High Affinity Calmodulin Binding Research Articles

Calmodulin-Cork Model of Gap Junction Channel Gating-One Molecule, Two Mechanisms.

The Calmodulin-Cork gating model is based on evidence for the direct role of calmodulin (CaM) in channel gating. Indeed, chemical gating of cell-to-cell channels is sensitive to nanomolar cytosolic calcium concentrations [Ca2+]i. Calmodulin inhibitors and inhibition of CaM expression prevent chemical gating. CaMCC, a CaM mutant with higher Ca2+-sensitivity greatly increases chemical gating sensitivity (in CaMCC the NH2-terminal EF-hand pair (res. 9–76) is replaced by the COOH-terminal pair (res. 82–148). Calmodulin colocalizes with connexins. Connexins have high-affinity CaM binding sites. Several connexin mutants paired to wild-type connexins have a high gating sensitivity that is eliminated by inhibition of CaM expression. Repeated transjunctional voltage (Vj) pulses slowly and progressively close a large number of channels by the chemical/slow gate (CaM lobe). At the single-channel level, the chemical/slow gate closes and opens slowly with on-off fluctuations. The model proposes two types of CaM-driven gating: “Ca-CaM-Cork” and “CaM-Cork”. In the first, gating involves Ca2+-induced CaM-activation. In the second, gating takes place without [Ca2+]i rise. The Ca-CaM-Cork gating is only reversed by a return of [Ca2+]i to resting values, while the CaM-Cork gating is reversed by Vj positive at the gated side.

Two EF-hand motifs in ryanodine receptor calcium release channels contribute to isoform-specific regulation by calmodulin

The mammalian ryanodine receptor Ca2+ release channel (RyR) has a single conserved high affinity calmodulin (CaM) binding domain. However, the skeletal muscle RyR1 is activated and cardiac muscle RyR2 is inhibited by CaM at submicromolar Ca2+. This suggests isoform-specific domains are involved in RyR regulation by CaM. To gain insight into the differential regulation of cardiac and skeletal muscle RyRs by CaM, RyR1/RyR2 chimeras and mutants were expressed in HEK293 cells, and their single channel activities were measured using a lipid bilayer method. All RyR1/RyR2 chimeras and mutants were inhibited by CaM at 2μM Ca2+, consistent with CaM inhibition of RyR1 and RyR2 at micromolar Ca2+ concentrations. An RyR1/RyR2 chimera with RyR1 N-terminal amino acid residues (aa) 1–3725 and RyR2 C-terminal aa 3692–4968 were inhibited by CaM at <1μM Ca2+ similar to RyR2. In contrast, RyR1/RyR2 chimera with RyR1 aa 1–4301 and RyR2 4254–4968 was activated at <1μM Ca2+ similar to RyR1. Replacement of RyR1 aa 3726–4298 with corresponding residues from RyR2 conferred CaM inhibition at <1μM Ca2+, which suggests RyR1 aa 3726–4298 are required for activation by CaM. Characterization of additional RyR1/RyR2 chimeras and mutants in two predicted Ca2+ binding motifs in RyR1 aa 4081–4092 (EF1) and aa 4116–4127 (EF2) suggests that both EF-hand motifs and additional sequences in the large N-terminal regions are required for isoform-specific RyR1 and RyR2 regulation by CaM at submicromolar Ca2+ concentrations.

Evaluation of the calmodulin-SOX9 interaction by "magnetic fishing" coupled to mass spectrometry.

Disruption of calmodulin (CaM)-based protein interactions has been touted as a potential means for modulating several disease pathways. Among these is SOX9, which is a DNA binding protein that is involved in chrondrocyte differentiation and regulation of the hormones that control sexual development. In this work, we employed a "magnetic fishing"/mass spectrometry assay in conjunction with intrinsic fluorescence to examine the interaction of CaM with the CaM-binding domain of SOX9 (SOX-CAL), and to assess the modulation of this interaction by known anti-CaM compounds. Our data show that there is a high affinity interaction between CaM and SOX-CAL (27±9 nM), and that SOX-CAL bound to the same location as the well-known CaM antagonist melittin; unexpectedly, we also found that addition of CaM-binding small molecules initially produced increased SOX-CAL binding, indicative of binding to both the well-known high-affinity CaM binding site and a second, lower-affinity binding site.

