title: The effects of deletions in the central helix of calmodulin on enzyme activation and peptide binding.
abstract: 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 CaM delta 84, Glu-84 removed; CaM delta 83-84, Glu-83 and Glu-84 removed; and CaM delta 81-84, 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. Vm 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 Kact or Kd 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, Kd values for binding of the synthetic peptide are increased 4-7-fold, and Kact values for calcineurin are increased only 1-3-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, Kact values for the bacterial control protein are increased 4-fold relative to values for bovine calmodulin, and Vm values are increased by 50%; with myosin light chain kinase, Kact values are increased 2-fold and Vm values are decreased 10-15% 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 residues, Ser-81 through Glu-84, are not critical in the calmodulin-target complexes we have evaluated. Formation of these calmodulin-target complexes also does not appear to be greatly affected by the global alterations in the structure of calmodulin that are associated with the deletions. In models in which the central helix is maintained in the altered calmodulins, each deleted residue causes the two lobes of calmodulin to be twisted 100 degrees relative to one another and brought 1.5 A closer together.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Biology, University of Virginia, Charlottesville 22901.
authors.forename: A
authors.initials: A
authors.lastname: Persechini
authors.forename: D K
authors.initials: DK
authors.lastname: Blumenthal
authors.forename: H W
authors.initials: HW
authors.lastname: Jarrett
authors.forename: C B
authors.initials: CB
authors.lastname: Klee
authors.forename: D O
authors.initials: DO
authors.lastname: Hardy
authors.forename: R H
authors.initials: RH
authors.lastname: Kretsinger
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D019703:Calcineurin; D002147:Calmodulin / Q000378:metabolism* / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D002417:Cattle; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D009154:Mutation; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor); D010944:Plants / Q000201:enzymology; D011487:Protein Conformation; D011817:Rabbits; D013329:Structure-Activity Relationship
chemical_list: D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D010455:Peptides; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C021844:NAD kinase; D009219:Myosin-Light-Chain Kinase; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases