No abstract

binding to the C-domain of CaM. Specifically, PEP-19 accelerates the rates of both association and dissociation of Ca 2ϩ without greatly affecting the overall K Ca of the C-domain (5). RC3 accelerates the rate of Ca 2ϩ dissociation from CaM, but has a lesser effect on the association rate, thereby decreasing the affinity of binding Ca 2ϩ to the C-domain of CaM (6). Importantly, both PEP-19 and RC3 exert these effects even when CaM is bound to CaM-dependent protein kinase II (CKII␣) (5, 6).These results suggest that PEP-19 and RC3 could have broad extrinsic effects on CaM-related signaling pathways by modulating the Ca 2ϩ binding properties of free or enzyme-bound CaM. This is consistent with the phenotype of RC3 knock-out mice, which show defects in synaptic plasticity (7), attenuated phosphorylation of hippocampal protein kinase A and C substrates (8), and altered Ca 2ϩ dynamics in cortical neurons (9). Both PEP-19 and RC3 contain an IQ motif. This rather loose consensus sequence (IQXXXRGXXXR) was first identified as the light chain binding site in conventional myosins, but was subsequently recognized as a CaM binding sequence in numerous other proteins (10). IQ motif proteins exhibit diverse modes of interaction with CaM that include Ca 2ϩ -dependent or independent binding (10), binding to both or only one domain of CaM (5, 11-13), binding multiple CaMs to multiple IQ motifs (14), and exchange of CaM between the IQ motif and other sites in the same protein (15, 16).These intriguing structure-function relationships of IQ motifs led us to identify amino acids in PEP-19 that are required to modulate Ca 2ϩ binding to CaM. We show here that the consensus IQ CaM binding motif is necessary, but not sufficient to mimic the effect of intact PEP-19 on CaM. An adjacent highly acidic amino acid sequence acts in synergy with the IQ motif to modulate Ca 2ϩ binding to the C-domain of CaM. We propose that this acidic/IQ sequence constitutes a new CaM regulatory motif. EXPERIMENTAL PROCEDURESRecombinant Proteins and Peptides-Recombinant CaM, CaM(K75C), CaM(T110C), CaM(T34C), CaM(T34C,T110C), PEP19, and RC3 were cloned, expressed, and purified as described previously (5, 6, 16 -18). The expression plasmid for the C-domain of CaM (residues 78 -148) was a generous gift * This work was supported in part by National Institutes of Health Grants GM069611 and NS038310 and Robert A. Welch Foundation Grant AU1144. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. -bound calmodulin; CaM ACR , acrylodan labeled CaM(K75C); CaM DANS , IAEDANS labeled CaM(K75C); CKII, CaM-dependent protein kinase II; RC3, neurogranin; FRET, fluorescence resonance energy transfer; MOPS, 4-morpholinepropanesulfonic acid; acrylodan, 6-acryloyl-2-dimethylaminonaphthalene; IAEDANS, 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid; DDPM,maleimide; HPLC, high performance liq...