Synergetic Effect of Recoverin and Calmodulin on Regulation of Rhodopsin Kinase

Phosphorylation of photoactivated rhodopsin by rhodopsin kinase (RK or GRK1), a first step of the phototransduction cascade turnoff, is under the control of Ca2+/recoverin. Here, we demonstrate that calmodulin, a ubiquitous Ca2+-sensor, can inhibit RK, though less effectively than recoverin does. We have utilized the surface plasmon resonance technology to map the calmodulin binding site in the RK molecule. Calmodulin does not interact with the recoverin-binding site within amino acid residues M1-S25 of the enzyme. Instead, the high affinity calmodulin binding site is localized within a stretch of amino acid residues V150-K175 in the N-terminal regulatory region of RK. Moreover, the inhibitory effect of calmodulin and recoverin on RK activity is synergetic, which is in agreement with the existence of separate binding sites for each Ca2+-sensing protein. The synergetic inhibition of RK by both Ca2+-sensors occurs over a broader range of Ca2+-concentration than by recoverin alone, indicating increased Ca2+-sensitivity of RK regulation in the presence of both Ca2+-sensors. Taken together, our data suggest that RK regulation by calmodulin in photoreceptor cells could complement the well-known inhibitory effect of recoverin on RK.

Interdomain Interactions Control Ca2+-Dependent Potentiation in the Cation Channel TRPV4

Several Ca2+-permeable channels, including the non-selective cation channel TRPV4, are subject to Ca2+-dependent facilitation. Although it has been clearly demonstrated in functional experiments that calmodulin (CaM) binding to intracellular domains of TRP channels is involved in this process, the molecular mechanism remains elusive. In this study, we provide experimental evidence for a comprehensive molecular model that explains Ca2+-dependent facilitation of TRPV4. In the resting state, an intracellular domain from the channel N terminus forms an autoinhibitory complex with a C-terminal domain that includes a high-affinity CaM binding site. CaM binding, secondary to rises in intracellular Ca2+, displaces the N-terminal domain which may then form a homologous interaction with an identical domain from a second subunit. This represents a novel potentiation mechanism that may also be relevant in other Ca2+-permeable channels.

Structural requirements for calmodulin binding to membrane‐associated guanylate kinase homologs

Effector molecules such as calmodulin modulate the interactions of membrane-associated guanylate kinase homologs (MAGUKs) and other scaffolding proteins of the membrane cytoskeleton by binding to the Src homology 3 (SH3) domain, the guanylate kinase (GK) domain, or the connecting HOOK region of MAGUKs. Using surface plasmon resonance, we studied the interaction of members of all four MAGUK subfamilies--synapse-associated protein 97 (SAP97), calcium/calmodulin-dependent serine protein kinase (CASK), membrane palmitoylated protein 2 (MPP2), and zona occludens (ZO) 1--and calmodulin to determine interaction affinities and localize the binding site. The SH3-GK domains of the proteins and derivatives thereof were expressed in E. coli and purified. In all four proteins, high-affinity calmodulin binding was identified. CASK was shown to contain a Ca2+-dependent calmodulin binding site within the HOOK region, overlapping with a protein 4.1 binding site. In ZO1, a Ca2+-dependent calmodulin binding site was detected within the GK domain. The equilibrium dissociation constants for MAGUK-calmodulin interaction were found to range from 50 nM to 180 nM. Sequence analyses suggest that binding sites for calmodulin have evolved independently in at least three subfamilies. For ZO1, pulldown of GST-calmodulin was shown to occur in a calcium-dependent manner; moreover, molecular modeling and sequence analyses predict conserved basic residues to be exposed on one side of a helix. Thus, calmodulin binding appears to be a common feature of MAGUKs, and Ca2+-activated calmodulin may serve as a general regulator to affect the interactions of MAGUKs and various components of the cytoskeleton.

