Abstract:
:Stabilization of the plasma membrane Ca-ATPase (PMCA) in an inactive conformation upon oxidation of multiple methionines in the calcium regulatory protein calmodulin (CaM) is part of an adaptive cellular response to minimize ATP utilization and the generation of reactive oxygen species (ROS) under conditions of oxidative stress. To differentiate oxidant-induced structural changes that selectively modify the amino-terminal domain of CaM from those that modulate the conformational coupling between the opposing domains, we have engineered a tetracysteine binding motif within helix A in the amino-terminal domain of calmodulin (CaM) that permits the selective and rigid attachment of the conformationally sensitive fluorescent probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethanedithiol)(2) (FlAsH-EDT(2)). The position of the FlAsH label in the amino-terminal domain provides a signal for monitoring its binding to the CaM-binding sequence of the PMCA. Following methionine oxidation, there is an enhanced binding affinity between the amino-terminal domain and the CaM-binding sequence of the PMCA. To identify oxidant-induced structural changes, we used frequency domain fluorescence anisotropy measurements to assess the structural coupling between helix A and the amino- and carboxyl-terminal domains of CaM. Helix A undergoes large amplitude motions in apo-CaM; following calcium activation, helix A is immobilized as part of a conformational switch that couples the opposing domains of CaM to stabilize the high-affinity binding cleft associated with target protein binding. Methionine oxidation disrupts the structural coupling between opposing globular domains of CaM, without affecting the calcium-dependent immobilization of helix A associated with activation of the amino-terminal domain to promote high-affinity binding to target proteins. We suggest that this selective disruption of the structural linkage between the opposing globular domains of CaM relieves steric constraints associated with high-affinity target binding, permitting the formation of new contact interactions between the amino-terminal domain and the CaM-binding sequence that stabilizes the PMCA in an inhibited conformation.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Chen B,Mayer MU,Squier TCdoi
10.1021/bi0474113subject
Has Abstractpub_date
2005-03-29 00:00:00pages
4737-47issue
12eissn
0006-2960issn
1520-4995journal_volume
44pub_type
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