On the mechanism of interaction between calmodulin and calmodulin-dependent proteins (original) (raw)
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Proceedings of the Japan Academy. Ser. B: Physical and Biological Sciences, 1995
Chimeric proteins from chicken and yeast calmodulin were prepared, and roles of three structural domains, N-domain (1-72), central helix (73-87) and C-domain (88-148), were evaluated. Mutants with the chicken-type C-domain activated the cyclic nucleotide phosphodiesterase with small values of Kact (the concentration of calmodulin giving a half maximal activation), a property of chicken calmodulin. On the other hand mutants with the yeast-type C-domain activated the phosphodiesterase with µ M range of Kact, a property of yeast calmodulin. In activation of myosin light chain kinase, introduction of the yeast-type C-domain into chicken calmodulin increased the Kact value by more than 1000-fold with a dramatic decrease in the maximum activity (Vmax). On the other hand introduction of the chicken-type C-domain led to a profile with lower Kact and higher Vmax. Minor effects on Vmax and Kact were observed by substitution of the central helix. Although various small contributions of the N-domain were observed, a common role of the chicken-type C-domain was suggested to catch and maintain the high-affinity interaction with target enzymes. Key words : Calmodulin; Ca2+ binding protein; cyclic nucleotide phosphodiesterase; myosin light chain kinase; calmodulin-dependent enzymes. Calmodulin (CaM)**) is a ubiquitous Ca2+-binding protein which binds four Ca2+ and modulates activities of its multiple target enzymes in a Ca2tdependent manner. 1) Recent x-ray and NMR analyses have revealed the structure of CaM consisting of both the N-and C-terminal globular domain, each containing a pair of EF-hand Ca2+-binding sites).2)-5) These terminal domains are connected by a flexible 3-to 4-turn a helical region which can possibly work as a flexible tether to adjust relative positions of the terminal domains and possibly plays an important role for activation of a variety of target proteins.6~ Calmodulins from wide variety of species are *) This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (No. 03241201, and No.
Topographical mapping of calmodulin-target enzyme interaction domains
Journal of Biological Chemistry, 1989
Calmodulin derivatives, specifically biotinylated in domains I and 111, were synthesized to address the structures of calmodulin necessary for binding to its target enzymes in active conformations. By binding avidin to these biotinylated calmodulins, the role of specific sequences of the calmodulin molecule in target enzyme interactions could then be evaluated. The role of domain I in these interactions was assessed by biotinylation of Cys-27 of wheat germ calmodulin with N-ethylmaleimidobiotin. This modification did not affect the ability of this calmodulin to activate 3'-5'cyclic pucleotide phosphodiesterase (PDE) or human erythrocyte Ca2+-M&+ ATPase. The addition of avidin to form a stable calmodulin-avidin complex also did not affect activation. Bovine testes calmodulin was biotinylated on Lys-94 by calcium-dependent reaction with N-hydroxysuccinimido ester-biotin at pH 6.0. This derivative was used to probe the Ca+" binding region of domain 111. The incorporation of biotin at Lys-94 of bovine calmodulin did not affect calmodulin activation of PDE. However, compared to unmodified calmodulin, a 4-fold higher concentration of this derivative was required to fully activate the ATPase. The addition of excess avidin to this derivative abolished all activation for both PDE and the ATPase. Sites of modification were determined by sequence analysis of labeled peptides.
Functional significance of the central helix in calmodulin
Journal of Biological Chemistry, 1988
The 3-A crystal structure of calmodulin indicates that it has a polarized tertiary arrangement in which calcium binding domains I and I1 are separated from domains I11 and IV by a long central helix consisting of residues 66-92. To investigate the functional significance of the central helix, mutated calmodulins were engineered with alterations in this region. Using oligonucleotide-primed site-directed mutagenesis, Thr-79 was converted to Pro-79 to generate CaMPM. CaMPM was further mutated by insertion of Pro-Ser-Thr-Asp between Asp-78 and Pro-79 to yield CaMIM. Calmodulin, CaMPM, and CaMIM were indistinguishable in their ability to activate calcineurin and Ca2+-ATPase. All mutated calmodulins would also maximally activate cGMP-phosphodiesterase and myosin light chain kinase, however, the concentrations of CaMPM and CaMIM necessary for half-maximal activation (ICaet) were 2-and 9-fold greater, respectively, than CaM23. Conversion of the 2 Pro residues in CaMIM to amino acids that predict retention of helical secondary structure did not restore normal calmodulin activity. To investigate the nature of the interaction between mutated calmodulins and target enzymes, synthetic peptides modeled after the calmodulin binding region of smooth and skeletal muscle myosin light chain kinase were prepared and used as inhibitors of calmodulin-dependent cGMP-phosphodiesterase. The data suggest that the different kinetics of activation of myosin light chain kinase by CaM23 and CaMIM are not due to differences in the ability of the activators to bind to the calmodulin binding site of this enzyme. These observations are consistent with a model in which the length but not composition of the central helix is more important for the activation of certain enzymes. The data also support the hypothesis that calmodulin contains multiple sites for protein-protein interaction that are differentially recognized by its multiple target proteins. Calmodulin (CaM)' is a member of a growing family of