Role of calmodulin methionine residues in mediating productive association with cardiac ryanodine receptors

Calmodulin (CaM) binds to the cardiac ryanodine receptor Ca2+ release channel (RyR2) with high affinity and may act as a regulatory channel subunit. Here we determine the role of CaM Met residues in the productive association of CaM with RyR2, as assessed via determinations of [3H]ryanodine and [35S]CaM binding to cardiac muscle sarcoplasmic reticulum (SR) vesicles. Oxidation of all nine CaM Met residues abolished the productive association of CaM with RyR2. Substitution of the COOH-terminal Mets of CaM with Leu decreased the extent of CaM inhibition of cardiac SR (CSR) vesicle [3H]ryanodine binding. In contrast, replacing the NH2-terminal Met of CaM with Leu increased the concentration of CaM required to inhibit CSR [3H]ryanodine binding but did not alter the extent of inhibition. Site-specific substitution of individual CaM Met residues with Gln demonstrated that Met124 was required for both high-affinity CaM binding to RyR2 and for maximal CaM inhibition. These results thus identify a Met residue critical for the productive association of CaM with RyR2 channels.

Ca2+-dependent and Ca2+-independent Calmodulin Binding Sites in Erythrocyte Protein 4.1: IMPLICATIONS FOR REGULATION OF PROTEIN 4.1 INTERACTIONS WITH TRANSMEMBRANE PROTEINS

In vitro protein binding assays identified two distinct calmodulin (CaM) binding sites within the NH(2)-terminal 30-kDa domain of erythrocyte protein 4.1 (4.1R): a Ca(2+)-independent binding site (A(264)KKLWKVCVEHHTFFRL) and a Ca(2+)-dependent binding site (A(181)KKLSMYGVDLHKAKDL). Synthetic peptides corresponding to these sequences bound CaM in vitro; conversely, deletion of these peptides from a 30-kDa construct reduced binding to CaM. Thus, 4.1R is a unique CaM-binding protein in that it has distinct Ca(2+)-dependent and Ca(2+)-independent high affinity CaM binding sites. CaM bound to 4.1R at a stoichiometry of 1:1 both in the presence and absence of Ca(2+), implying that one CaM molecule binds to two distinct sites in the same molecule of 4.1R. Interactions of 4.1R with membrane proteins such as band 3 is regulated by Ca(2+) and CaM. While the intrinsic affinity of the 30-kDa domain for the cytoplasmic tail of erythrocyte membrane band 3 was not altered by elimination of one or both CaM binding sites, the ability of Ca(2+)/CaM to down-regulate 4. 1R-band 3 interaction was abrogated by such deletions. Thus, regulation of protein 4.1 binding to membrane proteins by Ca(2+) and CaM requires binding of CaM to both Ca(2+)-independent and Ca(2+)-dependent sites in protein 4.1.

A Mechanism for Calmodulin (CaM) Trapping by CaM-kinase II Defined by a Family of CaM-binding Peptides

Autophosphorylation of Ca2+/calmodulin (CaM)-dependent protein kinase II (CaM-kinase II) induces a striking >1,000-fold increase in its affinity for CaM, which has been called CaM trapping. Two peptides modeled after the CaM binding domain of CaM-kinase II were previously shown to kinetically resemble CaM binding to phosphorylated and dephosphorylated forms of the enzyme, thus providing a model system with which to define the molecular basis of CaM trapping. In this report, the specific contribution of each amino acid to the rates of association and dissociation, and the overall Kd of CaM binding to CaM-kinase II was determined using an overlapping peptide family, and a fluorescently labeled CaM. The association rate constants were similar for the entire family of peptides and ranged from 8 x 10(7) to 32 x 10(7) M-1 s-1. In contrast, the dissociation rate constants for the peptides varied by >3500-fold and ranged from 0.26 to 7 x 10(-5) s-1. These rate constants yield overall Kd values for binding CaM to the peptides that range from 2 x 10(-9) M to 2 x 10(-13) M. Extending the low affinity CaM-binding peptide, CKII(296-312), to include 293Phe-Asn-Ala295 provided the single largest contribution to the decreased dissociation rate constant, 1,300-fold. It was further shown using Ala-substituted peptides that the basic residues 296Arg-Arg-Lys299 were also essential for slow CaM dissociation; however, their contribution was realized only when 293Phe-Asn-Ala295 were present. These results suggest a plausible model in which autophosphorylation of CaM-kinase II leads to a conformational change in the region of 293Phe-Asn-Ala295 which makes these residues accessible for binding to CaM. As a consequence of these changes, further CaM contacts with 296Arg-Arg-Lys299 are established leading to high affinity CaM binding or "CaM trapping."

