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We have used circular dichroism and frequency-domain fluorescence spectroscopy to determine how the site-specific substitution of Tyr138 with either Phe138 or Gln138 affects the structural coupling between the opposing domains of calmodulin (CaM). A double mutant was constructed involving conservative substitution of Tyr99 --> Trp99 and Leu69 --> Cys69 to assess the structural coupling between the opposing domains, as previously described [Sun, H., Yin, D., and Squier, T. C. (1999) Biochemistry 38, 12266-12279]. Trp99 acts as a fluorescence resonance energy transfer (FRET) donor in distance measurements to probe the conformation of the central helix. Cys69 provides a reactive group for the covalent attachment of 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS), which functions as a FRET acceptor and permits the measurement of the rotational dynamics of the amino-terminal domain. These CaM mutants demonstrate normal calcium-dependent gel-mobility shifts and changes in their near-UV CD spectra, have similar secondary structures to wild-type CaM following calcium activation, and retain the ability to fully activate the plasma membrane Ca-ATPase. The global folds, therefore, of both the carboxyl- and amino-terminal domains in these CaM mutants are similar to that of wild-type CaM. However, in comparison to wild-type CaM, the substitution of Tyr138 with either Phe138 or Gln138 results in (i) alterations in the average spatial separation and increases in the conformational heterogeneity between the opposing globular domains and (ii) the independent rotational dynamics of the amino-terminal domain. These results indicate that alterations in either the hydrogen bond between Tyr138 and Glu82 or contact interactions between aromatic amino acid side chains have the potential to initiate the structural collapse of CaM normally associated with target protein binding and activation.

To investigate the structural linkage between the opposing globular domains in vertebrate calmodulin (CaM), we have constructed a CaM mutant (CaMX(145)) deficient in the last four amino acids between Met(145) and Lys(148) at the carboxyl terminal. Circular dichroism and fluorescence spectroscopic measurements were used to detect changes in the average secondary and tertiary structure of CaMX(145) in comparison to full-length CaM. Complementary measurements of the maximal calcium-binding stoichiometry and ability to activate the plasma membrane (PM) Ca-ATPase permit an assessment of the functional significance of observed structural changes. In comparison with native CaM, we find that CaMX(145) exhibits (i) a large reduction in alpha-helical content, (ii) a dramatic decrease in the average spatial separation between the opposing globular domains, (iii) the loss of one high-affinity calcium-binding site, and (iv) a diminished binding affinity for the PM-Ca-ATPase. Thus, the sequence near the carboxyl terminus functions to stabilize high-affinity calcium binding at one site and facilitates important intramolecular interactions that maintain CaM in an extended conformation. However, despite the large conformational changes resulting from deletion of the last four amino acids at the carboxyl terminal, CaMX(145) can fully activate the PM-Ca-ATPase. These results indicate that target protein binding can restore the nativelike structure critical to function, emphasizing that the structure of the central helix is not critical to CaM function under equilibrium conditions. Rather, the central helix functions to maintain the spatial separation between the opposing domains in CaM that may be critical to high-affinity binding and the rapid activation of the PM-Ca-ATPase, which are necessary for optimal calcium signaling. Thus, following initial association between CaM and target proteins, structural changes involving the carboxyl-terminal sequence have the potential to play an important role in triggering the structural collapse of CaM that facilitates the rapid and cooperative binding of the opposing globular domains with target proteins, which is important to high-affinity binding and rapid enzyme activation.

We have investigated the ability of methionine sulfoxide reductase (MsrA) to maintain optimal calmodulin (CaM) function through the repair of oxidized methionines, which have been shown to accumulate within CaM in senescent brain [Gao, J., Yin, D. H., Yao, Y., Williams, T. D., and Squier, T. C. (1998) Biochemistry 37, 9536-9548]. Oxidatively modified calmodulin (CaMox) isolated from senescent brain or obtained by in vitro oxidation was incubated with MsrA. This treatment restores the functional ability of CaMox to activate the plasma membrane (PM) Ca-ATPase, confirming that (i) the decreased ability of CaM isolated from senescent animals to activate the PM Ca-ATPase results solely from methionine sulfoxide formation and (ii) MsrA can repair methionine sulfoxides within cytosolic proteins. We have used electrospray ionization mass spectrometry to investigate the extent and rates of methionine sulfoxide repair within CaMox. Upon exhaustive repair by MsrA, there remains a distribution of methionine sulfoxides within functionally reactivated CaMox, which varies from three to eight methionine sulfoxides. The rates of repair of methionine sulfoxides within individual tryptic fragments of CaMox vary by a factor of 2, where methionine sulfoxides located within hydrophobic sequences are repaired in preference to methionines that are more solvent accessible within the native structure. However, no single methionine sulfoxide is completely repaired in all CaM oxiforms. Decreases in the alpha-helical content and a disruption of the tertiary structure of CaM have previously been shown to result from methionine oxidation. Repair of selected methionine sulfoxides in CaMox by MsrA results in a partial refolding of the secondary structure, suggesting that MsrA repairs methionine sulfoxides within unfolded sequences until native-like structure and function are re-attained. The ability of CaMox isolated from senescent brain to fully activate the PM Ca-ATPase following repair by MsrA suggests the specific activity of MsrA is insufficient to maintain CaM function in aging brain. These results are discussed in terms of the possible regulatory role MsrA may play in the modulation of CaM function and calcium homeostasis under conditions of oxidative stress.