Journal of Molecular Recognition, 1995
Calmodulin, similarly to many other Ca"-activated proteins, undergoes considerable conformational changes in the presence of Ca2+ ions. These changes were followed using specific monoclonal antibodies against calmodulin. Since calmodulin is a poor immunogen due to its high phylogenetic conservancy, glutaraldehyde-crosslinked bovine brain extract, which contains a considerable amount of functionally active calmodulin complexed with its target proteins, was used as an antigen. Out of nine anti-calmodulin mAbs isolated, three (namely, CAMl, CAM2 and CAM4) were purified and characterized. MAb CAMl was identified as an IgGl while mAbs CAM2 and CAM4 belong to IgM class. Additivity ELISA showed that mAb CAMl binds to an epitope located remote from the epitopes recognized by the other two mAbs, while mAbs CAM2 and CAM4 recognize close epitopes. MAb CAMl was found to be especially sensitive to the conformational state of calmodulin in the presence of CaZ+ ions. The interactions of mAbs CAM2 and CAM4 with calmodulin are only slightly affected by Ca" removal. In addition mAb CAMl failed to recognize other calmodulin molecules, such as spinach and various plant recombinant calmodulins, while mAbs CAM2 and CAM4 share common epitopes with the above molecules
The effects of deletions in the central helix of calmodulin on enzyme activation and peptide binding
The Journal of Biological Chemistry, 1989
Using site-directed mutagenesis we have expressed in Escherichia coli three engineered calmodulins (CaM) containing deletions in the solvent-exposed region of the central helix. These are CaMA84, Glu-84 removed; CaMA83-84, Glu-83 and Glu-84 removed; and CaMA81-84, Ser-81 through Glu-84 removed. The abilities of these proteins to activate skeletal muscle myosin light chain kinase, plant NAD kinase, and bovine brain calcineurin activities were determined, as were their abilities to bind a synthetic peptide based on the calmodulin-binding domain of skeletal muscle myosin light chain kinase. Similar results were obtained with all three deletion proteins. V , values for enzymes activated by the deletion proteins are all within 10-20% of those values obtained with bacterial control calmodulin. Relative to bacterial control values, changes in K,,, or K,j values associated with the deletions are all less than an order of magnitude: KaCt values for NAD kinase and myosin light chain kinase are increased 5-7-fold, K d values for binding of the synthetic peptide are increased 4-7-fold, and K,,, values for calcineurin are increased only 1-%fold. In assays of NAD kinase and myosin light chain kinase activation some differences between bovine calmodulin and bacterial control calmodulin were observed. With NAD kinase, K,,, values for the bacterial control protein are increased 4-fold relative to values for bovine calmodulin, and V , values are increased by 50%; with myosin light chain kinase, K,,, values are increased 2fold and V , values are decreased 10-15'70 relative to those values obtained with bovine calmodulin. These differences between bacterial control and bovine calmodulins probably can be attributed to known differences in postranslational processing of calmodulin in bacterial and eucaryotic cells. No differences between bovine and control calmodulins were observed in assays of calcineurin activation or peptide binding. Our observations indicate that contacts with the deleted The nucleotide sequence(s) reported in thispaper has been submitted 504 729.
Intra- and Interdomain Effects Due to Mutation of Calcium-binding Sites in Calmodulin
Journal of Biological Chemistry, 2010
The IQ-motif protein PEP-19, binds to the C-domain of calmodulin (CaM) with significantly different k on and k off rates in the presence and absence of Ca 2؉ , which could play a role in defining the levels of free CaM during Ca 2؉ transients. The initial goal of the current study was to determine whether Ca 2؉ binding to sites III or IV in the C-domain of CaM was responsible for affecting the kinetics of binding PEP-19. EF-hand Ca 2؉binding sites were selectively inactivated by the common strategy of changing Asp to Ala at the X-coordination position. Although Ca 2؉ binding to both sites III and IV appeared necessary for native-like interactions with PEP-19, the data also indicated that the mutations caused undesirable structural alterations as evidenced by significant changes in amide chemical shifts for apoCaM. Mutations in the C-domain also affected chemical shifts in the unmodified N-domain, and altered the Ca 2؉ binding properties of the N-domain. Conversion of Asp 93 to Ala caused the greatest structural perturbations, possibly due to the loss of stabilizing hydrogen bonds between the side chain of Asp 93 and backbone amides in apo loop III. Thus, although these mutations inhibit binding of Ca 2؉ , the mutated CaM may not be able to support potentially important native-like activity of the apoprotein. This should be taken into account when designing CaM mutants for expression in cell culture.
