The actin-activated MgATPase and motor properties of SMM and its C-terminally truncated subfragment, HMM, are activated by phosphorylation of the RLC at Ser19 by the Ca2+
/calmodulin-dependent myosin light chain kinase [1
]. The RLC is a calmodulin-like structure that binds largely through hydrophobic interactions with the heavy chain helix. The N-lobe of the RLC (excluding the N-terminal domain see below) binds to a prominent bend in the heavy chain at the head-tail junction and the C-lobe binds further toward the heavy chain N-terminus (toward the motor domain). Phosphorylation of the RLC on each of the two head domains is sufficient to activate the ATPase by ~1000 fold [2
] and to enable the molecule to move actin filaments, whereas the unphosphorylated state is inactivated in both respects. Since the RLC is located at the head-rod junction [4
], it is unlikely that its phosphorylation site, which remains crystallographically undetermined, can interact directly with the ATP or actin binding sites more than ~10 nm away. The mechanism by which phosphorylation of S19 accelerates the rate of phosphate release from the active site [3
], thus activating the ATPase, remains unknown.
The RLC has been the focus of numerous previous mutagenesis studies related to phosphorylation-dependent regulation of SMM [5
]. Several unifying themes have emerged. Coupling of the ATPase to movement is mediated by the RLC [9
]. The ATPase activity of RLC-deficient SMM is much higher than unmodified up-SMM. However, RLC-deficient SMM cannot move actin in an in vitro motility assay. With 26 residues truncated from the C-terminus of the RLC, the molecule behaves exactly like the LC deficient myosin regardless of the state of phosphorylation [9
]. Without these residues, the dephosphorylated form of the protein has a high ATPase activity both in the absence and presence of actin. Therefore, the C-terminal lobe of the RLC is involved in interactions that regulate myosin's on-off switch, both in terms of inhibition and activation. Although the C-terminal domain is clearly critical for regulation of the ATPase and motor function, there have been no site-directed mutagenesis studies addressing the role of specific residues. Many of the studies were done with deletions, which severely weaken the binding of the RLC to the heavy chain, raising the possibility for global changes in structure.
The extreme N-terminal region of the RLC containing the phosphorylated Ser-19 is critical for regulation [5
]. Sequential deletion of N-terminal residues 1-16 from this phosphorylation domain incrementally increases the unphosphorylated and decreases the phosphorylated actin-activated ATPase activities, whereas activities remain relatively low in the absence of actin. This behavior might be expected if the entire region provides stabilization energy toward the switched-off and switched-on states, respectively. The phosphorylation domain can sense the nucleotide state of the active site but only in the context of a regulated construct, i.e. double-headed HMM, but not in the unregulated S1 head construct [12
]. Unfortunately, no electron density has been observed for the first 24 amino acids of the RLC in any of the myosin X-ray structures. A region of the domain, 4
, can be cross-linked to Cys-108 in the C-terminal lobe of the other head in the up-state, but not in the p-state [13
]. These residues lie within a region that probably constitutes an independent flexible domain [14
]. It is possible that the phosphorylated Ser-19 coordinates with a residue(s) in this domain but this has not been determined. Although this important domain has been extensively studied, nothing is known about its effects upon the motor function.
Previous work has shown that another important functional region of the RLC is the central linker between the N- and C-lobes. It has been proposed that a putative hinge region between the helixes (D and E) that connects the two lobes of the RLC, Gly95
, plays a significant role in phosphorylation-dependent regulation [16
]. However, certain questions remain unanswered because most mutants were again deletions. Indeed the effect of these deletions on the motor properties were similar to the above-mentioned RLC-deficient SMM, that is the mechanochemical coupling of the ATPase to motility was broken. This raises the possibility that the phenotype was due to improper binding of the RLC to the HC, and not to the specific importance of the hinge itself. The one mutation that was a substitution gave motility and ATPase characteristics that are difficult to reconcile considering the known parameters that control actin sliding velocity (see Discussion).
The present study represents initial work to address the above-mentioned deficiencies and uncertainties in our understanding of the role of the C-terminal, N-terminal, and central hinge domains of the RLC with respect to regulation by phosphorylation. Although prior studies in this area represent founding and significant work, many questions remain because of factors that make study-to-study comparisons ambiguous. For example, studies were done with different protein preparations. Those include full length tissue-purified SMM, tissue purified HMM and expressed HMM. ATPase assays were performed under different conditions and some work was completed prior to the common use of the in vitro motility assay to assess motor function. Importantly, ATPase assay results were often reported for only one actin concentration that was well below the Vmax
raising concerns over Km
) effects. Usually, the steady-state method of assay was used which can overestimate the ATPase activity of the inhibited state [3
]. In earlier reports, the heat-mediated [6
] or trifluoroperazine-mediated [9
] RLC exchange procedures were used to reconstitute mutant RLC with the heavy chain/ELC, which often resulted in mixtures of molecules with different RLC subunits. Also, many mutants severely weakened the binding affinity of the RLC for the heavy chain. These two latter problems often dictated that ATPase activities be adjusted by calculation. More recent work addressing other portions of the SMM molecule, for example the rod and motor domains, have successfully expressed HMM-like constructs using the baculovirus expression system [17
], thus avoiding many of the aforementioned problems. We have used that approach here. Also, we have used the single-turnover method of ATPase assay and determined the Vmax
for each construct, in addition to assessing motor function by the in vitro motility assay.
We have expressed selected smooth muscle HMM constructs containing RLC mutations in the 3 critical domains discussed above, some of which had been previously investigated, that we felt merited further study for reasons outlined above. K12 in the N-terminal phosphorylation domain is of interest due to recent modeling work showing that this residue lies in a critical location within the phosphorylation domain that undergoes a large conformational change upon phosphorylation [14
]. The GTDP/AAAA mutation in the central hinge is of interest due to the above-mentioned unusual properties, allowing for robust motility with low ATPase. R160 in the C-terminal lobe is of interest due to previous work suggesting that it may coordinate the negative charges of phosphorylated Ser-19. The motor properties and actin-activated Mg2+
-ATPase activity of the mutants were evaluated by motility and transient kinetic single turnover of Mant-ATP, respectively. We found that the actin sliding velocity and ATPase kinetics of HMM with the R160C and GTDP/AAAA mutations on the RLC are similar to WT in both up- and p-states, suggesting that the putative phospho-binding residue, Arg160
, is not important for phosphate coordination, and the Gly95
native sequence constituting the hinge region between the N- and C-lobes of the RLC is not required for phosphorylation-dependent regulation of SMM. In addition, we show that in the p-state a K12E mutation modestly inhibits the actin-activated ATPase and the actin-sliding motor activity, suggesting that the negative charge may destabilize but not prevent the proposed phosphorylation-dependent ordering of the phosphorylation domain.