Calcium-Dependent Structural Coupling between Opposing Globular Domains of Calmodulin Involves the Central Helix

Abstract

We have used fluorescence spectroscopy to investigate the average structure and extent of conformational heterogeneity associated with the central helix in calmodulin (CaM), a sequence that contributes to calcium binding sites 2 and 3 and connects the amino- and carboxyl-terminal globular domains. Using site-directed mutagenesis, a double mutant was constructed involving conservative substitution of Tyr(99) --> Trp(99) and Leu(69) --> Cys(69) with no significant effect on the secondary structure of CaM. These mutation sites are at opposite ends of the central helix. Trp(99) acts as a fluorescence resonance energy transfer (FRET) donor in distance measurements of the conformation of the central helix. Cys(69) provides a reactive group for the covalent attachment of the FRET acceptor 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS). AEDANS-modified CaM fully activates the plasma membrane (PM) Ca-ATPase, indicating that the native structure is retained following site-directed mutagenesis and chemical modification. We find that the average spatial separation between Trp(99) and AEDANS covalently bound to Cys(69) decreases by approximately 7 +/- 2 A upon calcium binding. However, irrespective of calcium binding, there is little change in the conformational heterogeneity associated with the central helix under physiologically relevant conditions (i.e., pH 7.5, 0.1 M KCl). These results indicate that calcium activation alters the spatial arrangement of the opposing globular domains between two defined conformations. In contrast, under conditions of low ionic strength or pH the structure of CaM is altered and the conformational heterogeneity of the central helix is decreased upon calcium activation. These results suggest the presence of important ionizable groups that affect the structure of the central helix, which may play an important role in mediating the ability of CaM to rapidly bind and activate target proteins.

Ordered and Cooperative Binding of Opposing Globular Domains of Calmodulin to the Plasma Membrane Ca-ATPase

“…A fragment corresponding to amino acids 78 -148 (CaMC) was obtained following trypsin digestion of CaM. In all cases, CaM or its individual domain elements were purified using weak anion exchange HPLC as described previously (30). Erythrocyte ghost membranes containing the PM Ca-ATPase were purified as described previously (21,31).…”

Monitoring Neural Activity and [Ca2+] with Genetically Encoded Ca2+Indicators

“…B, Structural transitions of free-CaM. The occupation of Ca 2ϩ -binding sites adds stability to the CaM structure, causing reorientation of the N-and C-terminal domains and increases ␣-helical content of central helix (Sun et al, 1999). This will cause an elongation of the free-CaM molecule after Ca 2ϩ loading (increase in both the maximum size and radius of gyration; see Discussion for details).…”

Helix A Stabilization Precedes Amino-Terminal Lobe Activation upon Calcium Binding to Calmodulin

“…Prior measurements have demonstrated that calcium binding modulates the structural coupling between the opposing domains of CaM, acting to stabilize the helical structure of the interfacial residues between Met-76 and Ser-81 (13,14,22,23). Indeed, occupancy of carboxylterminal calcium binding sites induces conformational changes in the amino-terminal domain, which is apparent from both changes in fluorescence signals of reporter moieties and from increased rates of proteolysis at selected sites in the aminoterminal lobe (14,24,25).…”

“…Indeed, occupancy of carboxylterminal calcium binding sites induces conformational changes in the amino-terminal domain, which is apparent from both changes in fluorescence signals of reporter moieties and from increased rates of proteolysis at selected sites in the aminoterminal lobe (14,24,25). Further, the structural coupling between the opposing domains of CaM is stabilized at physiological ionic strengths, which acts to diminish the calcium affinity of sites in the amino-terminal lobe (13,26). These latter results suggest an important role for calciumdependent structural changes involving interfacial amino acids in the structural coupling between the opposing aminoand carboxyl-terminal lobes of CaM that acts to modulate calcium binding.…”