Different Conformational Switches Underlie the Calmodulin-Dependent Modulation of Calcium Pumps and Channels

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• 作者：Curt B. Boschek ; Hongye Sun ; Diana J. Bigelow ; Thomas C. Squier
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：February 12, 2008
• 年：2008
• 卷：47
• 期：6
• 页码：1640 - 1651
• 全文大小：382K
• 年卷期：v.47,no.6(February 12, 2008)
• ISSN：1520-4995

文摘

We have used fluorescence spectroscopy to investigate the structure of calmodulin (CaM)bound with CaM-binding sequences of either the plasma membrane Ca-ATPase or the skeletal muscleryanodine receptor (RyR1) calcium release channel. Following derivatization with N-(1-pyrene)maleimideat engineered sites (T34C and T110C) within the N- and C-domains of CaM, contact interactions betweenthese opposing domains of CaM resulted in excimer fluorescence that permits us to monitor conformationalstates of bound CaM. Complementary measurements take advantage of the unique conserved Trp withinCaM-binding sequences that functions as a hydrophobic anchor in CaM binding and permits measurementsof both a local and global peptide structure. We find that CaM binds with high affinity in a collapsedstructure to the CaM-binding sequences of both the Ca-ATPase and RyR1, resulting in excimer formationthat is indicative of contact interactions between the N- and the C-domains of CaM in complex with theseCaM-binding peptides. There is a 4-fold larger amount of excimer formation for CaM bound to the CaM-binding sequence of the Ca-ATPase in comparison to RyR1, indicating a closer structural coupling betweenCaM domains in this complex. Prior to CaM association, the CaM-binding sequences of the Ca-ATPaseand RyR1 are conformationally disordered. Upon CaM association, the CaM-binding sequence of theCa-ATPase assumes a highly ordered structure. In comparison, the CaM-binding sequence of RyR1 remainsconformationally disordered irrespective of CaM binding. These results suggest an important role forinterdomain contact interactions between the opposing domains of CaM in stabilizing the structure of thepeptide complex. The substantially different structural responses associated with CaM binding to Ca-ATPase and RyR1 indicates a plasticity in their respective binding mechanisms that accomplishes differentphysical mechanisms of allosteric regulation, involving either the dissociation of a C-terminal regulatorydomain necessary for pump activation or the modulation of intersubunit interactions to diminish RyR1channel activity.