Calmodulin-dependent enzymes undergo a protein-induced conformational change that is associated with their interactions with calmodulin.
The anionic hydrophobic (amphipathic) fluorescent probe 2-(p-toluidinyl)-naphthalene-6-sulfonate was used to investigate the surface hydrophobic properties of calmodulin (CaM)-dependent enzymes as follows: calcineurin, myosin light chain kinase, cyclic nucleotide phosphodiesterase, CaM-dependent protein kinase II, and the gamma-subunit of phosphorylase kinase. We found that certain domains of these enzymes that interacted with 2-(p-toluidinyl)-naphthalene-6-sulfonate were exposed by a transient proton (H+) increase within the neutral pH range. This H(+)-induced exposure, which could be caused either by direct addition of H+ or by the release of H+ from metal chelators upon their binding of Ca2+, seemed to be more closely linked with a change in pH value (i.e. transient H+ increase) than with the actual equilibrium pH value of the system. Unlike the case with CaM-dependent enzymes, the H(+)-induced conformational change was uncommon in CaM-independent enzymes. When CaM-binding domains were removed from calcineurin and smooth muscle myosin light chain kinase, the resultant enzymes no longer exposed new domains in response to H+ increase. Using dansylated CaM to monitor the formation of CaM-enzyme complexes, we found that complex formation occurred with an uptake of H+ from solution. When CaM-dependent enzymes were evaluated at suboptimal concentrations of Ca2+, addition of H+ enhanced both the formation of CaM-enzyme complexes and the CaM-dependent catalytic activities, but this synergistic H+ effect occurred within only a narrow range of Ca2+ concentrations. These findings suggest that the H(+)-exposed domains in CaM-dependent enzymes are involved in the binding of CaM and that both conformational changes in CaM and its enzyme targets are necessary for complex formation. Further, the findings are consistent with the notion that CaM-binding domains are masked in the nonactivated (uncomplexed) conformations of CaM-dependent enzymes. The interplay between H+ and Ca2+ is discussed in relation to other systems that display interdependent effects of these two ions.
Huang
S
S
Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, Tennessee 38105.
Carlson
G M
GM
Cheung
W Y
WY
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011522:Protons
D002147:Calmodulin; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D011522:Protons; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D010727:Phosphoric Diester Hydrolases