skRLC and smRLC suggest that the structural basis for the loss of this regulation is within the C-terminal half of the RLC (Trybus, K. M., and Chatman, T. A.(1993) J. Biol. Chem. 268, 4412-4419). The purpose of this study is to delineate the structural elements within the C-terminal half of the smRLC that are absent in the skRLC and are necessary for regulation. By sequence comparison, six residues, Arg-103, Arg-123, Met-129, Gly-130, Arg-143, and Arg-160, which are conserved in regulated myosin RLCs but missing in nonregulated myosin RLCs, were identified in smRLC. To test whether these amino acids provide the missing structural elements necessary for phosphorylation-mediated regulation, a skRLC was engineered that replaced the corresponding skRLC amino acids (positions 100, 120, 126, 127, 140, and 157, respectively) with their smRLC counterparts. Using a newly developed RLC exchange procedure, the purified mutant protein was evaluated for its ability to regulate chicken gizzard smooth muscle myosin. Substitution of the six conserved amino acids into the skRLC completely restored phosphorylation-mediated regulation. Thus, a subset of these amino acids, including four basic arginine residues located in the E, F, G, and H helices which are missing in skRLC, may be the structural coordinates for the phosphorylserine in the N terminus. Based on this result, the regulation of glycogen phosphorylase is discussed as a model for the regulation of smooth muscle myosin.
Unlike striated muscle, the initiation of smooth muscle
contraction must be preceded by serine phosphorylation of the myosin
RLC ()which is accomplished by a
calcium/calmodulin-dependent myosin light chain kinase in the presence
of ATP(1) . Although the RLC is also reversibly phosphorylated
in vertebrate striated muscle (at a homologous serine), this
phosphorylation simply modulates contractile activity(2) . The
loss of myosin regulation in striated muscle is due to undetermined
alterations in the myosin heavy chain. Additionally, the smooth muscle
and skeletal muscle RLCs are not functionally equivalent, even though
the RLCs from skeletal muscle and smooth muscle are highly homologous
(71% similarity and 53% identity in sequence). The RLC from skeletal
muscle myosin (skRLC) is unable to confer regulation to smooth muscle
myosin (a phosphorylation-regulated myosin) and locks the myosin in the
``off'' state when substituted for the endogenous smooth
muscle RLC (smRLC)(3) . Additionally, the smRLC can confer
calcium sensitivity to scallop muscle myosin (a
Ca
-regulated myosin), while the skRLC fails to do
so(4) . However, no RLC can inhibit the activity of vertebrate
skeletal muscle myosin.
The fact that the skeletal muscle RLC has retained the structural elements to lock a phosphorylation-regulated myosin (smooth muscle myosin) into an inactive state that is not activated via RLC phosphorylation, presents an opportunity to use directed mutagenesis to gain insight into the mechanism of phosphorylation-dependent regulation. In the best understood example of phosphorylation-dependent enzyme regulation, that of glycogen phosphorylase(5) , loss of ability to activate enzyme activity via phosphorylation could be achieved by loss of the two arginine residues that coordinate the phosphoserine. The purpose of this study was to ascertain if the loss of function of the skRLC could be restored by insertion of arginine residues that may function in a putative coordination of the phosphoserine.
The phosphorylated serine residue
is near the N terminus of the RLC. Studies of chimeric RLCs comprised
of the N- or C-terminal half of each skRLC and smRLC indicate that it
is the C-terminal half of skRLC that lacks structural elements
necessary for phosphorylation-mediated regulation(3) . By
sequence comparison (Fig. 1A), four arginine residues
(Arg-103, Arg-123, Arg-143, and Arg-160; corresponding to amino acids
100, 120, 140, and 157, respectively, in the rabbit skRLC sequence) are
present in the C-terminal half of the smRLC which are conserved in
regulated myosin RLCs but missing in nonregulated myosin RLCs. The four
arginines are located in four different domains (the E, F, G, and H
helices, using calmodulin nomenclature for the helices(6) ; Fig. 1B), as revealed in the crystal structures of
chicken skeletal myosin S1 (7) and the scallop myosin
regulatory domain(8) . In addition to these arginines, the C
terminus of the skRLC was examined for other missing conserved amino
acids. A striking substitution was for glycine 130 and methionine 129
(amino acids 127 and 126, respectively, in the rabbit skRLC) in the
loop between the F and G helices, which is absolutely conserved in
regulated myosins (Fig. 1A). The location of this
glycine in the loop between two helices may confer flexibility that is
necessary for the helices to translate relative to each other upon
phosphorylation. (This glycine provides a different function in
Ca regulation in scallop muscle(8) ). A
functional role in regulation for a subset of the missing arginine
residues and for interhelical flexibility is postulated by analogy to
the case of glycogen phosphorylase, wherein coordination of the
phosphoserine is by arginines residing in different helices. Global
rearrangement of helices is associated with the coordination of the
phosphoserine.
Figure 1: A, sequence comparisons between chicken smooth muscle myosin regulatory light chain and rabbit fast skeletal muscle regulatory light chain. Amino acid alterations in the mutant rabbit skRLC (and position number in the rabbit skRLC sequence) are denoted by arrows beneath the sequence (R for arginine, M for methionine, G for glycine). B, ribbon diagram of regulatory domain of chicken skeletal muscle myosin indicating position of mutations (based on the structural determination of Rayment et al.(7) ). The four arginine mutations (amino acids 100, 120, 140, and 157) are shown as black balls, the Met-Gly pair of mutations (amino acids 126 and 127, respectively) as smaller gray balls. Each of the four arginines is located in a different helix (the E, F, G, and H helices, using calmodulin nomenclature(6) ). The first 18 residues, including the phosphorylatable serine 13 (serine 15 in case of rabbit skRLC) of the N terminus of the skRLC, are missing in the crystal structure.