Characterization of mutant Bordetella pertussis adenylate cyclase toxins with reduced affinity for calmodulin. Implications for the mechanism of toxin entry into target cells.

Bordetella pertussis secretes a calmodulin-stimulated adenylate cyclase toxin (CyaA) that is one of the major virulence factors of this organism. The toxin is able to enter various types of eukaryotic cells where, upon activation by calmodulin, it catalyzes the production of non-physiological amounts of cyclic AMP. The mechanism of toxin entry into target cells is unknown, although it has been shown that it does not involve receptor-mediated endocytosis. The adenylate cyclase toxin exhibits a very high affinity for calmodulin, and it has been proposed that the energy of calmodulin-binding to CyaA might be required for the entry of the toxin into the target cells [Oldenburg, D.J., Gross, M. K., Wong, C. S. & Storm, D. R. (1992) Biochemistry 31, 8884-8891]. In the present study, we have reexamined this issue by analyzing the cytotoxicity of various modified CyaA toxins that have altered calmodulin affinity. We show that despite their low affinity for calmodulin (at least 1000-times less than that of the wild type CyaA), these toxins were able to efficiently deliver their catalytic domain into the cytoplasm of the target cells, erythrocytes. These results demonstrate that high-affinity calmodulin binding is not required for the entry of B. pertussis adenylate cyclase into eukaryotic cells. However, the high-affinity of CyaA for calmodulin is crucial for an efficient synthesis of cAMP within the target cells.

The calcium-dependent transient inactivation of recombinant NMDA receptor-channel does not involve the high affinity calmodulin binding site of the NR1 subunit

N-methyl- d-aspartate (NMDA) receptor function can be regulated by direct binding of calmodulin to a low and high affinity (C1 exon cassette) site in the C-terminal region of the NR1 subunit. To evaluate the involvement of the high affinity binding site in the transient inactivation of the NMDA receptor-channels by intracellular calcium, several splice variants of the NR1 subunit have been individually co-transfected with the NR2A subunit in HEK 293 cells. The transient Ca 2+ induced inactivation (40–50%) of the heteromeric receptors was similar whether the NR1 variants contained (NR1–1a, 1b) or lacked (NR1–2a, 2b, 4a, 4b) the C1 exon cassette bearing the high affinity binding site for calmodulin. This demonstrates that this site is not involved in the Ca 2+ dependent transient inactivation of NMDA receptors.

Ca2⁺-Calmodulin Binds to the Carboxyl-terminal Domain of Dystrophin

The unique COOH-terminal domain of dystrophin (mouse dystrophin protein sequences 3266-3678) was expressed as a chimeric fusion protein (with the maltose-binding protein), and its binding to calmodulin was assessed. This fusion protein, called DysS9, bound to calmodulin-Sepharose, bound biotinylated calmodulin, caused characteristic changes in the fluorescence emission spectrum of dansyl-calmodulin, and had an apparent affinity for dansyl-calmodulin of 54 nM. Binding in each case was Ca2+-dependent. The maltose-binding protein does not bind calmodulin, and thus binding resides in the dystrophin-derived sequences. Deletion mutation experiments further localize the high affinity calmodulin binding to mouse dystrophin protein sequences 3293-3349, and this domain contains regions with chemical characteristics found in the calmodulin-binding sequences in other proteins. The COOH-terminal domain provides sites of attachment of dystrophin to membrane proteins, and calmodulin binding may modulate these interactions.