PEP-19 is a small calmodulin (CaM)-binding protein that greatly increases the rates of association and dissociation of Ca2؉ from the C-domain of CaM, an effect that is mediated by an acidic/IQ sequence in PEP-19. We show here using NMR that PEP-19 is an intrinsically disordered protein, but with residual structure localized to its acidic/IQ motif. We also show that the k on and k off rates for binding PEP-19 to apo-CaM are at least 50-fold slower than for binding to Ca 2؉ -CaM. These data indicate that intrinsic disorder confers plasticity that allows PEP-19 to bind to either apo-or Ca 2؉ -CaM via different structural modes, and that complex formation may be facilitated by conformational selection of residual structure in the acidic/IQ sequence. PEP-19 (purkinje cell protein 4, pcp4) and RC32 (neurogranin, Ng) are small IQ-motif calmodulin (CaM)-binding proteins originally identified in the central nervous system (for reviews see Refs. 1-4). They are present at high concentrations (up to 40 -50 M), but have no known activity other than binding to CaM in the presence or absence of Ca 2ϩ . We showed that PEP-19 and RC3 have unique effects on Ca 2ϩ binding to the C-domain of CaM. Specifically, PEP-19 greatly increases the Ca 2ϩ binding k on and k off rates, but does not alter Ca 2ϩ binding affinity (5), whereas RC3 increases k off but has a lesser effect on the k on thereby decreasing the affinity of Ca 2ϩ binding to the C-domain of CaM (6). These data point to PEP-19 and RC3 as modulators or regulators of CaM signaling that act at the level of Ca 2ϩ binding to CaM. PEP-19 is of particular interest because its expression is not restricted to the central nervous system, and its pattern of expression suggests a link to Ca 2ϩ metabolism. For example,the NCBI Gene Expression Omnibus shows PEP-19 is expressed in neuroendocrine organs such as prostate, uterus, and kidney, which require high Ca 2ϩ for secretory and transport functions. PEP-19 is induced in lactating breast (7) and during osteogenic differentiation of bone marrow stem cells (8), which also have a need for high Ca 2ϩ levels. A protective role for PEP-19 against high Ca 2ϩ levels is suggested by overexpression of PEP-19, which inhibits apoptosis in PC-12 cells (9), and protects HEK293T cells against death due to Ca 2ϩ overload (10). Indeed, a general protective role for PEP-19 was put forth previously based on the fact that purkinje cells of the cerebellum and granule-cell neurons in the dentate gyrus, which have high levels of PEP-19, are largely spared from the effects of Alzheimer disease, whereas PEP-19 negative cells are severely affected (11). Conversely, cell types that are most affected by Huntington disease exhibit a significant loss of expression of PEP-19 (11).A protective role for PEP-19 against the high Ca 2ϩ in normal or pathogenic conditions emphasizes the importance of characterizing its biophysical properties and its interaction with CaM. We show here that PEP-19 is intrinsically disordered, but with residual structure localized to the acidi...

No abstract

The ability to sensitize cardiac muscle to Ca 2ϩ would have promising therapeutic potential for the treatment for Ca 2ϩ desensitization that is associated with congestive heart failure due to acute myocardial infarction and associated ischemia (1). Ideally, the mechanism of sensitization would not involve altering Ca 2ϩ transients in myocardial cells that are already metabolically challenged. Regulatory proteins located on the thin filament of cardiac muscle are logical targets for such therapeutic compounds since they respond to cellular Ca 2ϩ levels but are not involved in modulation of Ca 2ϩ transients.Cardiac troponin C (cTnC) 1 is the EF-hand Ca 2ϩ binding receptor on the thin filament of slow skeletal and cardiac striated muscle. Cardiac muscle contraction is initiated when Ca 2ϩ binds to the N-terminal regulatory metal binding site II in cTnC. Muscle relaxation occurs upon release of Ca 2ϩ from this regulatory site. This central role for cTnC makes it an attractive target for putative Ca 2ϩ -sensitizing compounds designed to modify the Ca 2ϩ dependence of cardiac muscle contraction. Indeed, precedents have been established for both desensitization and sensitization of cardiac muscle to Ca 2ϩ via mechanisms that involve cTnC. Phosphorylation of Ser-22 and -23 cardiac troponin I (cTnI), which is constitutively associated with cTnC in the troponin complex, leads to a decrease in the Ca 2ϩ sensitivity of cardiac muscle fibers and myofibrils (2) and to a decrease in the affinity of site II in cTnC (3). In contrast, a variety of small hydrophobic compounds including the calmodulin antagonists bepridil (4 -6), trifluoperazine (TFP) (7), and calmidazolium (7-9) have the opposite effect of increasing the Ca 2ϩ sensitivity of cardiac muscle preparations. Bepridil has been shown to increase the affinity of cTnC for Ca 2ϩ by decreasing the Ca 2ϩ off-rate (5, 6). Such reports have led to a search for new generations of Ca 2ϩ -sensitizing compounds with greater specificity for cTnC (1). Knowledge of the structure of cTnC and identification of potential drug binding sites on this protein would help facilitate the design or selection of Ca 2ϩ -sensitizing compounds with desired pharmacological effects. Until recently, high resolution structural information was only available for the fast skeletal isoform of TnC (sTnC) (10, 11). Structural models for Ca 2ϩ -bound cTnC have been proposed based on the structures of sTnC and calmodulin. Not surprisingly, these models predict an open N-terminal regulatory domain of Ca 2ϩ -bound cTnC, with an exposed hydrophobic surface similar to the structure of sTnC and calmodulin. Existing models for drug binding to cTnC propose that these compounds bind to this exposed Nterminal hydrophobic pocket (5, 12, 13). Recently, the NMR solution structures of Ca 2ϩ -bound intact cTnC (14), and the apo and Ca 2ϩ -saturated N-terminal regulatory fragment (15) were reported. The most striking feature of these structures is that the Ca 2ϩ -bound N-terminal regulatory domain is partially closed, result...