To test whether all or a subset of the substituted amino acids (Arg-103, Arg-123, Met-129, Gly-130, Arg-143, and Arg-160) are structural elements necessary for conferring phosphorylation-mediated regulation to the RLC (via phosphorylation of serine 15), a skRLC was engineered that replaced the six altered skRLC residues with their smRLC counterparts (creating Arg-100, Arg-120, Met-126, Gly-127, Arg-140, and Arg-157 in the skRLC). Regulation was assayed following exchange of the mutant skRLC into chicken gizzard smooth muscle myosin.
Figure 2: Extent of exchange of mutant skRLC into gizzard smooth muscle myosin as assessed by 12% SDS-PAGE. Lane 1 contains the smRLC and smELC dissociated from gizzard smooth muscle myosin. Lane 2 contains the E. coli-expressed wild type rabbit skRLC. Lane 3 contains exchanged skRLC and endogenous smELC dissociated from chicken gizzard smooth muscle myosin following the RLC exchange procedure.
Trifluoperazine is a calmodulin antagonist
which inhibits the functions of calmodulin by formation of
antagonist-calmodulin complex. The crystal structure of this complex in
the presence of Ca indicates that the binding of TFP
induces conformational changes from an elongated dumbbell, with exposed
hydrophobic surfaces, to a compact globular form which can no longer
interact with its target enzymes(17, 18) . This
conformational change is similar to that seen in the calmodulin-target
peptide complex. Although the binding of TFP to RLC is not
characterized, it is likely that a similar mechanism applies to TFP-RLC
interactions. TFP may compete with the myosin heavy chain for the
hydrophobic pockets of the RLC, thereby dissociating the RLCs.
Previously, TFP has been used to extract troponin C (another member of
the calmodulin superfamily) from the troponin complex (19) and
to prepare RLC-deficient smooth muscle myosin through fast protein
liquid chromatography gel filtration (3) .
At a higher concentration (5 mM), TFP dissociates endogenous RLCs, but also interferes with the subsequent association of exogenous RLC. Presumably at high concentrations, TFP may effectively sequester the RLCs and prevent binding to the myosin heavy chain. On the other hand, lower TFP concentrations lead to a lower degree of exchange even at higher temperature (37 °C), but does not influence the binding of the exogenous RLC. Based on these observations, the two-step RLC exchange procedure that involves lowering of the TFP concentration (described in above) was developed.
Figure 3: Actin-activated MgATPase activities of dephosphorylated and phosphorylated native gizzard myosin and gizzard myosin reconstituted with either smRLC (wild type), skRLC (wild type), or mutant skRLC. Maximal actin-activated ATPase rates are for chicken gizzard myosin preparations without (open bars) phosphorylation or with (stippled bars) phosphorylation of the RLC. Standard deviations for different preparations (n = 3 in all cases) are indicated. The myosin was phosphorylated using rat skeletal myosin light chain kinase. Greater than 90% of the RLCs in each preparation were phosphorylated.
If phosphorylation of the RLC regulates myosin activity via a mechanism similar to that for the regulation of glycogen phosphorylase(5) , then it is likely that of the labeled residues in Fig. 1B, two of the arginines are required to provide structural coordinates for the phosphoserine. When the phosphorylatable N-terminal serine is phosphorylated, the mobile and disordered N terminus may become immobilized and ordered and bind to the surface of the interface of the C-terminal domain. Based on the orientation of the corresponding residues in chicken skeletal structure (Fig. 1B), only the arginines found in helices E (Arg-100) and H (Arg-157) would appear to be positioned appropriately. One or two of the engineered arginines probably coordinates the phosphate of the phosphoserine by hydrogen bonds. The coordination of the phosphoserine may cause winding or unwinding of the helices and the rearrangement of domains. These conformational changes likely lead to regulation of myosin through altered interactions that involve the two RLCs and the myosin heavy chain.
Based on this model, one would predict that loss of the conserved arginines in the E and H helices of the smooth muscle RLC would result in a loss of regulation. Indeed, when Ikebe et al.(20) substituted a region of the skeletal RLC H helix into the smooth RLC H helix, Arg-157 was lost and so was regulation. Furthermore, again based on considering the regulation of glycogen phosphorylase, the N-terminal basic residues may be involved in stabilizing the interaction of the phosphoserine with its coordination sites. Such a mechanism would explain the results of the study of Ikebe and Morita(21) , wherein cleavage of the smooth RLC removed Arg-13 and Arg-16 and generated a RLC that could not activate smooth muscle myosin (heavy meromyosin) even when serine 19 was phosphorylated. This hypothetical regulatory scheme also is supported by NMR results. The study of Levine et al.(22) suggests that the N-terminal region of both rabbit skRLC and gizzard smRLC exhibits segmental mobility independent of the rest of the molecule. When the RLC is phosphorylated, mobility of the N-terminal segment is diminished and the serine phosphate is influenced by neighboring positively charged side chains.
While the skeletal muscle RLC has maintained the ability to inhibit the activity of smooth muscle myosin, regulation (i.e. activation) of activity via phosphorylation of the RLC has been lost. Restoration of regulation results from the alteration of maximally six amino acids, and likely from a subset of the six, that are conserved in regulated myosins but absent in the vertebrate striated muscle RLCs. Additional mutagenesis, involving both the skeletal and smooth RLCs, will further delineate the necessary amino acids and the critical interactions involved in myosin regulation via phosphorylation. The regulation of glycogen phosphorylase should provide a useful framework for understanding the structural changes that accompany phosphorylation of the myosin RLC and which underlie the regulatory process.