The polymeric immunoglobulin receptor is the major high affinity calmodulin binding protein in rat liver endosomes

The polymeric immunoglobulin receptor is the major high affinity calmodulin binding protein in rat liver endosomes

The calmodulin binding domain of nitric oxide synthase and adenylyl cyclase.

Peptides corresponding to regions of the calmodulin-activated NO-synthase and of the calmodulin dependent adenylyl cyclase, which could represent the calmodulin binding domains of the two proteins, have been synthesized and tested for calmodulin binding. The chosen peptides were those in the sequence of the two proteins which most closely corresponded to the accepted general properties of the calmodulin binding domains, i.e., a hydrophobic sequence containing basic amino acids. In the case of the NO-synthase, the putative high-affinity calmodulin binding domain was identified by urea gel electrophoresis and fluorescence spectroscopy with dansylcalmodulin as peptide NO-30 (amino acids 725-754). The highest affinity calmodulin binding site of the calmodulin-dependent adenylyl cyclase was assigned to peptide AC-28 (amino acids 495-522) by titration with dansylcalmodulin and by the ability to inhibit the calmodulin-stimulated activity of purified calmodulin-stimulated adenylyl cyclase. The sequence 495-522 is located in the unit protruding into the cytosol from the sixth putative transmembrane domain of the molecule. It has the typical hydrophobic/basic composition of canonical calmodulin binding domains, and also contains, like most calmodulin binding domains, an aromatic amino acid in its N-terminal portion. It also contains two Cys residues in the central portion, which is an unusual feature of the calmodulin binding domain of this enzyme.

Virulence of a Bordetella pertussis strain expressing a mutant adenylyl cyclase with decreased calmodulin affinity

Bordetella pertussis, the pathogen responsible for whooping cough, produces a toxic calmodulin-sensitive adenylyl cyclase which enters animal cells and increases intracellular cAMP. A point mutant of B. pertussis with abolished adenylyl cyclase catalytic activity was over 1000-fold less pathogenic to newborn mice than wild-type bacteria, demonstrating the importance of the adenylyl cyclase for B. pertussis virulence (Gross et al. ). The B. pertussis adenylyl cyclase is highly sensitive to calmodulin with an apparent Kd for calmodulin of approximately 1 nM. The importance of this high-affinity calmodulin binding for virulence in vivo was examined by the creation of a B. pertussis point mutant (Trp-242 to Glu-242) with 200-fold lower calmodulin affinity than the native enzyme. This mutant B. pertussis strain retained its virulence in a newborn mouse model of pertussis, but the time course for establishment of a lethal infection in vivo was significantly delayed for the mutant strain. These data illustrate that high-affinity calmodulin binding is not obligatory for the activity of this toxin but is important for the rate for establishment of a lethal infection.

Regulation of peptide-calmodulin complexes by protein kinase C in vivo.

We used the freshwater protozoan Paramecium tetraurelia to investigate the potential regulation by protein kinase C of calmodulin interactions with binding peptides in intact cells. In these organisms, an action potential results in membrane depolarization and a period of backward swimming; repolarization and a return to forward swimming requires the presence of normal calmodulin. We postulated that injection of high-affinity calmodulin binding peptides might interfere with repolarization and thus prolong the period of membrane depolarization. Synthetic peptides spanning the protein kinase C phosphorylation site/calmodulin-binding domains of the myristoylated alanine-rich C-kinase substrate (MARCKS) and the MARCKS-related protein (also known as F52 or MacMARCKS) were injected into cells; these caused a 2- to 3-fold increase in the duration of backward swimming. Similar changes were seen with two other calmodulin-binding peptides. This behavioral response could be prevented by coinjecting calmodulin. Activation of Paramecium protein kinase C with an active phorbol ester completely reversed (within 3 min) the behavioral effects of the normal MARCKS and MARCKS-related protein peptides. Injection of a nonphosphorylatable peptide, in which alanines were substituted for serines, resulted in the usual behavioral response; however, this was not reversed by phorbol ester treatment. The corresponding aspartate-substituted peptide, which has a 10-fold lower affinity for calmodulin, did not prolong backward swimming. These data suggest that these peptides can form complexes with calmodulin at the calcium concentrations that prevail in intact Paramecium cells and that such complexes can be disrupted by protein kinase C phosphorylation of the peptides.