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 muscle ryanodine receptor (RyR1) calcium release channel. Following derivatization with N-(1-pyrene)maleimide at engineered sites (T34C and T110C) within the N- and C-domains of CaM, contact interactions between these opposing domains of CaM resulted in excimer fluorescence that permits us to monitor conformational states of bound CaM. Complementary measurements take advantage of the unique conserved Trp within CaM-binding sequences that functions as a hydrophobic anchor in CaM binding and permits measurements of both a local and global peptide structure. We find that CaM binds with high affinity in a collapsed structure to the CaM-binding sequences of both the Ca-ATPase and RyR1, resulting in excimer formation that is indicative of contact interactions between the N- and the C-domains of CaM in complex with these CaM-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 between CaM domains in this complex. Prior to CaM association, the CaM-binding sequences of the Ca-ATPase and RyR1 are conformationally disordered. Upon CaM association, the CaM-binding sequence of the Ca-ATPase assumes a highly ordered structure. In comparison, the CaM-binding sequence of RyR1 remains conformationally disordered irrespective of CaM binding. These results suggest an important role for interdomain contact interactions between the opposing domains of CaM in stabilizing the structure of the peptide 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 different physical mechanisms of allosteric regulation, involving either the dissociation of a C-terminal regulatory domain necessary for pump activation or the modulation of intersubunit interactions to diminish RyR1 channel activity.

To investigate the role of hydrophobic interactions involving methionine side chains in facilitating the productive association between calmodulin (CaM) and the plasma membrane (PM) Ca-ATPase, we have substituted the polar amino acid Gln for Met at multiple positions in both the amino- and carboxyl-terminal domains of CaM. Conformationally sensitive fluorescence signals indicate that these mutations have little effect on the backbone fold of the carboxyl-terminal domain of CaM. The insertion of multiple Gln in either globular domain results in a decrease in the apparent affinity of CaM for the PM-Ca-ATPase. However, despite the multiple substitution of Gln for four methionines at positions 36, 51, 71, and 72 in the amino-terminal domain or for three methionines at positions 124, 144, and 145 in the carboxyl-terminal domain, these mutant CaMs are able to fully activate the PM-Ca-ATPase. Thus, although these CaM mutants have a decreased affinity for the CaM-binding site on the Ca-ATPase, they retain the ability to fully activate the Ca-ATPase at saturating concentrations of CaM. The role of individual methionines in modulating the affinity between the carboxyl terminus and the PM-Ca-ATPase was further investigated through the substitution of individual Met with Gln. Upon substitution of Met(124) and Met(144) with Gln, there is a 5- and 10-fold increase in the amount of CaM necessary to obtain half-maximal activation of the PM-Ca-ATPase, indicating that these methionine side chains participate in the high-affinity association between CaM and the PM-Ca-ATPase. However, substitution of Gln for Met(145) results in no change in the apparent affinity between CaM and the PM-Ca-ATPase, indicating that in contrast to all other known CaM targets, Met(145) does not participate in the interaction between CaM and the PM-Ca-ATPase. These results emphasize differences in the binding interactions between individual methionines in CaM and different target enzymes, and suggest that hydrophobic interactions between methionines in CaM and the binding site on the PM-Ca-ATPase are not necessary for enzyme activation. Calculation of the binding affinities of individual CaM domains associated with activation of the PM-Ca-ATPase suggests that mutations of methionines located in either domain of CaM can decrease the initial high-affinity association between CaM and the PM-Ca-ATPase, but have little effect upon the subsequent binding of the opposing globular domain. These results suggest that the initial associations between CaM and the CaM-binding sequence in the PM-Ca-ATPase are guided by nonspecific hydrophobic interactions involving both domains of CaM.

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.