Experimental and computational approaches for the study of calmodulin interactions
Phytochemistry, 2011
Ca 2+ , a universal messenger in eukaryotes, plays a major role in signaling pathways that control many growth and developmental processes in plants as well as their responses to various biotic and abiotic stresses. Cellular changes in Ca 2+ in response to diverse signals are recognized by protein sensors that either have their activity modulated or that interact with other proteins and modulate their activity. Calmodulins (CaMs) and CaM-like proteins (CMLs) are Ca 2+ sensors that have no enzymatic activity of their own but upon binding Ca 2+ interact and modulate the activity of other proteins involved in a large number of plant processes. Protein-protein interactions play a key role in Ca 2+ /CaM-mediated in signaling pathways. In this review, using CaM as an example, we discuss various experimental approaches and computational tools to identify protein-protein interactions. During the last two decades hundreds of CaM-binding proteins in plants have been identified using a variety of approaches ranging from simple screening of expression libraries with labeled CaM to high-throughput screens using protein chips. However, the high-throughput methods have not been applied to the entire proteome of any plant system. Nevertheless, the data provided by these screens allows the development of computational tools to predict CaM-interacting proteins. Using all known binding sites of CaM, we developed a computational method that predicted over 700 high confidence CaM interactors in the Arabidopsis proteome. Most (>600) of these are not known to bind calmodulin, suggesting that there are likely many more CaM targets than previously known. Functional analyses of some of the experimentally identified Ca 2+ sensor target proteins have uncovered their precise role in Ca 2+-mediated processes. Further studies on identifying novel targets of CaM and CMLs and generating their interaction network-''calcium sensor interactome''-will help us in understanding how Ca 2+ regulates a myriad of cellular and physiological processes.
Production of Reagents and Optimization of Methods for Studying Calmodulin-Binding Proteins
Protein Expression and Purification, 1999
Owing to subtle but potentially crucial structural and functional differences between calmodulin (CaM) of different species, the biochemical study of low-affinity CaM-binding proteins from Dictyostelium discoideum likely necessitates the use of CaM from the same organism. In addition, most of the methods used for identification and purification of CaM-binding proteins require native CaM in nonlimiting biochemical quantities. The gene encoding D. discoideum CaM has previously been cloned allowing production of recombinant protein. The present study describes the expression of D. discoideum CaM in Escherichia coli and its straightforward and rapid purification. Furthermore, we describe the optimization of a complete palette of assays to detect as little as nanogram quantities of proteins binding CaM with middle to low affinities. Purified CaM was used to raise high-affinity polyclonal antibodies suitable for immunoblotting, immunofluorescence, and immunoprecipitation experiments. The purified CaM was also used to optimize a specific and sensitive nonradioactive CaM overlay assay as well as to produce a high-capacity CaM affinity chromatography matrix. The effectiveness of this methods is illustrated by the detection of potentially novel D. discoideum CaM-binding proteins and the preparatory purification of one of these proteins, a short tail myosin I.
Ca 2+ Binding and Conformational Changes in a Calmodulin Domain �
Biochemistry Usa, 1998
Calcium activation of the C-terminal domain of calmodulin was studied using 1 H and 15 N NMR spectroscopy. The important role played by the conserved bidentate glutamate Ca 2+ ligand in the binding loops is emphasized by the striking effects resulting from a mutation of this glutamic acid to a glutamine, i.e. E104Q in loop III and E140Q in loop IV. The study involves determination of Ca 2+ binding constants, assignments, and structural characterizations of the apo, (Ca 2+ ) 1 , and (Ca 2+ ) 2 states of the E104Q mutant and comparisons to the wild-type protein and the E140Q mutant [Evenäs et al. (1997) Biochemistry 36, 3448-3457]. NMR titration data show sequential Ca 2+ binding in the E104Q mutant. The first Ca 2+ binds to loop IV and the second to loop III, which is the order reverse to that observed for the E140Q mutant. In both mutants, the major structural changes occur upon Ca 2+ binding to loop IV, which implies a different response to Ca 2+ binding in the N-and C-terminal EF-hands. Spectral characteristics show that the (Ca 2+ ) 1 and (Ca 2+ ) 2 states of the E104Q mutant undergo global exchange on a 10-100 µs time scale between conformations seemingly similar to the closed and open structures of this domain in wild-type calmodulin, paralleling earlier observations for the (Ca 2+ ) 2 state of the E140Q mutant, indicating that both glutamic acid residues, E104 and E140, are required for stabilization of the open conformation in the (Ca 2+ ) 2 state. To verify that the NOE constraints cannot be fulfilled in a single structure, solution structures of the (Ca 2+ ) 2 state of the E104Q mutant are calculated. Within the ensemble of structures the precision is good. However, the clearly dynamic nature of the state, a large number of violated distance restraints, ill-defined secondary structural elements, and comparisons to the structures of calmodulin indicate that the ensemble does not provide a good picture of the (Ca 2+ ) 2 state of the E104Q mutant but rather represents the distance-averaged structure of at least two distinct different conformations.