Analysis by mutagenesis of the ATP binding site of the gamma subunit of skeletal muscle phosphorylase kinase expressed using a baculovirus system.

Active gamma subunit of skeletal muscle phosphorylase kinase has been obtained by expression of the rat soleus cDNA in a baculovirus system. The protein exhibited the expected pH 6.8/8.2 activity ratio of 0.6, and its activity was insensitive to Ca2+ addition, indicating that it was free gamma subunit and not a gamma subunit-calmodulin complex. It was stimulated approximately 2-fold by Ca(2+)-calmodulin addition, demonstrating that it had retained high-affinity calmodulin binding. By site-directed mutagenesis, we have examined the role of six of the amino acids that constitute the consensus ATP binding site of the protein kinase, which in the gamma subunit is represented by the sequence 26Gly.Arg.Gly.Val.Ser.Ser.Val.Val33. Changes were evaluated by the kinetic determination of the dissociation constants of gamma-ATP, gamma-ADP, gamma-AMP.PCP, and gamma-phosphorylase and the maximum catalytic activity. The mutants Ser26-gamma, Ser29-gamma, Phe30-gamma, and Gly31-gamma each exhibited an essentially identical dissociation constant for gamma subunit phosphorylase, indicating that these mutations had not caused a global alteration in the protein structure but were limited to changes in the nucleotide binding site domain. Substitution of either Val33 (by Gly) or Gly28 (by Ser), two of the most conserved residues in all protein kinases, resulted in enzyme with marginally detectable activity. In noted contrast, the Ser26 mutant, which substituted the first glycine of the consensus glycine trio motif, and which is also very highly conserved, retained at least 25% of the enzymatic activity. The Gly31 substitution, which restored a glycine to a position characteristic for most protein kinases, had little overall effect upon the maximum rate of catalysis. Restoration of Ser30 to the more typical phenylalanine, which is present in most protein kinases, had minimal effect on catalysis. These data provide the first direct evaluation of the roles that different residues play within this consensus glycine trio/valine motif of the protein kinases, which up to now have only been surmised to be of importance because of their conservation. Two unexpected findings are that for one residue that is very conserved (Gly26) there is some flexibility of substitution not apparent from the evolutionary conservation and that a second quite conserved residue in protein kinases (equivalent to Gly at position 31) does not produce a protein optimized for nucleotide binding.

High-affinity calmodulin binding is required for the rapid entry of Bordetella pertussis adenylyl cyclase into neuroblastoma cells.

Bordetella pertussis produces a calmodulin-stimulated adenylyl cyclase that invades animal cells and raises intracellular cAMP levels [Confer, D. L., & Eaton, J. W. (1982) Science 217, 948-950; Shattuck, R. L., & Storm, D. R. (1985) Biochemistry 24, 6323-6328]. The mechanism for invasion of animal cells by this enzyme has not been defined, but there is considerable evidence that it does not enter by receptor-mediated endocytosis [Gordon, V. M., Leppla, S. H., & Hewlett, E. L. (1988) Infect. Immun. 56, 1066-1069; Donovan, M. G., & Storm, D. R. (1990) J. Cell. Physiol. 145, 444-449]. In this study, the importance of high-affinity calmodulin (CaM) binding for entry of the enzyme into neuroblastoma cells was evaluated using a mutant enzyme that has significantly lower affinity for calmodulin than the wild-type enzyme. Oligonucleotide-directed site-specific mutagenesis was used to create a point mutant at a critical tryptophan residue (Trp-242) within the proposed CaM binding domain of the B. pertussis adenylyl cyclase. Substitution of Trp-242 with Glu lowered the apparent affinity of the enzyme for calmodulin by 250-fold; however, the maximal enzyme activity in the presence of saturating calmodulin was equivalent to the wild-type enzyme. The Glu-242 mutant adenylyl cyclase was returned to B. pertussis by homologous recombination, and the enzyme produced by this strain was examined for invasion of neuroblastoma cells. Although the mutant enzyme stimulated the production of intracellular cAMP in neuroblastoma cells, the rate of cAMP accumulation was at least 10-fold lower than that caused by the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

