Shaowei Ni

University of Nevada School of Medicine, Reno, Nevada, United States

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Publications (5)21.51 Total impact

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    ABSTRACT: We examined the regulatory importance of interactions between regulatory light chain (RLC), essential light chain (ELC), and adjacent heavy chain (HC) in the regulatory domain of smooth muscle heavy meromyosin. After mutating the HC, RLC, and/or ELC to disrupt their predicted interactions (using scallop myosin coordinates), we measured basal ATPase, Vmax, and KATPase of actin-activated ATPase, actin-sliding velocities, rigor binding to actin, and kinetics of ATP binding and ADP release. If unphosphorylated, all mutants were similar to wild type showing turned-off behaviors. In contrast, if phosphorylated, mutation of RLC residues smM129Q and smG130C in the F-G helix linker, which interact with the ELC (Ca2+ binding in scallop), was sufficient to abolish motility and diminish ATPase activity, without altering other parameters. ELC mutations within this interacting ELC loop (smR20M and smK25A) were normal, but smM129Q/G130C-R20M or -K25A showed a partially recovered phenotype suggesting that interaction between the RLC and ELC is important. A molecular dynamics study suggested that breaking the RLC/ELC interface leads to increased flexibility at the interface and ELC-binding site of the HC. We hypothesize that this leads to hampered activation by allowing a pre-existing equilibrium between activated and inhibited structural distributions (Vileno, B., Chamoun, J., Liang, H., Brewer, P., Haldeman, B. D., Facemyer, K. C., Salzameda, B., Song, L., Li, H. C., Cremo, C. R., and Fajer, P. G. (2011) Broad disorder and the allosteric mechanism of myosin II regulation by phosphorylation. Proc. Natl. Acad. Sci. U.S.A. 108, 8218–8223) to be biased strongly toward the inhibited distribution even when the RLC is phosphorylated. We propose that an important structural function of RLC phosphorylation is to promote or assist in the maintenance of an intact RLC/ELC interface. If the RLC/ELC interface is broken, the off-state structures are no longer destabilized by phosphorylation.
    Journal of Biological Chemistry 05/2012; 287(26):22068-79. DOI:10.1074/jbc.M112.343491 · 4.57 Impact Factor
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    Biophysical Journal 01/2010; 98(3). DOI:10.1016/j.bpj.2009.12.2236 · 3.97 Impact Factor
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    Biophysical Journal 01/2010; 98(3). DOI:10.1016/j.bpj.2009.12.2938 · 3.97 Impact Factor
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    ABSTRACT: To understand the importance of selected regions of the regulatory light chain (RLC) for phosphorylation-dependent regulation of smooth muscle myosin (SMM), we expressed three heavy meromyosins (HMMs) containing the following RLC mutants; K12E in a critical region of the phosphorylation domain, GTDP(95-98)/AAAA in the central hinge, and R160C a putative binding residue for phosphorylated S19. Single-turnover actin-activated Mg(2+)-ATPase (V(max) and K(ATPase)) and in vitro actin-sliding velocities were examined for both unphosphorylated (up-) and phosphorylated (p-) states. Turnover rates for the up-state (0.007-0.030 s(-1)) and velocities (no motion) for all constructs were not significantly different from the up-wild type (WT) indicating that they were completely turned off. The apparent binding constants for actin in the presence of ATP (K(ATPase)) were too weak to measure as expected for fully regulated constructs. For p-HMM containing GTDP/AAAA, we found that both ATPase and motility were normal. The data suggest that the native sequence in the central hinge between the two lobes of the RLC is not required for turning the HMM off and on both kinetically and mechanically. For p-HMM containing R160C, all parameters were normal, suggesting that R160C is not involved in coordination of the phosphorylated S19. For p-HMM containing K12E, the V(max) was 64% and the actin-sliding velocity was approximately 50% of WT, suggesting that K12 is an important residue for the ability to sense or to promote the conformational changes required for kinetic and mechanical activation.
    Biochimica et Biophysica Acta 08/2009; 1794(11):1599-605. DOI:10.1016/j.bbapap.2009.07.009 · 4.66 Impact Factor
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    ABSTRACT: A current popular model to explain phosphorylation of smooth muscle myosin (SMM) by myosin light-chain kinase (MLCK) proposes that MLCK is bound tightly to actin but weakly to SMM. We found that MLCK and calmodulin (CaM) co-purify with unphosphorylated SMM from chicken gizzard, suggesting that they are tightly bound. Although the MLCK:SMM molar ratio in SMM preparations was well below stoichiometric (1:73+/-9), the ratio was approximately 23-37% of that in gizzard tissue. Fifteen to 30% of MLCK was associated with CaM at approximately 1 nM free [Ca(2+)]. There were two MLCK pools that bound unphosphorylated SMM with K(d) approximately 10 and 0.2 microM and phosphorylated SMM with K(d) approximately 20 and 0.2 microM. Using an in vitro motility assay to measure actin sliding velocities, we showed that the co-purifying MLCK-CaM was activated by Ca(2+) and phosphorylation of SMM occurred at a pCa(50) of 6.1 and at a Hill coefficient of 0.9. Similar properties were observed from reconstituted MLCK-CaM-SMM. Using motility assays, co-sedimentation assays, and on-coverslip enzyme-linked immunosorbent assays to quantify proteins on the motility assay coverslip, we provide strong evidence that most of the MLCK is bound directly to SMM through the telokin domain and some may also be bound to both SMM and to co-purifying actin through the N-terminal actin-binding domain. These results suggest that this MLCK may play a role in the initiation of contraction.
    Journal of Molecular Biology 06/2009; 390(5):879-92. DOI:10.1016/j.jmb.2009.05.033 · 4.33 Impact Factor