Abstract

Nitric oxide and nitrovasodilators induce vascular smooth muscle cell relaxation in part by cGMP-dependent protein kinase I (PKG-Ialpha)-mediated activation of myosin phosphatase (MLCP). Mechanistically it has been proposed that protein-protein interactions between the N-terminal leucine zipper (LZ) domain of PKG-Ialpha ((PKG-Ialpha(1-59)) and the LZ and/or coiled coil (CC) domain of the myosin binding subunit (MBS) of MLCP are localized in the C terminus of MBS. Although recent studies have supported these interactions, the critical amino acids responsible for these interactions have not been identified. Here we present structural and biophysical data identifying that the LZ domain of PKG-Ialpha(1-59) interacts with a well defined 42-residue CC motif (MBS(CT42)) within the C terminus of MBS. Using glutathione S-transferase pulldown experiments, chemical cross-linking, size exclusion chromatography, circular dichroism, and isothermal titration calorimetry we identified a weak dimer-dimer interaction between PKG-Ialpha(1-59) and this C-terminal CC domain of MBS. The K(d) of this non-covalent complex is 178.0+/-1.5 microm. Furthermore our (1)H-(15)N heteronuclear single quantum correlation NMR data illustrate that this interaction is mediated by several PKG-Ialpha residues that are on the a, d, e, and g hydrophobic and electrostatic interface of the C-terminal heptad layers 2, 4, and 5 of PKG-Ialpha. Taken together these data support a role for the LZ domain of PKG-Ialpha and the CC domain of MBS in this requisite contractile complex.

Highlights

  • Vascular smooth muscle cell tone in blood vessels is clearly coupled to the phosphorylation state of the myosin light chain

  • Using glutathione S-transferase (GST) pulldowns, chemical cross-linking, size exclusion chromatography (SEC), circular dichroism (CD), and isothermal titration calorimetry (ITC) we identified and characterized this interaction between PKG-I␣1–59 and MBSCT42 that supports the formation of a dimer1⁄7dimer interaction complex

  • Expression of recombinant GSTPKG-I␣1–59 (33.4 kDa) was identified by a band corresponding to this molecular mass

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Summary

EXPERIMENTAL PROCEDURES

Expression and Purification of PKG-I␣ LZ Domain (PKGI␣1–59) and MBS LZ Domains—Plasmids comprised of the PKG-I␣ LZ domain (PKG-I␣1–59) and the C-terminal 100 amino acids (residues 930 –1030) of MBS (MBSCT100) were used for this investigation. We performed experiments in which MBSCT100 (160 ␮M) was titrated with PKG-I␣1–59 using identical acquisition parameters These data were analyzed using a non-linear least square program (Microcal, Origin) and fit to a single site or sequential two-site binding model permitting us to determine a dissociation binding constant (Kd), change in enthalpy (⌬H), and/or change in entropy (⌬S). Chemical shift perturbation studies were carried out by collecting a series of two-dimensional 1H-15N HSQC spectra at 25 °C on uniformly 15N-labeled PKG-I␣1–59 as a function of increasing MBSCT42 concentrations until a stoichiometric ratio of 1:2 was reached. Beyond this ratio, no further chemical shift change or peak intensity perturbation was noticed. Figures were generated using MOLMOL [23]

RESULTS
Circular Dichroism
DISCUSSION
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