High and intermediate affinity calmodulin binding domains of the alpha and beta subunits of phosphorylase kinase and their potential role in phosphorylation-dependent activation of the holoenzyme.

Phosphorylase kinase is a calcium-regulated multimeric enzyme of composition (alpha beta gamma delta)4, which contains calmodulin as the integral delta subunit and also is activated further by addition of extrinsic calmodulin. Previous studies by Dasgupta, M., Honeycutt, T., and Blumenthal, D.K. ((1989) J. Biol. Chem. 264, 17156-17163) have identified gamma 302-326 and gamma 342-366 as two calmodulin binding regions. Using peptides that were synthesized based on alpha and beta primary structure and that were predicted to contain the basic amphiphilic alpha-helix motif thought important for calmodulin binding, four additional potential calmodulin binding domains have now been identified: one of high affinity, beta 770-794; two of intermediate affinity, beta 5-28 and beta 920-946; and one with marginally low affinity, alpha 1070-1093. Peptide beta 770-794 was of higher calmodulin affinity than either gamma 302-326 or gamma 342-366; it was of higher affinity than the model synthetic peptide IV defined by O'Neil, K.T., and DeGrado, W.F. ((1990) Trends Biochem. Sci. 15, 59-64); and it is currently the most potent calmodulin-binding peptide so far described. Correlated with their affinity for calmodulin, all six phosphorylase kinase-derived peptides and several other established calmodulin-binding peptides inhibited phosphorylase kinase previously activated by cAMP-dependent phosphorylation, reducing its activity to the level of the nonactivated enzyme. However, these peptides did not inhibit (and some peptides slightly activated) the nonphosphorylated enzyme. Even in the presence of these peptides both activated and nonactivated enzyme remained fully Ca(2+)-dependent. The beta 770-794 peptide has at least a 5-fold greater calmodulin binding affinity than the holo-phosphorylase kinase. This, and its higher affinity for calmodulin than either of the sites on the gamma subunit, raises the possibility that in the native enzyme it may be involved in binding the intrinsic delta subunit. Further, inhibition of activated but not nonactivated enzyme by calmodulin-binding peptides would suggest that the phosphorylation-dependent activation of phosphorylase kinase may be mediated by changes in the binding interactions of the intrinsic calmodulin delta subunit.

Basic residues are important for Ca2+/calmodulin binding and activation but not autoinhibition of rabbit skeletal muscle myosin light chain kinase

Several allosterically modulated protein kinases have been shown to be regulated by an autoinhibitory domain located within the kinase molecules. The inhibitory domain has been proposed to act as a "pseudosubstrate" inhibitor binding to the substrate binding site of the kinase, thereby blocking the binding of the enzyme's true substrate. In this report, site-directed mutagenesis has been used to further investigate the mechanism of activation of the inhibitory domain of rabbit skeletal muscle myosin light chain kinase. Basic residues within the pseudosubstrate domain (572-573, 577-579, 580-581), which are analogous to the important substrate determinants of the myosin light chain, were found not to be required in order to maintain the kinase in an inhibited state. Two groups of these residues (577-579 and 581-582) were, however, found to be important for high affinity calmodulin binding to the kinase. These data suggest that the autoinhibitory domain of myosin light chain kinase may not function by directly mimicking the light chain substrate.