DYNAMIC STRUCTURE OF THE CALMODULIN-BINDING DOMAIN OF THE PLASMA-MEMBRANE CA-ATPASE IN NATIVE ERYTHROCYTE GHOST MEMBRANES

We have used frequency-domain fluorescence resonance energy transfer ( FRET) and anisotropy measurements to identify the structural propertie s of wheat germ calmodulin (CaM) bound to either the plasma membrane C a-ATPase (PM-Ca-ATPase) in native erythrocyte ghost membranes or a pep tide (C25W) that has an identical sequence to the CaM-binding domain o n the PM-Ca-ATPase, Cross-linking experiments using benzophenone label ed CaM in conjunction with immunoblots using antibodies specific for e ither CaM or the PM-Ca-ATPase indicate that one molecule of CaM select ively binds one PM-Ca-ATPase polypeptide chain in native erythrocyte g host membranes, There are no other proteins in the erythrocyte membran e that bind CaM with high affinity, permitting the measurement of the structural properties of CaM bound to the PM-Ca-ATPase in native eryth rocyte ghost membranes. FRET measurements between the fluorophore pyre ne maleimide (PMal) located at Cys(27) in calcium binding loop I and n itrotyrosine(139) in calcium binding loop IV on wheat germ CaM indicat e that the average spatial separation and conformational heterogeneity associated with the two opposing globular domains of CaM are virtuall y identical upon CaM binding to either the PM-Ca-ATPase or C25W. Measu rements of the solvent accessibility and segmental rotational dynamics of PMal-CaM bound to either the PM-Ca-ATPase or C25W further indicate that the local environment around the pyrene label located at Cys(27) is very similar, However, the overall rotational dynamics of CaM boun d to the PM-Ca-ATPase is much slower (phi 2 = 83 +/- 14 ns) than obser ved when CaM binds C25W (phi(2) = 10.3 +/- 0.5 ns), This implies that CaM is tightly associated with the CaM-binding domain of the PM-Ca-ATP ase and that the observed rotational motion of pyrenylmaleimide labele d CaM is characteristic of the global motion of the CaM-binding domain on the PM-Ca-ATPase. The similar conformational heterogeneity and loc al environment of CaM bound to either the PM-Ca-ATPase or C25W indicat es that CaM binds to a contiguous sequence of amino acids on the Ca-AT Pase that are analogous to C25W and that there are no significant inte ractions with other structural elements within the PM-Ca-ATPase. The r ate of rotational motion associated with CaM bound to the PM-Ca-ATPase is consistent with hydrodynamic calculations in which the calmodulin binding domain located at the carboxyl-terminus of the PM-Ca-ATPase ha s a stable and defined tertiary structure that is independent of the o ther cytoplasmic domains of the PM-Ca-ATPase.