Two fragments of the C-terminal tail of the ␣ 1 subunit (CT1, amino acids 1538 -1692 and CT2, amino acids 1596 -1692) of human cardiac L-type calcium channel (Ca V 1.2) have been expressed, refolded, and purified. A single Ca 2؉ -calmodulin binds to each fragment, and this interaction with Ca 2؉ -calmodulin is required for proper folding of the fragment. Ca 2؉ -calmodulin, bound to these fragments, is in a more extended conformation than calmodulin bound to a synthetic peptide representing the IQ motif, suggesting that either the conformation of the IQ sequence is different in the context of the longer fragment, or other sequences within CT2 contribute to the binding of calmodulin. NMR amide chemical shift perturbation mapping shows the backbone conformation of calmodulin is nearly identical when bound to CT1 and CT2, suggesting that amino acids 1538 -1595 do not contribute to or alter calmodulin binding to amino acids 1596 -1692 of Ca V 1.2. The interaction with CT2 produces the greatest changes in the backbone amides of hydrophobic residues in the N-lobe and hydrophilic residues in the C-lobe of calmodulin and has a greater effect on residues located in Ca 2؉ binding loops I and II in the N-lobe relative to loops III and IV in the C-lobe. In conclusion, Ca 2؉ -calmodulin assumes a novel conformation when part of a complex with the C-terminal tail of the Ca V 1.2 ␣ 1 subunit that is not duplicated by synthetic peptides corresponding to the putative binding motifs.Calmodulin (CaM), 1 a ubiquitous Ca 2ϩ sensor, directly or indirectly regulates excitation-contraction coupling and other important physiological functions in cardiac myocytes (3-7). Cardiac L-type Ca 2ϩ channels (Ca V 1.2) are modulated by the interaction of the channel ␣ 1 subunit C-terminal tail with CaM (8), such that CaM binding to this region is required for both Ca 2ϩ -dependent inactivation (CDI) and Ca 2ϩ -dependent facilitation (CDF) of cardiac L-type Ca 2ϩ channels (9 -14). CDI is the process whereby the entry of Ca 2ϩ enhances channel closing during a maintained depolarization (15, 16), whereas CDF is the process whereby increased basal Ca 2ϩ or repeated transient depolarizations leads to increased channel opening (17). CDI of Ca V 1.2 appears to be driven by Ca 2ϩ binding to the C-lobe of CaM and is unaltered by the presence of intracellular Ca 2ϩ buffers (18). Although a number of sequences within the C-terminal tail of the ␣ 1 subunit appear to be capable of binding CaM (1, 9, 19 -21), it is unclear which actually contribute to CaM binding in the native channel. A sequence designated the IQ motif is required for both CDI and CDF, but the precise roles of other neighboring sequences remain to be elucidated. Peterson et al. (13) maintained that CDI of Ca V 1.2 did not require the EF hand motif. Zü hlke and Reuter (21) suggested that CDI was a cooperative process involving three noncontiguous amino acid sequences: the EF hand motif, two hydrophilic residues (asparagine and glutamic acid, residues 1630 and 1631), and the IQ motif. To compli...

Compounds that sensitize cardiac muscle to Ca 2؉ by intervening at the level of regulatory thin filament proteins would have potential therapeutic benefit in the treatment of myocardial infarctions. Two putative Ca Congestive heart failure results in desensitization of the myocardium to Ca 2ϩ as well as depressed cardiac contractility. A promising approach to the treatment of congestive heart failure is the development of positive inotropic Ca 2ϩ

No abstract